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INFECTIOUS DISEASES DIAGNOSTICS (B) SUBCELLULAR

PATHOGENS(9 CE Hours)

Learning objectives ! List the steps in the initial diagnostic overview. ! Explain the importance of a local

epidemiological background and considerations.

! Identify how and where humans and animals can become infected.

! Explain how to diagnose and monitor the progress of infectious diseases.

! List the contraindications and differential diagnostic considerations.

IntroductionThe initial triage establishes facts about the animal’s ease of breathing, appetite, fever, anemia (color of the gums, conjunctivae), debilitation, weakness, level of awareness, muscular coordination, gait, posture and mobility, gastrointestinal difficulties (vomiting, diarrhea, appearance of vomit and stool), distension of abdomen and the ability to urinate (volume and coloration). It should include the animal’s history, contacts with other animals, travels and present medication.

The initial impression a diseased animal presents must be placed in the present-day’s regional epidemiological context, such as whether it is mosquito season, diseases in the neighborhood, and the presence and prevalence of vectors or carriers of potential infectious agents.

Initial sample collection for diagnosis should include samples for the study of its microbiology, serology, pathology, hematology, and clinical biochemistry aspects. Samples should be kept at refrigerator temperature rather than frozen, provided they can reach the investigating laboratory within less than 72 hours.

After categorizing the disease presentation based on its history and initial triage, our assumptions must be confirmed and verified. Isolate and identify the causative pathogen: viruses and aberrant proteins known as prions, their antigens and the antibody induced by these antigens as well as the general immune responsiveness of the patient and the likely effectiveness of available treatment.

Because the number of global zoonoses is overwhelming, only viral and subviral pathogens reported in the United States are included here.

Safety concerns More than 60 percent of emerging human diseases are zoonoses, i.e., derived from animals. It is therefore critical that all laboratory workers concerned with early diagnostic processes be aware and trained in proper safety procedures.

Facilities should be provided with airlocks and negative internal air pressure. All surfaces should be nonporous and easily sterilizable. Protective clothing, including booties, when entering and the removal of protective covers when exiting the work area is absolutely essential. All protective laboratory clothing, unless disposable, should be autoclaved before going through the standard laundering process. Technicians should wear face masks, goggles, head cover and gloves; and eating, drinking or mouth-pipetting should not be allowed.

Ideally, laminar flow hoods under negative pressure should be available to protect from aerosols produced by homogenization and sonication of samples being processed. Particular care should be taken with the manipulation, shipment, storage and disposal of fresh and often highly infectious test samples stored for cataloguing and reference purposes. As long as it does not interfere with testing purposes, samples should be preserved in disinfecting preservatives, such as 10 percent buffered formalin or similar.

PathogensThe parasites considered here are viral and subviral pathogens. The parasite lives in symbiosis with its host organism and benefits from it by exploiting it for food, habitat and spreading its kind. In the ideal parasite world, both host and parasite benefit and need each other to survive. If the parasite lives to kill its host, it terminates its own chance of survival. This review only covers pathogenic parasites that will harm their hosts, weaken or kill them.

Viruses are about 1/100 the size of a bacterium, and are obligatory intracellular organisms usurping live cells for their replication and growth. They enter the cell through endocytosis. The type of cell substrate required for their growth, be it in cell culture or in the laboratory animal, and the appearance of damage produced is usually descriptive for the virus species involved.

They do not metabolize by themselves, but must instruct the host cell to synthesize and assemble new virus. While multiplying rapidly within their host cells, changes or flaws in the virus’s genetic make-up, i.e. mutations, will occur and be expressed immediately in their offspring. Any

chance of success of the mutant form depends on the host environment.

Virions consist of: ■ Their genetic matter, either DNA or RNA,

carrying the genetic information for their make-up.

■ A protein shell, surrounding the genes. ■ Occasionally, a lipid envelope surrounding

the protein coat.

They vary in shape: helical, polyhedral (icosahedron), enveloped and pleomorphic.

Viruses are spread by many ways, through blood-sucking insects (vectors), aerosols, coughing and sneezing, the fecal-oral route, consumption of contaminated food and water, and by transmission from person to person via direct contact (sex).

Prions (proteinaceous infection) are very small, infectious abnormal proteins, obligatorily intracellular, do not contain nucleic acids and cause diseases like scrapie, bovine spongiform encephalopathy and Creutzfeldt–Jakob disease. Once inside the cell, it co-opts the protein manufacturing mechanism to reproduce proteins like its own. They affect neural tissues exclusively. All are untreatable and fatal.

CharacterizationArenaviridaeArenaviridae are single-stranded spherical RNA viruses about 110 to 130 nm in diameter, enveloped in a lipid coat. Their genome is made up of two segments, one long and one short. They enter the host cell as they attach to its receptors via their envelope glycoprotein and are taken up into the cell through endocytosis – invagination of the plasma membrane with the virus membrane fusing to the membrane of the resulting cell vesicle.

The arenaviridae are grouped into two serocomplexes:

■ The Old World complex, which includes lymphocytic choriomeningitis virus and Lassa virus, among others (Ippy virus, Lujo virus, Mobala virus, Mopeia virus) not usually found in the U.S.

■ The New World, Tacaribe virus complex, which includes Whitewater Arroyo virus and Tamiami virus. There are many others not found in the U.S.: Amapari virus, Chapare virus, Flexal virus, Guanarito virus (Venezuelan hemorrhagic fever), Junin virus (Argentine hemorrhagic fever), Latino

Arenaviridae: Group V/(-)ssRNA/2 segments/lipid envelope/spherical/110-130nm

Serocomplex Virus Distribution Host/reservoir Vector Disease in man

Old World Lymphocytic choriomeningitis virus

Worldwide Rodents, guinea pigs, hamsters, dogs, monkeys

Secretions, droppings Fever, meningitis, lymphocytic infiltration in CSF

Lassa virus West Africa Mouse (Mastomys natalensis)

Secretions, droppings Lassa fever, Viral hemorrhagic fever

New World White Water Arroyo virus Southwest U.S. Woodrats, cotton rat Secretions, droppings Hemorrhagic fever

Tamiami Southern U.S. Cotton rat Secretions, droppings Hemorrhagic fever

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virus, Machupo virus (Bolivian hemorrhagic fever), Oliveros virus, Paraná virus, Pichinde virus, Pirital virus, Sabiá virus (Brazilian hemorrhagic fever) and Tacaribe virus.

When they infect man, they produce severe hemorrhagic fever involving the visceral organs, edema, acute respiratory disease and hypovolemic shock. If left untreated, the fatality rate reaches as many as one-third of those infected.

Lymphocytic choriomeningitis virus, maintained in wild mice as its reservoir, infects mice, rats and hamsters, dogs, monkeys and man. Transmission is via the placenta, aerosol, urine, feces, and the saliva and nasal secretions of infected animals. Surviving animals may be shedding the virus as long as they live.

In the subclinical phase, there may be viremia, chronic wasting, convulsions, depression, decreased fertility and renal failure. Liver, spleen, kidneys and lymph nodes may be enlarged. There is no treatment.

In man, infection is significant with influenza-like symptoms: high fever, malaise, sore throat, loss of appetite, headache and photophobia, nausea and weakness. After subsiding for a few days, the flu-like symptoms may recur, and fingers,

knuckles, and testicles become inflamed, swollen and painful. There may be headache and stiffness of the neck often caused by meningitis, and the hair may fall out.

Most individuals with meningitis will recover, but headache and fever may recur occasionally. Treatment is symptomatic only and necessary usually only for a couple of weeks. Congenital malformations have been reported following infection with the virus.

Lassa virus is endemic in West Africa. It causes viral hemorrhagic fever in up to a half-million people, with a mortality rate of close to 5,000 per year. Its transmission is via ingestion or inhalation of dried rodent feces dust and urine found in grain storage areas and it can enter through broken skin and via infected tissues and meat. Man to man infection is through exchange of infected body fluids and dirty needles.

It is a systemic disease and dispersed throughout the body via mucosae, intestine, and the pulmonary, urinary and the vascular systems. The symptoms include fever, sore throat, chest pain, myalgia, and swelling of the head and neck. There may be proteinuria, bleeding of the mucosae, encephalitis and hearing loss.

Arenaviridae

Molecular biology

VIRION

Enveloped, spherical. Diameter from 60 to 300 nm.

REPLICATIONCYTOPLASM1. Virus attaches to host receptors though GP glycoprotein and is endocytosed into vesicles in the

host cell. 2. Fusion of virus membrane with the vesicle membrane; ribonucleocapsid is released in the

cytoplasm. 3. Sequential transcription, viral mRNAs are capped in the cytoplasm. 4. Replication presumably starts when enough nucleoprotein is present to encapsidate neo-

synthetized antigenomes and genomes. 5. The ribonucleocapsid interacts with the Z protein under the plasma membrane and buds,

releasing the virion.6. Membrane, releasing the virion.

While it can be a severe and often deadly disease, only about one of five infected individuals will develop the more severe effects of the disease. The other 80 percent may show a mild fever, and few, if any noticeable symptoms. Of the individuals requiring hospitalization, 15 to 20 percent are likely to die. The overall mortality of all those infected is about 1 percent. The highest death rates are found in pregnant women in their last trimester, and nearly 95 percent of fetuses from infected mothers die in utero. Those surviving the disease may develop varying degrees of deafness, and there may be spontaneous abortions.

Ribavirin and supportive care have shown some effect. Rodent control and rodent-proofing storage areas and containers are essential efforts.

Other arena viruses causing disease in man are Tamiami, Bear Canyon and Whitewater Arroyo virus. They cause hemorrhagic fever and are carried by the woodrat (Neotoma species). They are found in the Southwestern United States. Because of the quick onset and severity of the disease, they are considered of potential bioterror importance. Although there seems to be some cross immunity, there is no vaccine.

FlaviviridaeThe Flaviviridae are glycoprotein enveloped, icosahedral, 40 to 60 nm in diameter and consist of single-stranded linear RNA. They are arthropod-borne (ticks, mosquitoes) and affect animals as well as man. Their geographic distribution is controlled by the availability of arthropod carriers.

Mosquito-borne flaviviruses include, among many others not existing in the U.S., flaviviruses with yellow fever virus, West Nile virus, St. Louis encephalitis virus, and dengue fever virus; hepacivirus (hepatitis C virus); and pestivirus (bovine viral diarrhea virus, classical swine fever).

The viruses are enveloped, spherical, and about 50 nm in diameter. The surface proteins are arranged in an icosahedral-like symmetry.

There are seven that are tick-borne, among them tick-borne encephalitis virus, Powassan virus and Louping Ill virus.

After outbreaks in the early part of last century, yellow fever has not played a major role in the United States. In Nigeria, more than 60 percent of ruminants tested had experienced yellow fever as confirmed by the presence of antibody. Antibody was found in 26 percent of the camels tested, 20 percent of sheep, 18 percent of goats and 6 percent of cattle.

In man, it causes an acute viral hemorrhagic disease, producing jaundice in some, loss of appetite, fever and chills, headache, muscle pain, backache, nausea and vomiting. The symptoms may disappear after three to four days. About one to two out of 10 individuals may enter a second, more severe stage of the disease a day later, with high fever, rapid jaundice, abdominal pain, vomiting, blood coming from all orifices, kidney failure and death in up to half of the patients going through the severe stage of the disease.

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The West Nile virus, usually mosquito-borne, can be found in horses, birds and man in all 48 contiguous states of the United States. According to the Centers for Disease Control and Prevention the incidence of West Nile virus disease in the United States in 2009 was a total of 720 infected people, of which 373 exhibited meningitis or encephalitis, 322 had fever, and 25 had unspecified complaints. Overall, there were 32 fatalities or 4.4 percent.

Symptoms range from mild influenza-like symptoms to inflammation of the brain and sometimes to death in man and horse. A few

people of all those infected, about one out of five, develop West Nile fever, usually within two to 14 days after infection.

In immuno-suppressed individuals, it may take even longer for symptoms to appear. They include fever, headache, fatigue, skin rash, swollen lymph nodes, and occasionally, eye pain. The neuroinvasive West Nile virus infection results in febrile headache, aseptic meningitis and encephalitis not unlike other viral-induced central nervous system complications. About seven of 10 people with the neuroinvasive infection develop

encephalitis or meningoencephalitis (altered mental state, lethargy, confusion, coma, limb paralysis, cranial nerve palsies and tremors) while the other three show signs of meningitis (headache, fever and stiff neck) without encephalitis.

Another expression of West Nile virus infection is asymmetric limb paralysis described as West Nile poliomyelitis. Other than supportive care, there is no treatment for this disease.

St. Louis encephalitis virus is transmitted like all the other mosquito-borne viruses during the mosquito season. Birds appear as intermediate hosts, amplifying the virus population in their blood without showing signs of the disease themselves.

There have been several St. Louis encephalitis virus epidemics in the past. In 2008, the Centers for Disease Control and Prevention reported eight confirmed and probable St. Louis encephalitis virus neuroinvasive disease cases and five confirmed and probable St. Louis encephalitis virus non-neuroinvasive disease cases.

It is often asymptomatic, but when illness does occur, it is more severe and more frequent in older people than in the young. Fatality rates in individuals over 50 years of age are 7-24 per 100, while they are less than five in 100 in those younger. Individuals surviving the more severe disease may show long-term damage to the central nervous system as well as paralysis, memory loss, and loss of fine motor skills. There is no vaccine and no cure available for this disease.

Dengue virus: There are four antigenically related strains of the virus-causing disease, but their antigenic relationship is not sufficient to cross-protect against each other. However, previous infection with one strain will provide life-long protection against the same strain.

Flaviviridae: Group IV/(+)ssRNA/glycoprotein envelope/icosahedral/40-60 nm

Serocomplex Virus Distribution Host/reservoir Vector Disease in man

Flavivirus Yellow fever Worldwide Primates, ruminants mosquitoes Mosquito Hemorrhagic fever

West Nile Worldwide Horses, birds Mosquito Hemorrhagic fever

St. Louis encephalitis North and South America Birds Mosquito CNS infection

Dengue Tropics Mosquitoes Mosquito Hemorrhagic fever

Hepacivirus Hepatitis C Worldwide Man to man Needles Hepatitis

Pestivirus Tick-borne encephalitis Europe, Asia Rodents, Ixodes tick Ixodes Meningoencephalitis

Louping Ill Europe Sheep, grouse, hares I. ricinus Influenza-like

Powassan Canada, U.S., Northeast Asia Woodchuck, ticks I. cookei Encephalitis

Flaviviridae

Molecular biology

VIRION

Enveloped, spherical, about 50 nm in diameter. The surface proteins are arranged in an icosahedral-like symmetry. Source: Zhang et al (Pubmed)

REPLICATIONCYTOPLASM1. Virus attaches to host receptors and is endocytosed into vesicles in the host cell. 2. Fusion of virus membrane with the vesicle membrane; RNA genome is released into the

cytoplasm. 3. The positive-sense genomic ssRNA is translated into a polyprotein, which is cleaved into all

structural and non structural proteins. 4. Replication takes place at the surface of endoplasmic reticulum. A negative-sense

complementary ssRNA is synthesized using the genomic RNA as a template. 5. New genomic RNA is synthesized using the negative-sense RNA as a template. 6. Virus assembly occurs at the endoplasmic reticulum. The virion buds at the endoplasmic

reticulum, is transported to the Golgi apparatus, and then bud from the cell membrane.

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Dengue viruses cause dengue fever and dengue hemorrhagic fever and are a major cause of disease and death in the tropics and subtropics. Dengue virus infections have been reported in more than 100 countries, and the World Health Organization estimates that 50 million to 100 million infections occur yearly, including 500,000 dengue hemorrhagic fever cases and 22,000 deaths, mostly among children.

In the United States, the Centers for Disease Control and Prevention reported 368 cases of dengue fever and four of dengue hemorrhagic fever in the first 43 weeks of 2010. Among travelers returning to the United States from tropical countries, dengue is, after malaria, the most common cause of hospitalization. It should always be considered in the diagnosis of febrile events in recent arrivals from the tropics or subtropics.

Symptoms range from light, undifferentiated fever to severe headache and retro-orbital pain, joint pain, muscle pain, bone pain, macular rash, nose- or gum bleed, petechiae, easy bruising and low white-cell count. In younger children, the illness is less severe than in older ones and adults.

Occasionally, in about one of 100 dengue fever patients, there is an interim decline in the disease followed by hemorrhage, severe abdominal pain, nausea and persistent vomiting, vascular leakage and thrombocytopenia, skin erythemias, superficial bleeding and petechiae. The patient will bring up blood, produce black and bloody stools, exhibit drowsiness, dyspnea, cold clammy skin and irritability. In this appearance of the dengue shock syndrome, medical care becomes a matter of extreme urgency.

There is no vaccine available. The best defenses remain staying away from mosquitoes, using insect repellent, protective clothes and mosquito netting, and an early response to the disease.

The Hepacivirus, hepatitis C virus is a single virus entity and is not of zoonotic interest, although it will infect monkeys and primates. Transmission is strictly man-to-man via exchange of bodily fluids, dirty needles, blood and blood products by transfusion and transplantation.

About one of five infected individuals show signs of the disease. However, the disease may appear decades after the first contact. The initial symptoms are ill defined and minor. They are often influenza-like and last for a couple of months: fatigue, loss of appetite, nausea and vomiting and diarrhea. Yet, while there may be no open signs of the disease, the viral damage to the liver is progressive and becomes increasingly more severe. Eventually there will be headache, pain over the liver area, jaundice, slowed urination and discoloration of urine (darker) and stool (grayish, pale), weakness, fatigue, confusion, irritability and difficulty concentrating.

Hepatitis C infections become chronic in more than three-quarters of those infected. It can be a decade-long process. The liver becomes cirrhotic, replacing active parenchymal tissue with fibrous scar tissue and loss of function in the

process. Suggestions of this process taking place are ascites, fatigue, ongoing jaundice, loss of appetite and progressive weight loss, itchy skin, sleeplessness, vomiting and bringing up blood. There may be mental symptoms, such as lethargy, sleepiness, confusion and hallucinations.

About 4 percent of individuals with chronic liver disease will develop liver cancer, and as many will die from the disease or cancer.

The Pestivirus group is the third group of Flaviviridae, it includes bovine viral diarrhea virus (BVDV-1, BVDV-2), border disease virus of sheep and classical swine fever virus. The latter used to be called hog cholera virus. None of them is considered of zoonotic interest.

Tick-borne encephalitis is a viral disease affecting the central nervous system. It does not exist in the U.S. There is a western tick-borne encephalitis virus in Europe, Siberian tick-borne encephalitis virus in Siberia, and Far Eastern tick-borne encephalitis virus, what used to be called Russian spring summer encephalitis virus.

It is expressed initially by mild fever, malaise, anorexia, headache, myalgia, nausea and vomiting. In about one-quarter to one-third of the infected individuals, this initial stage is followed by meningitis (headache, fever, stiff neck), encephalitis (drowsiness, confusion, paresis, aberrant behavior) and meningoencephalitis. About one to two of 10 infected individuals

exhibit long-term neuro-psychiatric aftereffects, and about one or two of 100 will die.

Other than supportive care, there is no known treatment, and there is no vaccine available in the United States. Prevention, like with all arbovirus infections, rests on arthropod repellent and elimination and the use of protective clothing.

Ixodes ticks are a reservoir for the virus as well as the transmitting vector. Small rodents are the main host, with man being accidental hosts. Other animals may feed the ticks but do not contribute to the viral reproductive cycle. The virus cycle proceeds through the tick transovarially or through its various developmental stages.

There is no person-to-person transmission in man. Congenital transmission from mother to fetus, however, has been reported. Transmission increases in parallel with the tick activity in spring, summer and early fall. Transmission is also possible through milk from infected goats, sheep or cows.

Louping Ill is a tick-borne zoonosis severely affecting sheep and other cloven-hoofed animals as well as smaller mammals (wood mice, shrews, voles, lagomorphs) and grouse on the European continent. It causes disease of the central nervous system and may kill over half the flock, provided the flock had no prior experience. In endemic areas, where you would expected a base level of immunity, the mortality remains below 10 percent

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and mostly in lambs older than 6 months or so after maternal antibody protection has waned.

It does infect man as an accidental host and produces minor influenza-like or neurological symptoms, but it rarely kills.

The Powassan virus is also a tick-borne encephalitis. Its reservoirs are the deer tick or blacklegged tick (Ixodes scapularis) and small mammals. In man, the virus causes little or no symptoms, sometimes meningoencephalitis (swelling of brain and meninges): headache, fever, nausea and vomiting, weakness, confusion, memory loss, aberrant speech and loss of coordination. Mortality rate is about one in 10, and survivors may experience long-term aftereffects.

However, its overall impact is minor; in the past 50 years, fewer than 50 cases have been reported in Canada and the United States.

The only preventive measures are tick repellents (DEET or permethrin) and protective clothing.

BunyaviridaeThe family Bunyaviridae contains among others (Hantaan virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, tomato spotted wilt virus) the California serogroup. Members of that group have been found to produce acute encephalitis in horses and infect cats and man.

In the United States, the incidence of the human disease ranges from 29 to 167 cases per year. The neuroinvasive disease has an incubation period of one to two weeks. Most of these cases are caused by the La Crosse virus.

Because the symptoms of this disease mimic those of other encephalitic virus diseases, such as herpes and enteroviruses, it may be somewhat underreported. The virus causes encephalitis in children and should be considered whenever there is a case of unexplained meningoencephalitis appearing in summer and fall in the Midwestern and Mid-Atlantic states. Signs include headache, fatigue, nausea, vomiting, abdominal pain, and occasionally, disorientation and seizures.

Aftereffects may include epilepsy, cognitive and behavioral changes, and hemiparesis in 6 to 15 percent of those who had been diseased. Fatality is about 2 percent.

It is carried by chipmunks and squirrels and passed on by mosquitoes. Mosquito control (removing breeding grounds, such as standing surface water) and protection from mosquito bites (fly screens, appropriate clothing, mosquito

repellent) are the recommended means of prevention. The incidence of rates of infection is a function of mosquito season and activity.

Hantavirus produces the deadly hantavirus pulmonary syndrome. Sin Nombre virus – the no-name virus in Spanish – is a member of the same group of viruses often implicated in North America. In contrast to other members of this family, this virus is transmitted through inhalation of dust from droppings and secretions of infected rodents in old deserted houses, sheds or barns. It can also enter through broken skin.

It exists throughout the United States as well as in Scandinavia, Korea and Russia. Hantavirus carrying rodents are thought to exist in every

national park. Campers and hikers are more likely to get infected than urban dwellers.

It has been a notifiable disease in the United States since 1995, and by the end of 2009, a total of 537 cases had been reported with a mortality of 36 percent. More than 90 percent of those cases occurred in individuals 17 and older. Only four children younger than 10 years old developed the disease.

Although uncommon in children, the clinical picture is similar to that in adults. There are usually two to six days of nonspecific, influenza-like, viral disease followed by shortness of breath and the sudden eruption of acute respiratory symptoms and respiratory failure.

Bunyaviridae

Molecular biology

VIRION

Enveloped, spherical. Diameter from 80 to 120nm.

REPLICATIONCYTOPLASMIC1. Virus attaches to host receptors though Gn-Gc glycoprotein dimer and is endocytosed into

vesicles in the host cell. 2. Fusion of virus membrane with the vesicle membrane; ribonucleocapsid segments are released

in the cytoplasm. 3. Transcription, viral mRNAs are capped in the cytoplasm. 4. Replication presumably starts when enough nucleoprotein is present to encapsidate neo-

synthetized antigenomes and genomes. 5. The ribonucleocapsids migrate under the plasma membrane and buds, releasing the virion.

Bunyaviridae: Group V/(-)ssRNA/envelope/helical/40-60 nm/tripartite genomes

Serocomplex Virus Distribution Reservoir Vector Disease in man

California group La Crosse virus United States Horses, cats Mosquito Encephalitis

Hantavirus Pulmonary syndrome virus United States Rodents, deer mouse

Feces, urine Hemorrhagic fever with renal syndrome

Crimean-Congo hemorrhagic fever CCHF virus East and West Africa Small rodents, hedgehog

Hyalomma tick Hemorrhagic fever

Rift Valley fever Rift Valley fever virus Africa, Yemen, Saudi Arabia

Aedes eggs Mosquito Hemorrhagic fever

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The blood-cell picture shows thrombocytopenia, elevated white blood cell count and pulmonary infiltrates. The hematocrit reading is elevated in maybe a quarter of the cases. The differential diagnosis of unexplained acute respiratory distress in children following contact with rodents should always include the possibility of this disease.

Vaccination does not exist. Antivirals have limited effect, although the renal syndrome of the disease seems to respond to it by shortening the disease and reducing mortality. Other complications include kidney failure and cardiorespiratory insufficiency.

Basically, treatment consists of supportive care and treating the symptoms. Prevention requires effective rodent control in and around playgrounds and residential areas and the avoidance of rodent contact and rodent infested areas.

Another hot virus of this family is the Crimean-Congo hemorrhagic fever virus with very high morbidity and mortality rates. It is found in Asia, Eastern Europe and the Near East and Africa. The virus infects ruminants (cattle, sheep, goats), rats, hedgehogs and ostriches, rarely eliciting major symptoms. Small mammals (hares, hedgehogs, rats) serve as reservoir for the blood-sucking tick.

In man, the virus kills about one-third of those infected. An incubation period of less than one week after exposure to the virus is followed by the appearance of minor influenza-like disease that will resolve itself within another week in one-quarter of infected cases. The majority of cases, however, develops to full-blown hemorrhagic fever with mucosal petechiae, nosebleeds, blood in vomits, stool and urine as well as acute respiratory dystress, hepatomegaly, acute kidney failure and shock with death in about 30 percent of those diseased.

Prevention has to concentrate on tick control, de-ticking animals before moving them into or out of a herd. Insect repellent, protective garb

and frequent inspection for ticks is important. Ribavirin has been used with some effectiveness.

Rift Valley fever causes severe disease in animals and man. It occurs in tropical areas all over Africa, the Yemen and Saudi Arabia.

It is transmitted by bloodsucking flies and mosquitoes. Aedes mosquitoes are the main culprit, either by direct transfer from an infected animal to a new host or through their offspring, which receive the virus through the eggs from which they developed.

This seems also to be one of the important reservoirs for the virus. Mosquito eggs can survive for years in a dry environment, and heavy rainfalls will allow those eggs to hatch and produce an active and highly infectious population of mosquito vectors.

While sheep are the more susceptible host animal, it will infect other ruminants.

The fatality rate is highest in the very young; nine out of 10 lambs infected will die, while adult sheep have a mortality of about one out of 10. Almost every pregnant ewe will abort when infected with the Rift Valley virus. An explosion of abortions in a flock of sheep suggests the possibility of a Rift Valley fever epidemic.

Humans are infected mostly through handling infected animals and animal matter. Butchers, farmers, veterinarians and individuals disposing of infected animal carcasses are the high-risk populations. Broken skin, careless handling of equipment (needles, knives), aerosols produced by homogenization and sonication of infectious samples, and the ingestion of raw milk have all been implicated. Of course, mosquitoes also do play an important role in transmitting the disease to man.

In man, the disease often produces no symptoms at all or an indistinct, general influenza-like disease with headache, light fever, a stiff neck and muscle and joint pain. There may be loss of appetite and vomiting.

In a few cases, however it will progress to more severe complications:

■ In 0.5 to 2 percent of the cases, the eyes are affected, including blurred vision; when lesions involve the macula, half the cases may retain permanent vision loss.

■ It also can affect the central nervous system; in less than 1 percent of the cases, there will be symptoms of meningoencephalitis (severe headache, disorientation, confusion, memoryloss, vertigo, convulsions and coma). It is rarely fatal, but there may be residual neurological sequelae.

■ In fewer than 1 percent of the cases, hemorrhagic fever will develop within two to four days after the appearance of the disease; severe hepatitis and jaundice, mucosal bleedings and ecchymosis, uterine bleeding, blood in stool and vomit may be present. It is fatal in about half of the hemorrhagic fever patients.

The overall death rate of Rift Valley fever in man is less than 1 percent, and most of them are due to hemorrhagic icterus. Because the disease in most cases remains mild if not asymptomatic and resolves itself within a week or two, no special treatment is required. However, people with fever for more than 48 hours should urgently seek attention.

There is no vaccine in use for man. Live attenuated and inactivated virus vaccines are employed to prevent outbreaks in animals. The live virus vaccine requires only one shot, allowing the attenuated virus to replicate and increase its antigenicity as it multiplies. Virus growth, of

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course, will be self-limiting as it stimulates an immune response blocking its own progress.

Several doses of the inactivated virus are needed to achieve the required antigenic boost over time. Where a quick response is necessary, as in breaking endemics, the live vaccine would be the better option. On the other hand, it has been reported that the live vaccine virus may induce abortions.

Once the virus has appeared in a herd, all vaccination should be stopped to avoid cross-contamination by the process as viremia may develop before there are any open signs of the disease. Standard herd management procedures must be observed when moving animals between infected and non-infected areas.

Common sense should guide people who are around or deal with infected animals directly or indirectly, including not ingesting unprocessed meat or milk from infected animals and wearing protective garb when handling them. Because there is no disease without mosquitoes, mosquito control is of the essence: use of larvicides, insect repellents and protective clothing is recommended.

PoxviridaeThe family of Poxviridae includes double-stranded DNA viruses, parapox viruses among them, which are included under global zoonoses.

They are enveloped, brick-shaped or ovoid virions, 220-450 nm long and 140-260 nm wide. The surface membrane displays surface tubules or surface filaments.

There are two types of infectious virus particles: the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). They produce acute viral diseases affecting birds, animals and man, producing generalized lesions on skin and mucosae developing from papules to vesicles and pustules, eventually overcrusting and healing. Poxviruses are the only DNA viruses replicating exclusively in the cytoplasma, without cell nucleus involvement.

Smallpox, an orthopoxvirus, is no longer of concern, having been eradicated in 1979. It was a curse even as late as the first half of the 20th century when 50 million cases occurred annually, and 300 million to one-half billion people died during that century. The eradication of the disease was based on a well-coordinated global vaccination program and the immunity of potential target populations.

The United States stopped vaccinating in 1972, and in 1980, the World Health Organization

suggested no more vaccinations worldwide. The world today is one whole unprotected target population, unvaccinated and non-immune. Consideration must therefore be given to the early recognition of smallpox infection and an effective response to it.

The incubation period after infection and before symptoms appear is 12 to 14 days. The virus is fairly stable for 24 hours and could easily

be spread by aerosolization. In bedclothes and blankets, it may stay alive for much longer.

Our armed forces personnel leaving the contiguous United States are still being vaccinated as a matter of course. First responders, who used to be protected by vaccination, are no longer vaccinated because of possible side effects, such as spreading the live vaccine virus to those in weakened condition and hospitalized,

Poxviridae: Group I/dsDNA/complex coats/140-260 nm x 220-450 nm/one linear, double-stranded segment

Serocomplex Virus Distribution Host/reservoir Vehicle

Orthopox Smallpox virus Eradicated

Vaccinia virus India Ruminants and man Direct contact

Monkeypox virus West Africa, Central African rainforest Ruminants, monkeys and man Direct contact

Parapox Pseudocowpox virus Ruminants and man Direct contact

Bovine papular stomatitis Worldwide Ruminants and man Direct contact

Orf Ruminants and man Direct contact

Poxviridae

Molecular biology

VIRION

Enveloped, brick-shaped or ovoid virion, 220-450 nm long and 140-260 nm wide. The surface membrane displays surface tubules or surface filaments. Two distinct infectious virus particles exists: the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV).

GENOMELinear, dsDNA genome of 130-375 kb. The linear genome is flanked by inverted terminal repeat (ITR) sequences which are covalently-closed at their extremities.

GENE EXPRESSIONREPLICATIONCYTOPLASMIC1. Virus attaches to glycosaminoglycans (GAGs) on the surface of the target cell or by

components of the extracellular matrix, triggering membrane fusion and release of the virus core into the cytoplasm.

2. Early phase: early genes are transcribed in the cytoplasm by viral RNA polymerase. Early expression begins at 30 minutes post-infection.

3. Core is completely uncoated as early expression ends, viral genome is now free in the cytoplasm.

4. Intermediate phase: Intermediate genes are expressed, triggering genomic DNA replication at approximately 100 minutes post-infection.

5. Late phase: Late genes are expressed from 140 min to 48 hours post-infection, producing all structural proteins.

6. Assembly of progeny virions starts probably in association with internal membranes of the infected cell, producing an spherical immature particle. This virus particle matures into brick-shaped intracellular mature virion (IMV).

7. IMV virion can be released upon cell lysis, or can acquire a second double membrane from trans-Golgi and bud as external enveloped virion (EEV).

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and to the immunosuppressed and pregnant women. Although the risk is minuscule, those around the vaccinated may develop infections of the respiratory tract, pericarditis, myocarditis. On rare occasions, permanent neurological damage has been observed.

The present plan of action considered by the Centers for Disease Control and Prevention includes vaccination of first responders and the expectation that vaccination within three days of exposure will prevent or significantly lessen the severity of smallpox symptoms in the vast majority of people. Vaccination four to seven days after exposure likely offers some protection from disease or may modify the severity of disease.

Vaccinia virus is an orthopoxvirus. It was the vaccine that allowed eradication of smallpox. Having lost some of its image after the elimination of smallpox, it is being re-discovered in view of the potential of smallpox as a bioterrorism agent. Smallpox virus is considered a Category A bioterrorism agent. Category A is the highest priority of bioterrorism concern.

Vaccinia virus is genetically and antigenically related to variola (smallpox) and cowpox virus. This specific antigenicity is the basis for its use as smallpox vaccine. Infections with vaccinia may produce generalized vaccinia, eczema vaccinatum, progressive vaccinia and roseola vaccinia. It is typically mild and usually without symptoms, although it may produce fever and a minor rash.

Of course, any live virus vaccine may produce detrimental complications in the immunocompromised individual. There have been reports of the occasional fatality after vaccination (1 in 1 million vaccinees). Almost 200 million doses of a new vaccine (ACAM2000) are stockpiled for use in case of a bioterrorism attack.

Monkey pox virus is an orthopox virus. It is found in monkeys, rodents and squirrels in West and Central Africa near the rainforest and has been found in the United States in sick prairie dogs. It is transmitted to man by direct contact when handling, killing and consuming infected squirrels and monkeys.

In man, the virus produces a papular rash similar to smallpox, swollen cervical and inguinal lymph nodes, fever, headaches, muscle and backaches and exhaustion. In Africa, as many as 10 percent of infected individuals will die.

Transmission is through bite or contact with infected matter (blood, body fluids, lesions, contaminated clothes). Man-to-man transmission has been reported in less than 30 percent of observed cases. The disease is rare: during the 16

years between 1970 and 1986, the World Health Organization reported 400 cases worldwide.

There is no treatment for monkey pox. Smallpox vaccine tends to protect from the disease.

Pseudo cowpox, a disease caused by the Pseudocowpox virus, a parapox virus, is also called Milker’s nodules. It occurs worldwide in cattle, producing ring-shaped or horseshoe-shaped scabbing sores on the teats of cows and ulcerating stomatitis in calves suckling from those teats.

Control of the disease in the herd is limited. The sores will heal in a matter of weeks. Basic treatment should consist of local sanitary measures: rinsing with virus-killing, iodophor disinfectants; disinfectant dips for teats; and careful disinfection of milking equipment and hands to prevent passing the virus from one cow to the next.

Solid immunity does not seem to follow infection. Re-infection is possible and relatively frequent. Farmers or veterinarians inspecting these sores may acquire itchy red to purple lesions on their hands and face. The lesions will develop into tender nodules and sometimes become infected bacterially, generating reddened lymphatic channels up the arm and enlarged lymph nodes. There may be generalization and the eruption of pustules and blisters on hands arms, neck and legs, which fade within about a week or two.

Bovine papular stomatitis virus occurs worldwide in cattle. It produces red ulcerating lesions on oral and nasal mucosae, particularly in younger cattle (less than 2 years old), sheep and goats. It may produce nodules on hands of milkers and animal handlers.

Immunity after infection is only short term, and re-infection is possible.

Another parapox virus, Orf, produces sore mouth infection on the lips, muzzle, oral mucosa and urogenital areas in sheep, goats, and even reindeer. It can spread to lower legs and teats.

The infection is passed on by crusty lesions falling off and contaminating soil and feed, by direct contact with infected animals (animal shows and markets), and infected trucks and tools. Infected animals usually recover within four weeks.

It is common worldwide. Farmers, animal and meat handlers develop itchy, weeping, red sores on hands and fingers and on other areas of the skin touched by hand. They heal spontaneously within six weeks or so.

RhabdoviridaeThe family of Rhabdoviridae are bullet-shaped, enveloped single-stranded RNA viruses (75 by 150 nm in size) with five structural proteins:

glycoprotein, large protein, phosphoprotein, matrix protein and nucleoprotein.

The glycoprotein facilitates entrance into the host cell, attachment to the cell wall and endocytosis into host cell vesicles, vesicle membrane fusing with virus membrane and releasing the ribonucleocapsid into the cytoplasm, sequential transcription, encapsidation of the new product and eventually budding from the plasma membrane and releasing the new virus.

The Rhabdoviridae include lyssa virus, producing rabies and vesicular stomatitis virus, which infects horses, cattle and swine among others as well as insects.

Rabies affects all mammals, but particularly carnivores and bats, causing acute viral encephalomyelitis. Once clinical symptoms have appeared, death is certain.

Reservoir animals for rabies, which occurs worldwide, vary by regions. In the United States in 2008, 93 percent of all reported cases came from wildlife animals: raccoons (34.9 percent), bats (26.4 percent), skunks (23.2 percent), foxes (6.6 percent) and other animals, including rodents, hares and rabbits. Domestic animals represent only 7 percent of all rabid animals reported.

Rabies in man in the United States is usually caused by bat rabies. Virus is found in rabid cat saliva and rabid cat bites have produced rabies in man. In the United States, more cats are reported with the disease than dogs.

Transmission may also occur via broken skin and mucosae, but this is unusual, as animal bites seem to be the predominant course of infection. High-dose aerosols in bat caves have occasionally produced rabies.

Early treatment and an extended incubation period will improve chances of post-exposure effectiveness of rabies immune gamma globuline. Annual death rates in man have declined from hundreds in the early last century to two or three a year today, thanks to effective animal and human vaccines and immunoglobulin.

The early signs of rabies are not very descriptive: influenza-like, headache, general discomfort and malaise, and fever. At the bite site, there may be an itching sensation, then progression to a central nervous system disease such as agitation, confusion, anxiety and hallucinations, behavior changes, insomnia and delirium.

The disease is almost always fatal. It is therefore crucial that the infection of the animal be identified early and treatment in the form of human immune globulin be commenced as soon as possible along with a course of rabies vaccination.

Rhabdoviridae: Group V/(-)ssRNA/lipid envelope/helical-bullet shaped/75 nm x 150 nm/five proteins

Serocomplex Virus Distribution Reservoir Vehicle Disease in man

Lyssavirus Rabies virus Worldwide Bats, raccoons, skunks Bites; aerosol Central nervous system; death

Vesiculovirus Vesicular stomatitis virus Worldwide Arthropods, mammals Culicoides midges, blackflies, sand flies

Influenza-like

Ephemerovirus Bovine ephemeral fever Tropics, subtropics Cattle Culicoides midges ?

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Vesicular stomatitis virus involves two serotypes, Indiana and New Jersey, and both are zoonotic, infecting arthropods, cattle, horses, pigs and man. It exists worldwide.

It progresses through two phases, the cytolytic infection in mammals, and the non-cytolytic one in insects (Culicoides midges, Simulium blackflies and Phlebotomine sandflies). Typical symptoms are ulcerating and crusting vesicles on the muzzle, oro-nasal mucosae, the epithelial surface of the tongue, on teats and coronary bands.

Antibody has been found in deer, primates, rodents, birds, dogs and others. While an entire herd of cattle may have antibody to the virus, only one or two of them may show signs of the disease. After an incubation of two to eight days, there may be fever,

excessive salivation and drooling (ptyalism). There may be evidence of ruptured vesicles in the oral cavity and ulceration, sloughing of the epithelium of the tongue and erosion at the cutaneous-mucosal interphase. Erosions may appear on the teats of cows and cause mastitis.

Except for the fact that it will affect horses, its symptoms look much like foot-and-mouth disease. In cattle, pigs and horses, there may be coronitis, with erosions at the coronary band and lameness. Horses show lesions on muzzle, abdomen, sheath and udder. There may be loss of appetite. All symptoms will pass quickly as the disease is self-limiting and will lead to full recovery within weeks. Antibody may persist for years but will not necessarily be immunizing or protect from re-infection.

The virus also infects man, producing influenza-like symptoms and, occasionally, oral vesicles, cervical lymphadenopathy, and temporary debilitating illness. Infection is through direct contact.

Ephemerovirus produces bovine ephemeral fever, an arthropod-borne disease of cattle (three-day sickness): fever, discharges from mouth, nozzle and eyes, stiffness, depression, loss of appetite and rumination, constipation.

TogaviridaeTogaviridae are single-stranded RNA viruses, spherical (65 to 70 nm in diameter), enveloped with an icosahedral capsid within. They contain glycoprotein spikes essential for attachment and entry into their host cell.

Togaviridae include (1) alphaviruses, such as Eastern Equine Encephalitis, Western Equine Encephalitis and Venezuelan Equine Encephalitis, as well as Sindbis virus (South and East Africa, Egypt, Israel, Philippines, Australia), Ross River virus (Australia, South Pacific Islands), O’nyong-nyong virus (Africa), and (2) Rubivirus with rubella virus its only representative. The primary reservoir of alphaviruses is birds. Horses and man are dead-end hosts. High infection rates in birds are followed by infections in horses and man, with mosquitoes being responsible for passing on the virus. Alphaviruses are among the most serious pathogens for horses. There is a variety of alphavirus species throughout the world, many with like symptomatology, but only a few are active in the United States.

Eastern equine encephalitis consists of two antigenic variants, the more pathogenic and more antigenically homogeneous one in the United States and Canada, and a somewhat less pathogenic and less antigenically homogeneous one in South America.

Like all the alphaviruses, the virus is transmitted by mosquitoes (Culiseta melanura, Culiseta morsitan). The virus enters the lymphatic system and lymph nodes and replicates in neutrophils and macrophages, destroying them in the process. As a result there will be lymphopenia, leucopenia and fever. As the virus becomes viremic, it will spread, enter other organs and the central nervous system.

Early symptoms appear within about five days after infection. They are neurological in nature: the horse becomes quiet and depressed, exhibits head pressing, aimless wandering, circling, ataxic gait, asymmetric paralysis, paresis and

Rhabdoviridae

Molecular biology

VIRION

Enveloped, bullet shaped. 180 nm long and 75 nm wide. Certain plant rhabdoviruses are bacilliform in shape and almost twice the length.

REPLICATIONCYTOPLASMIC1. Virus attaches to host receptors though G glycoprotein and is endocytosed into vesicles in the

host cell. 2. Fusion of virus membrane with the vesicle membrane; ribonucleocapsid is released into the

cytoplasm. 3. Sequential transcription, viral mRNAs are capped and polyadenylated in the cytoplasm. 4. Replication presumably starts when enough nucleoprotein is present to encapsidate neo-

synthetized antigenomes and genomes. 5. The ribonucleocapsid interacts with the matrix protein under the plasma membrane M protein

and buds from the plasma membrane, releasing the virion.

Togaviridae: Group IV/(+)ssRNA/envelope/icosahedral/spherical/65 nm – 70 nm

Serocomplex Virus Distribution Reservoir Vector Disease in man

Alphavirus Eastern equine encephalitis U.S., Canada, South America

Birds, rodents, mosquitoes

Mosquitoes Fever, rash, arthritis, encephalitis

Western equine encephalitis Plains of U.S. Birds Mosquito, tick CNS

Venezuelan equine encephalitis South and Central America Bats, birds, rodents, mosquitoes

Mosquitoes

Rubivirus Rubella

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convulsions. Death occurs usually within three days after the onset of the disease.

Alphaviruses cause disease in man. Like the horse, man is a dead-end host and is not an essential part of the transmission process. Birds, rodents and mosquitoes are the main reservoir for these viruses.

The symptoms in man, as a rule, include fever, rash, encephalitis and arthritis. Following the infected mosquito bite, the virus enters the lymphatic system and the bloodstream and disseminates throughout the body. It will enter the central nervous system to multiply in and destroy neurons, causing encephalitis and often death.

After entering the lymphatic system, alphaviruses will stimulate T cells and induce the production of interferon and antibody in order to limit virus progress. There are no alphavirus vaccines.

Removal of mosquito breeding grounds, stagnant surface water, public spraying and use of protective clothing are presently the best means of dealing with the disease progress.

Culiseta melanura appears to be the main vector for eastern equine encephalitis. Its importance in the transmission process is highlighted by the regional and seasonal occurrence of the disease: In Florida, for instance, where the mosquito season includes all four seasons, transmission occurs year-round with the summer as the peak, while in the more temperate climates transmission occurs during the late summer and fall until the first frost.

By contrast, the western equine encephalitis virus is transmitted mostly by Culex tarsalis and a tick Dermacentor andersoni to horses and human beings. Dependent on the presence of infected vectors, its incidence increases with early spring rains and a warm summer.

Only female Culex mosquitoes will pass on the disease. They bite animals and man as well as birds, and suck blood from the infected, viremic host and spread the virus by passing it on directly to its next meal donor. Infected birds, of course, are an excellent means of spread over wide areas. The female culex passes the virus on vertically through its eggs. It needs repeated feedings for the development of a batch of eggs. Infected Culex eggs survive for years in dry areas and will arise during rainy periods and floods.

The disease process in horses is like that for other alphaviruses, although there may be less of a viremia for this virus. Its main course of operation is in the central nervous system, brain and spinal cord. Immunologically active cells such as macrophages and neutrophils invade the brain tissue and perivascular regions, leading to neuronal destruction, spotty demyelination, focal necroses, thrombosis and endothelial proliferation.

Western equine encephalomyelitis in man is not very common: there were fewer than 700 cases in the United States during the past 46 years. Fewer than five cases are seen annually in the U.S., and usually in early summer. This may be because open symptomatic infections are rarely seen. When there are such, they appear within about a week to 10 days after the mosquito bite.

Togaviridae

Molecular biology

VIRION

Enveloped, spherical, icosahedral, 65-70 nm in diameter, capsid made of 240 monomers. The envelope contains 80 trimer spikes, each spike = 3 x E1/E2 heterodimers.

REPLICATIONCYTOPLASMIC1. Virus attaches to host receptors though E glycoprotein and is endocytosed into vesicles in the

host cell.2. Fusion of virus membrane with the vesicle membrane occurs; RNA genome is released into

the cytoplasm.3. The positive-sense genomic ssRNA is translated into a polyprotein, which is cleaved into non

structural proteins.4. Replication takes place at the surface of endoplasmic reticulum. A negative-sense

complementary ssRNA is synthesized using the genomic RNA as a template.5. New genomic RNA as well as subgenomic RNA are synthesized using the negative-sense RNA

as a template. Subgenomic RNA is translated in structurals proteins.6. Virus assembly occurs at the endoplasmic reticulum. The virion buds at the endoplasmic

reticulum, is transported to the Golgi apparatus, and then bud from the cell membrane.

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They bracket the spectrum of symptoms, from a light influenza-like picture, possibly with a headache and a light fever to an abrupt high fever, nausea and vomiting, encephalitis, coma and death. More infants and children get sick than adults, and about one out of three develop neurological consequences, including behavioral disorders, spasticity, seizures and retardation.

The mortality rate is about 4 percent, and it is mostly the elderly that will die. There is no treatment other than supportive care. The control of mosquitoes and mosquito breeding grounds and the avoidance of mosquito bites using insect repellent and protective garb are essential for prevention.

Venezuelan equine encephalitis affects all equines and man. It does not occur very often in the United States. In 1971, there was a major epidemic in horses in Texas, but only about 100 people were affected. Data from international outbreaks suggest that many more subclinical infections may have occurred.

The diseased animal may exhibit progressive disease of the central nervous system or it may die without any prodromal symptoms. The mortality rates reported reach as high as eight out of 10 infected animals.

The virus will infect and produce disease in humans, especially when they are in weakened condition or immunocompromised, and the very young and the elderly. It causes influenza-like symptoms, fever, headache, hypotension, stiff neck, altered behavior, coma and death.

During the course of three epidemics in Venezuela and Columbia in the last half of last century, more than 300,000 people were infected, more than 12,000 had severe neurological complications, and more than 2,000 died. In patients developing encephalitis, as many as one out of five may die.

Transmission again is via mosquito bite, feeding on an infected viremic animal then passing it on to a new host. Thus the effectiveness of spread is a direct function of the number of mosquitoes present, the viremic character of the virus and the access to a virus reservoir. The control of mosquitoes in the subtropics in large swamp-like regions is all but impossible.

The second group of the family of Togaviridae is the rubivirus. Rubella does not really fit into this review because it is not of zoonotic concern, man is its only host, and transmission is by direct contact without the need for animal or arthropod. When occurring during the early weeks of pregnancy, it

causes the congenital rubella syndrome with usually devastating effects on the fetus morphology.

ParamyxoviridaeThe Paramyxoviridae family is a mixed bag, very numerous and varied. Only a few are of zoonotic interest and will be included in this review.

Menangle virus, a Rubulavirus of the Paramyxoviridae family, will cause disease in bats, pigs and man. It is found in Australia and is transmitted by fruit bats and flying foxes, which appear to be the reservoir for the virus. The menangle virus interferes with the reproductive capacity of pigs and causes disease in humans.

Contact with infected body fluids or fetuses is the mode of transmission. In pig herds, it will produce central nervous system complications and will cross the placental barrier and induce deformities, mummified fetuses and stillbirths. In man, it causes serious influenza-like disease and stimulates the production of antibody.

Newcastle disease virus is an infectious disease of domestic fowl and wild birds. This disease occurs in many countries and has considerable economic impact on the poultry industry wherever it hits because the virus is highly contagious, especially

Paramyxoviridae: Group V/(-)ssRNA/envelope/spherical-pleomorph/spiked surface/150 nm – 300 nmSubfamily Genus Species Host ZoonosisParamyxovirus Respirovirus Parainfluenza Type 1 virus Man ?

Parainfluenza Type 3 virus Man ?Sendai virus Rodents ?Common cold virus Man ?

Rubulavirus Parainfluenza Type 2 virus Man ?Parainfluenza Type 4 virus Man ?Mumps virus Man ?Simian parainfluenza Type 5 Monkeys ?Menanglevirus Pigs, man Neurotropic; CNSTiomanvirus Pigs, bats ?Tohokuviruses Types 1 - 3 ? ?

Avulavirus Newcastle disease virus Domestic and wild birds Conjunctivitis, mild feverAvian paramyxovirus Types 2 through 9 Chicken, turkey ?Goose paramyxovirus ( ?: not confirmed) ? ?

Morbillivirus Measles virus Man ?Canine distemper virus Dog, ferret ?Rinderpest virus Cattle ?Phocine distemper virus Seals ?Peste des petits ruminants Goats, sheep ?

Henipavirus Hendravirus Horses, man Respiratory diseaseNipahvirus Pigs, man Encephalitis

TPMV-viruses Tupaia paramyxovirus ? ?Pneumovirinae Pneumovirus Human respiratory syncytial Man ?

Bovine respiratory syncytial Cattle ?Metapneumovirus Human meta pneumovirus Man ?

Avian pneumovirus Turkeys, chicken ?OTHERS: Fer-de-Lance virus, Nariva virus, Salem virus, J virus, Mossman virus, Beilong virus.? Denotes nothing of zoonotic importance is known, and subject will not be considered in this review.

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within the high population densities of today’s breeding establishments. It is transmitted through fecal matter and other dropping from infected birds, from their secretions from eyes, nose and mouth, and through contaminated feed and water.

Proper flock management includes special attention to procedures, the movement of birds and people, rodent control, and the sanitation of vehicles and equipment.

Parrots and other exotic birds may carry the virus and allow its importation into the country. Without exhibiting any symptoms, infected exotic birds have been shown to shed the virus for over a year.

The disease-producing virus strains vary from low virulence (few signs of the disease and no mortality) to intermediate infectivity (some respiratory problems, drop in egg production, 10 percent mortality or so), to highly infectious (severe respiratory and neurotropic distress, rapid spread and 90 percent mortality).

Two days to two weeks after initial exposure, you will notice initial symptoms such as respiratory distress, gasping cough, lack of appetite, drowsiness, drooping wings, head and neck contortions, circling and paralysis. There may be swelling of the head and neck and greenish watery discharge and diarrhea. The egg yield will drop, and there will be thin-shelled, misshaped eggs.

With the highly infectious strains of the virus, deaths may occur abruptly, even before the appearance of disease symptoms. Some may develop respiratory symptoms and others again may show signs of central nervous system involvement, including torticollis. There is no treatment for the disease other than prevention through vaccination and proper flock management.

In man, the virus produces flu-like symptoms and occasionally conjunctivitis.

Hendra virus produces respiratory and neurotropic disease in horses and man. It is another one of the recently identified zoonotic viruses in Australia, the Menanglevirus, Tiomanvirus and Nipahvirus. The Pteropus bat (flying fox) in Australia is the reservoir for the Hendravirus. Infections in man derive from direct contact with infectious matter and body fluids from diseased horses.

The disease is expressed as severe influenza-like respiratory disease followed by progressive encephalitis. Although there is not much evidence for the disease in human beings, two of the three individuals found infected by the virus had died.

Ribavirin has been demonstrated to be effective in cell cultures infected with the virus, however, there is not enough evidence yet to show its effectiveness in human patients.

Nipahvirus is the second member of the Henipahvirus genus. Nipahvirus exists on the Malaysia Peninsula and Singapore. It infects pigs, dogs, cats and man. It causes neurotropic disease, including fever, drowsiness, encephalitis, seizures, coma and respiratory disease.

Pig farmers and other individuals handling infected pigs are likely to become infected. Out of 477 infected cases 248 died (52 percent). Reservoir for the virus is thought to be the same species of bat that is involved with Hendravirus.

After an incubation period of three days to two weeks, there may be respiratory disease, headache, fever, disorientation, drowsiness, mental confusion and coma, all within less than two days. Aftereffects include personality changes and convulsions.

PicornaviridaeThe family of Picornaviridae is very extensive, consisting of 12 different genera with over 200 species. They are responsible for a variety of diseases, from mild to severe to deadly. They may be chronic in both man and animal, but there is relatively little crossover transmission from one to the other.

They disease syndrome usually is systemic and affects all visceral organs, the liver, the upper respiratory system, the central nervous system and the meninges, as well as the gastrointestinal tract and the naso-oral mucosae. Poliomyelitis virus, member of the family of human enterovirus C, still appears in South Asia, the Middle East and parts of Africa.

It is spread from person to person by the fecal-oral route and occasionally is imported through non-vaccinated travelers.

A large number of people infected by the virus show no symptoms (about 90 percent); some may exhibit symptoms of influenza-like upper respiratory tract infection and gastrointestinal disturbances, and about 3 percent show central nervous system (motor neuron) complication, one third of which may end up with acute flaccid paralysis.

Different forms of the disease follow the virus pathway in the body: spinal poliomyelitis (asymmetric paralysis), bulbar polio (difficulty speaking, breathing, swallowing) and bulbospinal polio combining the two. In infants, the disease sometimes appears as encephalitis, with confusion, altered mental state, headache and fever, and, occasionally seizures and paralysis.

Surviving the infection with a given serotype will provide immunity against it but not the other poliovirus serotypes. Therefore, the vaccine must include all three serotypes to provide protection.

To the Enterovirus genus belong the bovine enteroviruses, which exist worldwide in cattle. As a rule, they do not produce a disease but they are found in the feces of cattle and deer in large numbers. They are so widely distributed that they have been used as identifiable water pollution indicators.

Paramyxoviridae

Molecular biology

VIRION

Enveloped, spherical. Diameter from about 150 nm.

REPLICATIONCYTOPLASMIC1. Virus attaches to host cell surface receptors through HN, H or G glycoproteins. 2. Fusion with the plasma membrane; ribonucleocapsid is released in the cytoplasm. 3. Sequential transcription, viral mRNAs are capped and polyadenylated in the cytoplasm. 4. Replication presumably starts when enough nucleoprotein is present to encapsidate neo-

synthetized antigenomes and genomes. 5. The ribonucleocapsid interacts with the matrix protein under the plasma membrane and buds,

releasing the virion.

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Porcine enterovirus B, compared to the porcine Teschovirus below, seems to be relatively innocent. There are suggestions it may be associated with a reproductive disease in swine, producing infertility, embryonic death and fetus mummification as well as stillbirths.

Encephalomyocarditis virus is a member of the genus Cardiovirus. It occurs worldwide and infects swine as well as exotic mammals from free-ranging African elephants to lions and lemurs. The virus infects mice and rats and other rodents, which through their feces and urine as well as their carcasses pass on the virus. Pigs spread the virus through bodily secretions and excretions.

The virus is very hardy and can survive for months, even under adverse conditions. It is essentially a neurotropic and cardiotropic virus.

Death is most often ascribed to respiratory insufficiency (pulmonary edema and exudation) as well as myocardial distress and cardiac failure. There may be fever, loss of appetite, drowsiness, dyspnea, trembling, staggering gait and paralysis. Mortality can be 100 percent in

suckling piglets. As the animals get older, they become more resistant.

In swine, the virus will cross the placenta and produce reproductive failure, late-term abortion and mummified fetuses. Reproductive problems may persist for months in a given herd. Some virus strains cause pancreatitis and associated illness, possibly diabetes.

Man seems to be susceptible to the disease as shown by the frequent occurrence of antibody, and the virus has been isolated from man, but there are no known fatalities. Treatment is mainly of preventive and supportive nature, including rodent control, immediate and proper disposal of infected carcasses, and the appropriate use of antiviral disinfectants.

Killed vaccines are available and should be used in piglets 4 weeks of age and older, followed by a booster dose two to three weeks later and re-boosting in six-monthly intervals.

One of a number of strains is mengovirus, which infects vertebrate animals and has been isolated

from mice and other rodents. It has been reported to cause acute fever in humans.

Theilovirus, a cardiovirus as well, also infects mainly swine and rodents. Among a number of strains, including Theiler’s murine encephalomyelitis virus and rat encephalomyelitis virus, are strains that have received particular interest because they simulate human poliomyelitis and multiple sclerosis. There are suggestions that the Vilyuisk human encephalomyelitis virus is derived from a human with infectious neuro-degenerative disease.

Reports of cardiovirus isolations from humans have to be considered with a grain of salt, because they all were achieved by mouse-to-mouse passage, the natural host for such viruses and a potential silent carrier.

Saffold virus is another virus of the Cardiovirus genus. This virus has been isolated from children with respiratory disease. Isolation this time, bypassing the mouse, was achieved by passage through diploid fetal lung and diploid fetal kidney cell culture lines.

Picornaviridae: Group IV/(+)ssRNA/naked/icosahedral/protein shell/30 nmGenus Species Serotypes Host Disease in manEnterovirus Bovine enterovirus 2 Cattle ?

Human enterovirus A 21 ManHuman enterovirus B 59 ManHuman enterovirus C 19 Man PoliomyelitisHuman enterovirus D 3Porcine enterovirus B 2 Pigs ?Simian enterovirus 1 Monkeys ?Human rhinovirus A 75 ManHuman rhinovirus B 25 ManHuman rhinovirus C 7+ Man

Cardiovirus Encephalomyocarditis 1 Pigs, rodents, man Acute fever, (Mengovirus)Theilovirus 12 Tick, man, rodents Acute aseptic meningitis (Syr-Daria Valley fever).

Aphthovirus Foot-and-mouth disease 7 Artiodactyls, rodents, man Malaise, fever, vesicles on skin and mucosaeEquine rhinitis A virus 1 Horse, man AsymptomaticBovine rhinitis B virus 1 Cattle ?

Hepatovirus Hepatitis A virus 1 Man ?Parechovirus Human parechovirus 14 Man ?

Ljungan virus 4 Lab animals, man Fetal death, malformationsErbovirus Equine rhinitis B virus 3 Horses ?Kobuvirus Aichi virus 1 Oysters, man Gastroenteritis

Bovine kobuvirus 1 Cattle ?Teschovirus Porcine teschovirus 11 Pigs EncephalomyelitisSapelovirus Porcine sapelovirus 1 Pigs ?

Simian sapelovirus 3 Monkeys ?Avian sapelovirus 1 Ducks ?

Senecavirus Seneca Valley virus 1 ? Used in oncolytic studiesTremovirus Avian encephalomyelitis 1 Domestic, wild owl ?Avihepatovirus Duck hepatitis A virus 3 Ducks ?#: Number of strains of that species? Denotes nothing of zoonotic importance is known and the subject will not be considered in this review.

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Real-time reverse transcriptase PCR studies suggested that Saffold-like viruses might be found in man. The virus has been isolated from both stool samples and respiratory secretions. It seems that there is a global presence of these viruses and that human-to-human passage is probable.

Another Theilovirus strain has been isolated from man, in Kazakhstan from the cerebrospinal fluid of an individual with acute aseptic meningitis (Syr-Daria Valley fever virus).

Foot-and-mouth disease virus has been eliminated from many parts of the globe, but still is endemic in southern parts of what used to be the Soviet Union, in India and South East Asia, Iran, Korea and Africa. It infects artiodactyls, i.e. cloven hoofed, ungulated domestic and wildlife species.

It is highly contagious and will infect all of the reachable susceptible population. Its symptoms are fever, vesicles and pustules on mouth, muzzle, teats and feet. Rodents are susceptible to artificial infection.

There are seven antigenically distinct serotypes requiring the combination of at least two vaccines for effective immunization and protection.

Transmission is strictly by direct contact, aerosolized exhalate, exudation and excretion, milk, semen, equipment, vehicles of transport,

clothing and boots of animal handlers and visitors as well as pets, birds and rodents. It is complicated by the fact that virus shedding may start days before symptoms show up. The aerosol may travel considerable distance when environmental conditions are right.

The virus is not very resistant to environmental influences, drying, elevated temperatures, and disinfectants outside the normal range. However, it may find some protection when tied to animal protein, such as milk. On dry fecal matter it may survive for two weeks, longer if it is in a wet slurry. Cattle surviving infection and vaccinated animals that had live virus contact may carry the virus for years.

The virus enters through the oral or nasal mucosa, through breaks in the skin, and via the gastrointestinal tract. It is transported through the lymphatic system to its final destination in the epithelial cells of corona of hoof, mouth, muzzle and other stressed skin areas. After an incubation period of two days to two weeks, there may be high fever, loss of appetite, salivation and the development of vesicles at the exposed and often stressed areas. The cattle may stamp their feet and become restless. Newborns and the very young may die before the appearance of symptoms. Milk yields drop, development is

delayed and there may be general weakening of the diseased animal.

The acute disease is short; the vesicles will heal within days or a week. However, regaining weight will take some time, and there may be chronic lameness.

In pigs, symptoms usually are just as acute, and deaths in the very young as quick. In sheep and goats, the disease is less severe and the number of developing vesicles smaller.

It is of considerable economic importance for foot-and-mouth-disease-free countries to remain free from the disease, especially if their exports include beef and beef products. Slaughter of all infected animals and their susceptible contacts, restriction of travel and visits, movement of vehicle or animal, disinfection of premises, equipment and tools, appropriate and supervised disposal of carcasses must all be among their top priorities. Tracing and backtracking of animal, staff and vehicle movements before and after exposure are prerequisite for control.

The status of being FMDV-free and not allowing vaccination is the most sought-after in countries exporting cattle and cattle products; being FMDV-free and requiring vaccination is less so, and not being FMDV-free leaves you out of the export market altogether.

Human infection is rare. Direct contact with the infected animal as well as laboratory contact may be source of the infection and elicit fever, malaise, vesicular, and often ulcerating lesions of mucosae and skin. Aside from the deaths of two children in the late 19th century, no deaths have been reported.

Equine rhinitis A virus belongs, like the foot-and-mouth-disease virus, to the genus Aphthovirus. It occurs worldwide and produces fever, loss of appetite, mild to severe respiratory disease, nasal discharge, infection of the pharynx, and viremia and infection of the lymphatic system. The virus is shed persistently from the pharynx, feces and urine.

The development of antibody in veterinarians dealing with equine rhinitis A-infected horses suggest that man can become infected by this virus.

Human parechovirus usually produces minor respiratory or gastrointestinal disease but has also been reported to have caused encephalitis and myocarditis. Almost all humans, at least 95 percent, have had exposure to the disease.

Ljungan virus, initially isolated from voles, causes disease in laboratory animals as well as in animals in the wild. It has been found in Europe and America. It has been linked with diabetes and neurotropic disease as well intrauterine malformations, fetal death and sudden infant death. Exposure of Ljungan virus-infected rodents to stress has led to diabetes and to the study of that disease in this system.

Equine rhinitis B virus is found in Europe, the Near East, Canada, Australia and New Zealand with more than half their horse populations presenting evidence of infection with equine rhinitis B virus. The symptoms observed include

Picornaviridae

Molecular biology

VIRION

Non-enveloped, spherical, about 30 nm in diameter, composed of a protein shell surrounding the naked RNA genome. The capsid consists of a densely-packed icosahedral arrangement of 60 protomers, each consisting of 4 polypeptides, VP1, VP2, VP3 and VP4. VP4 is located on the internal side of the capsid.

REPLICATIONCYTOPLASMIC1. Virus attaches to host receptors, formation of a coated vesicle.2. Uncoating, and release of the viral genomic RNA into the cytoplasm possibly through the

formation of a pore in the host cell membrane.3. VPg is removed from the viral RNA, which is then translated into a processed polyprotein.4. In entero-, rhino, and aphtoviruses, shutoff of cellular cap-dependent translation through the

cleavage of the translation initiation factor eIF4G by viral protease.5. Replication of viral RNA takes place on membrane vesicles derived from the ER. A negative-

sense complementary ssRNA is synthesized using the genomic RNA as a template. 6. New genomic RNA synthesized using the negative-sense RNA as a template is believed to be

packaged into preformed procapsids. 7. Cell lysis and virus release.8. Maturation of provirions by an unknown host protease.

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fever, loss of appetite, indolence, acute respiratory illness, nasal discharge, edema of the legs and swollen regional lymph nodes. Asymptomatic infections have been recorded as well.

Aichi virus has been isolated from oysters and produces an acute gastroenteritis in man. In Japan, there is a high incidence of antibody in the human population; about eight out of 10 individuals 35 years and older have had experience with the virus. It has been found in travelers from Southeast Asia and in Pakistani children.

Porcine teschovirus contains 11 serotypes of this virus. They are widely distributed. Most of them cause little if any disease, and when they do, it is in weaned piglets and young growing pigs.

There are some more virulent strains that can cause the more severe Teschen disease, encephalomyelitis affecting only the motor nerves (poliomyelitis suum, benign enzootic paresis). However, outbreaks are rare.

Ingestion of even small doses guarantees infection in the new host. After multiplication in the gastrointestinal tract, the tonsils and the associated lymphatic system, the virus is shed in the feces in large numbers.

Animals that have overcome the disease may continue to shed the virus for weeks, up to two months. The shed virus is very hardy and may survive in liquid manure for many months.

Newborn piglets are protected by maternal immunity and colostral antibody. But after the piglet is weaned and the colostral antibody has waned, the virus can multiply and stimulate active immunity. This process tends to establish

immune pig populations and lower the disease-producing effects of the new infection.

When the mother has had no prior experience with the virus or when this process meets with an inefficient immune system or is complicated by some other disease, the virus can proceed to the central nervous system and produce poliomyelitis-like illness. Starting with fever, a temporary loss of appetite and depression, muscle tremor, stiffness, clonic spasms and anormal posture, difficulty walking, swaying gait, staying in sitting position, incoordination, the disease may lead to gradual loss of motor function of back legs and progress to complete paralysis, which may often be irreversible. The most virulent strain, PTV 1, may produce paralysis and death in less than a week.

Proper herd management is of importance in the prevention of this disease, including the hygiene of individuals handling pigs and their protective garb (boots and clothing) as well as appropriate sanitation of transportation and maintenance equipment, the avoidance of unnecessary fecal matter and the movement of animals that may be subclinical carriers.

Avian encephalomyelitis virus is a worldwide viral disease of domestic and wild fowl. It produces ataxia, tremors, paresis and unsteadiness, and paralysis in chicks seven to 10 days old. In adult birds, the infection is asymptomatic, although there may be a drop in egg production. The disease usually affects about 5 percent of the flock. Turkeys are less affected than chickens.

Vaccines are available and should be employed to reduce economic losses in egg

producing establishments. Proper flock management and destruction and disposal of affected chicks are important.

Duck hepatitis A virus exists worldwide. It is highly contagious and affects young ducklings less than 6 weeks of age. It appears suddenly and is often fatal. There are three antigenically different strains (DHAV-1, -2, -3) although the symptomatology of disease caused by each is similar.

The clinical disease consists of listlessness, ataxia, opisthotonus and falling over and death. Onset is sudden, and the course of entire disease process may last from mere hours to maybe three to four days, with most deaths around day two.

The liver is enlarged with hemorrhagic lesions expressed by ecchymoses and petechiae. There may be splenomegaly and swollen kidneys with congested renal vessels.

Adult birds may become infected, but they do not show symptoms above the age of 7 weeks.

Flock management and strict isolation must be a priority. Rodent control is also indicated, because rats have been reported to be the reservoir for the virus. Both live attenuated vaccines and inactivated vaccines are available and should be used.

HerpesviridaeHerpesviridae: there are only two herpesviruses that are known to have crossed the barrier between animal and man. One is Murine herpesvirus-68 (MHV-68) antibody, which has been found in laboratory technicians working with infected mice. The second one is

Herpesviridae: Group I/ dsDNA/Enveloped, spherical to pleomorphic/ 150-200 nm in diameterSubfamily Genus Species Host Disease in manAlphaherpesvirinae Iltovirus Gallid herpesvirus 1 Poultry ?

Psittacid herpesvirus 1 Psittacine birds ?Mardivirus Marek’s disease virus Chickens ?

Gallid herpesvirus 3 Chickens ?Herpesvirus of turkeys Turkeys ?

Simplexvirus Herpes simplex virus 1 Man Muco-epithelial blistersHerpes simplex virus 2 Man Muco-epithelial blisters

Varicellovirus Varicellavirus Man Chickenpox, shingles Siminan varicella virus Simians ?

Betaherpesvirinae Cytomegalovirus Human herpesvirus 5 Man Salivary glandsMuromegalovirus Murine herpesvirus 1, 2 Mouse, pig, horse, rat ?Proboscivirus Elephantid herpesvirus Elephant ?Roseolovirus Human herpesvirus 6, 7 Man Roseola infantum

Gammaherpesvirinae Lymphocryptovirus Epstein-Barr virus Man Mononucleosis, BurkittMacavirus Alcelaphine herpesvirus 1 Wildebeest ?

Alcelaphine herpesvirus 2 Hartebeest ?Bovine herpesvirus 6 Cattle ?Ovine herpesvirus 2 Sheep, ungulates ?

Percavirus Equid herpesvirus 2 Horses ?Equid herpesvirus 5 Horses ?Cercopithecine herpesvirus 1 Monkey: Macaca cynomolgus CNS; high mortality

Rhadinovirus Kaposi sarcoma herpes virus Man Kaposi sarcoma? Denotes nothing of zoonotic importance is known, and subject will not be considered in this review.

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cercopithecine herpesvirus-1, (monkey B virus), which is found in macaca and cynomolgus monkeys and has been shown to infect laboratory workers, often with deadly result.

The monkeys, natural hosts of the virus themselves, do not exhibit any or only minor symptoms of infection. It may take as long as one month after exposure for infected humans to show signs of the disease. Those signs include blistering lesions near the site of scratch or bite and involvement of the lymphatic system (lymphadenitis, lymphangitis).

The infection process is function of the dose of virus received and the location of entry. As a rule, there are signs of neurological complications in the same area, pain, itching or loss of sensation. There usually is fever, malaise, influenza-like

complaints, chills, enduring headaches and fatigue. Some patients have abdominal pain and are nauseated and vomiting. There may be respiratory involvement, dyspnea and loss of muscular coordination. Death can occur suddenly a day or two after onset of the disease, or it may take weeks.

Central nervous system involvement produces symptoms such as abnormal increase in sensitivity, loss of coordination, double vision, excitability and ascending flaccid paralysis. Prognosis upon the appearance of these systems is ominous, regardless of the use of antivirals and supportive therapy. Cause of death usually is ascending paralysis and respiratory failure.

Survivors may suffer severe neurological consequences. Asymptomatic infections of

laboratory workers have not been reported. Rabbits, guinea pigs and mice can be infected experimentally. Before monkeys are used in the laboratory or for vaccine production, they are subject to strict control prerequisites: one to three months of isolation and quarantine, physical examination, tuberculin testing, blood cell workup and antibody screening. Sick or dying recent imports must undergo necropsies and be reported to the Centers for Disease Control and Prevention.

The absolutely most important first step after scratch or bite by a monkey is to thoroughly cleanse and rinse the wound with lots of soap and water for at least 15 minutes while refraining from massaging it in even further. It only takes minutes for the virus to enter sensory nerve endings, from where it will no longer become accessible to physical removal and will be protected from the developing antibody.

ReoviridaeThe family of Reoviridae is a mixed bag of viruses. Named after respiratory enteric orphan viruses, they are subdivided into two subfamilies, based on their structure: Sedoreovirinae and Spinareovirinae. They are double-stranded RNA viruses with segmented genom (12 portions), non-enveloped, with an icosahedral capsid and an outer and inner protein shell, ranging from 55 nm to 70 nm in size.

Reoviridae infectiousness ranges the gamut of the biosphere, from bacteriophage, through plant, arthropod, crustacean, fish to man – they infect everything.

Existing obviously worldwide, some of them cause minor disease of the gastrointestinal system (Rotavirus) and the respiratory tract in man. They are easily isolated from feces, bodily fluids and other secretions.

Among the subfamily of Sedoreovirinae are six genera: Cardoreovirus (infecting crabs), Mimoreovirus (Micromonas algae), Orbivirus (ungulates, horses), Phytoreovirus (plants), Rotavirus (domestic animals, livestock, man), and Seadornavirus (pigs, cattle, man).

Blue tongue disease virus is a strain belonging to the genus Orbivirus. It exists worldwide, is arthropod-borne, with mosquitoes, midges, gnats, sandflies, ticks representing both host and vector. The virus infects cattle, sheep, goats and other ungulates, producing blue tongue disease.

Also called catarrhal fever, it is a serious disease, especially in sheep, with high morbidity and fatality rates. There frequently is high fever, and salivation, the face and tongue may swell up, and there will be cyanotic discoloration of the tongue, nasal discharge and heavy, labored breathing. There usually is coronitis, stamping of feet (dancing disease), lameness, knee-walking, opisthotonus and torticollis.

For those most severely affected, death may come within a week. For survivors, recovery is slow. The economic impact of this disease is considerable, especially in the more susceptible breeds of sheep.

Herpesviridae

Molecular biology

VIRION

Enveloped, spherical to pleomorphic, 150-200 nm in diameter, T=16 icosahedral symmetry. Capsid consists of 162 capsomers and is surrounded by an amorphous tegument. Glycoproteins complexes are embeded in the lipid envelope.

REPLICATIONNUCLEARLytic replication: 1. Virus attaches to host receptors through gB, gC, gD and gH .2. Fusion with the plasma membrane to release the core and the tegument proteins into the host

cytoplasm.3. The capsid is transported to the nuclear pore where the viral DNA is released into the nucleus.4. Transcription of immediate early genes which promote transcription of early genes.5. Transcription of early viral mRNA by host polymerase II, transport into the cytoplasm and

translation into early proteins.6. Early proteins are involved in replication of the viral DNA and are transported back into the

nucleus.7. Synthesis of multiple copies of viral DNA by the viral DNA-dependent DNA polymerase.8. Transcription of late mRNAs by host polymerase II, transport into the cytoplasm and translation

into late proteins.9. Late proteins are structural or core proteins and are transported back into the nucleus.10. Assembly of the virus and budding through the inner lamella of the nuclear membrane which

has been modified by the insertion of herpes glycoproteins, throughout the Golgi and final release at the plasma membrane.

Latent replication: replication of circular viral episome in tandem with the host cell DNA using the host cell replication machinery.

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Mortality in breeds may vary from none to 90 percent. In other ruminants, the disease is milder if not asymptomatic, except for red deer, in which the disease is as severe as in sheep.

Other orbiviruses affecting animals are the African horse sickness virus, equine encephalosis virus and the epizootic hemorrhagic disease virus in deer. None of these viruses is zoonotic.

Rotavirus is mostly associated with stomach flu and severe watery diarrhea in infants and children. It is found worldwide and causes millions of cases of gastrointestinal disease requiring hospitalization and leads to more than 600,000 deaths. In the United States, rotavirus gastroenteritis causes in excess of 3 million of cases of diarrhea and 500,000 doctor visits in children under 5 at an economic cost, including loss of work days for parents, of $1 billion dollars yearly.

At-risk populations are children in hospitals and day care centers and the elderly in old-age facilities. Nearly every child worldwide is thought to have had contact with the virus by the time he or she reaches school age. Transmission is by the fecal-oral route facilitated by careless sanitary hygiene and procedures.

Rotavirus also infects non-human vertebrates.

Rotavirus gastroenteritis, often only a mild disease, can appear with light fever, vomiting, a teeming diarrhea and dehydration. Symptoms appear about two days after infection and may last a week. If death occurs, it is usually due to dehydration. After recovery, re-infection is possible, but subsequent infections are usually accompanied by much reduced disease and symptomatology.

Infection in the newborn, still protected by maternal immunity, is mild if not nonexistent. Vaccines are available and recommended by the WHO to be included in all national immunization programs.

One species of the genus Seadornavirus is the Banna virus, which is distributed throughout Southeast Asia. The virus has been isolated from an individual with encephalitis and from others with febrile illness as well as from pigs and cattle in Southern China.

In man, its symptoms are influenza-like: fever, headache, muscle and joint pain, and encephalitis. The virus has also been found in mosquitoes. There seems to be no evidence of a zoonotic

relationship between the mosquito isolates and the one from man.

Among the Spinareovirinae group of nine genera are aquareovirus (infecting fish), coltivirus (humans, rodents, arthropods), cypovirus (arthropods), dinovernavirus (man?, mosquito cell lines), Fijivirus (plants, arthropods), idnoreovirus (hymenoptera), mycoreovirus (fungus), orthoreovirus (bird, baboon, reptile) and oryzavirus (plants, planthopper).

Coltivirus causes Colorado tick fever, which is found in the western United States and Canada. It infects man, arthropods and rodents as a rule at altitudes above 4,000 feet. Rodents are the reservoir and host, and wood ticks (Dermacentor andersoni) serve as vector. Populations at risk includes hikers, campers and outdoorsmen.

Colorado tick fever is an acute viral infection that causes fever, muscle pain, pain in the eyes and headache. There is no man-to-man transfer other than possibly iatrogenically in the course of blood transfusions or organ transplants. The virus replicates in hematopoietic cells, ending up in mature erythrocytes and thus becoming available to the vector tick for transmission to the new host.

After an incubation period of three to six days, an infected individual may go through headache, photophobia and painful eyes; fever, chills and sweating; and myalgia, malaise and nausea with vomiting and abdominal pain. Sometimes there will be light-colored rash.

Following a short period of apparent recovery, there may be a second stage of the disease with considerably higher fever and more severe symptoms. The viremic phase may last as long as 120 days. During this four-month period, the patient’s blood remains infective and must not be used for blood transfusion. Children are particularly susceptible and may require hospital care. Hemorrhagic fever, aseptic meningitis, encephalitis are possible but not very likely complications. There is no treatment for this disease other than supportive care.

To reduce the chance of tick bite, avoid tick-infested areas, wear light colored, protective clothing, long sleeved shirts, long slacks, high socks to tuck in pants. Use insect repellent (DEET, permethrin). Inspect exposed areas of skin frequently to remove ticks quickly. The prognosis is good, complications are rare and the disease will go away by itself.

FiloviridaeFiloviridae are threadlike viruses that cause hemorrhagic fever and are deadly. They belong to Group V (-) ssRNA, are 790 nm (Marburg virus) or 970 nm (Ebola virus) long with a diameter of 80 nm. Their reservoir is bats, man and primates.

They are categorized as biosafety level 4, requiring the highest biocontainment precautions. When the Marburg outbreak occurred in 1967, 31 people were infected and seven died. The Ebola virus outbreak happened nine years later, infected 500 people and killed 460. One species of this

Reoviridae

Molecular biology

VIRION

Rotavirus maturation schemes

Non enveloped, icosahedral virion with a double capsid structure (except for cypoviruses and dinovernaviruses which have only the equivalent of the inner capsid). The outer capsid has an icosahedral T=13 symmetry, the inner capsid an icosahedral symmetry T=2.

REPLICATIONCYTOPLASMIC1. Virus attaches to host receptors and is endocytosed into vesicles in the host cell.2. Particles are partially uncoated in endolysosomes, but not entirely, and penetrates in the

cytoplasm.3. Early transcription of the dsRNA genome by viral polymerase occurs inside this sub-viral

particle (naked core), so that dsRNA is never exposed to the cytoplasm. Full-length plus-strand transcripts from each of the dsRNA segments are synthesized. These plus-strand transcripts are used as templates for translation.

4. (+)RNAs are encapsidated in a sub-viral particle, inside which they are transcribed to give RNA (-) molecules with which they become base-paired to produce dsRNA genomes.

5. The capsid is assembled on the sub-viral particle.6. Mature virions are released presumably following cell death and associated breakdown of host

plasma membrane.

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group of viruses, isolated in Virginia, the Reston ebolavirus, caused no disease in man.

There is no treatment for Filoviridae, and there is no vaccine. Supportive care and the containment of secondary infection must be the main effort.

The virus attaches to the host cell, undergoes endocytosis by invagination with the host cell plasma membrane into a vesicle inside the cell and takes over virus manufacture.

The virus is zoonotic, and has been found in insectivorous bats and fruit bats without making them ill. These bats are potential reservoir and carrier. Contact with a diseased individual or his infected body fluids will transmit the infection. Hospitals and health care providers are possible sources. Aerosolized transmission has been shown in monkeys.

Symptoms of the disease for both species are high fever, sore throat, fatigue and body aches, maculopapular rash, nausea, vomiting, stomach pain and diarrhea. The virus destroys the endothelial cell lining of blood vessels and interferes with the coagulation process leading to internal and external bleeding. Death occurs through organ failure, fluid and blood loss, hypovolemic shock.

OrthomyxoviridaeOrthomyxoviridae are influenza viruses that are varied and variable in their antigenic composition. They belong to Group V; their characteristics are (-)ssRNA, enveloped, helical, and spherical, and they are 80-120 nm in diameter.

In the United States, the annual influenza season puts more than 200,000 people into the hospital and kills about 36,000. It has an economic toll of more than $10 billion.

There are five genera of influenza viruses: influenza virus A, influenza virus B, influenza virus C, is a virus (infecting salmon), and thogotovirus (mosquitoes, sea lice).

Influenza A virus infects wild aquatic birds, which are their natural host; some mammals; and man. Wild birds seem to be the reservoir for influenza A virus; all subtypes of that virus have been isolated from birds in the wild. Wild birds transmit the virus to domestic fowl. Strains of the virus vary in virulence and infectivity to chickens and can cause severe disease in chickens and occasionally in man.

Virus subtypes mutate easily and will develop different levels of pathogenicity to different types of host species – hence the influenza pandemics in man.

Influenza A is the most virulent and contagious of the influenza virus genera. In poultry, influenza A produces bird influenza. Depending on the severity of the disease in poultry. The avian influenza strains are divided into the highly pathogenic ones (HPAI) and the mildly pathogenic ones (MPAI).

HPAI are highly contagious, causing severe systemic disease involving multiple organs during the course of the disease, with high mortality rates. They used to be recognized as fowl plague. In the peracute events, birds may die suddenly without clinical symptoms or noticeable lesions. The acute course of the diseases involve swelling of the head, wattle, comb, legs and feet; cyanosis; and severe hemorrhages causing ecchymosis and petechiae on the skin and on the visceral organs and muscle tissue. Toward the end, there may be greenish, watery diarrhea.

In survivors, there are severe sequelae, suggesting central nervous system complications including incoordination, opisthotonus and torticollis. Pathology shows the evidence of hemorrhage, edema, petechiae, necrosis of visceral organs and central nervous system matter.

Most strains are in the MPAI category and produce asymptomatic infections in all domestic fowl. However, occasionally they cause sinusitis and swelling of infraorbital sinuses, nasal discharge and discharge from the eyes, tracheal and pulmonary inflammation and congestion, and renal failure and concomitant urate buildup on internal organs. Breeding and egg producing establishments will notice a drop in egg production, ova rupture and egg malformation. There are signs of mucosal edema and inflammation of the oviduct. These complications do not occur very often, and overall mortality is low.

Cats, which are possible carriers after contact with or ingesting infected birds, should be considered when setting up biosecurity procedures. Main sources of transmission are careless movement and disposal of infected birds, contaminated feces and nasal secretions, clothing and equipment used in flock management as well as windblown aerosols and dust.

The Office International des Epizootie (OIE) placed highly pathogenic avian influenza on List A, which comprises 15 communicable diseases that have the potential for very serious and rapid spread irrespective of national borders (e.g., foot-and-mouth disease or rinderpest), that are of serious socioeconomic or public health consequence, and that are of major importance in the international trade of animals and animal products.

Since the middle of the last century, there have been 18 outbreaks of the disease. The mildly pathogenic strain is found in clinically normal shorebirds, wild aquatic birds, and migratory waterfowl initiating occasional outbreaks in domestic poultry. While there is no known reservoir for the highly pathogenic strain, it is thought that it might rise out of the mildly pathogenic virus population through mutation and antigenic shift.

Filoviridae

Molecular biology

VIRION

Filamentous 790 nm long for Marburg virus and 970 nm long for Ebolavirus. Diameter is about 80 nm.

REPLICATIONCYTOPLASMIC1. Virus attaches to host receptors through GP glycoprotein and is endocytosed into vesicles in

the host cell. 2. Fusion of virus membrane with the vesicle membrane; ribonucleocapsid is released into the

cytoplasm. 3. Sequential transcription, viral mRNAs are capped and polyadenylated in the cytoplasm. 4. Translation of the mRNA into viral proteins occurs using the host cell’s machinery.5. Replication presumably starts when enough nucleoprotein is present to encapsidate neo-

synthetized antigenomes and genomes. 6. The ribonucleocapsid interacts with the matrix protein under the plasma membrane, buds from

the plasma.

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Careful biosecurity practices assuring the elimination of contact with the virus combined with eradication are essential preventive measures. Allowing a low infective presence in a flock to maintain active immunity is not recommended in view of these considerations. Vaccines are available.

Influenza virus B infects man, seal and ferret. It occurs less frequently than influenza A, and it mutates only at one-third the rate of influenza A. Accordingly, there is much less genetic diversity in influenza B than there is in influenza A.

This confirms the observation that immunity to a given influenza B strain can be acquired and last for some time. Still, mutational variability is efficient enough to overcome immunity after some period of time. This particularity of influenza type B promises the unlikelihood of a pandemic by this virus.

Influenzavirus C infects man, dogs and swine, occasionally producing disease and local epidemics. It causes only mild illness in children.

The symptomatology for all three types of virus is similar, although they do vary in severity, with influenza A the most severe, followed by Influenza B, and influenza C as the least severe. It includes general discomfort, fever, severe headache, chills, sore throat, coughing, myalgia, weakness and fatigue. Occasionally there are

symptoms of gastroenteritis with nausea and vomiting. Symptoms can progress to pneumonia, which may lead to death in the very young and very old.

Transmission occurs from person to person via droplet aerosol, direct contact and contact with contaminated surfaces, as well as bird droppings. Influenza viruses are susceptible to inactivation by sunlight, disinfectants, soap and water and detergents.

Seasonal epidemics and the frequent antigenic shift of epidemic-causing viruses make it important to get an updated vaccine before the influenza season starts. This is especially important for the elderly and people in weakened condition.

Seasonal epidemics lead to the death of one-quarter to one-half-million humans worldwide, and millions in pandemic years. More than 41,000 died in the United States annually during the period between 1979 and 2001. New virus strains also caused pandemics in the past century, killing tens of millions of people.

Neuraminidase inhibitors and other antiviral drugs can be considered for treatment.

Prions: Infectious proteins usurp the cell’s protein manufacturing mechanism and make it produce rogue proteins of its own configuration at the expense of the manufacturing cell.

They are cause of transmissible spongiform encephalopathies (TSEs), progressive degenerative brain diseases such as bovine spongiform encephalopathy, also known as mad cow disease (cattle); scrapie (sheep, goat); transmissible mink encephalopathy (mink); chronic wasting disease (white-tailed deer, elk, mule deer, moose); feline spongiform encephalopathy (cat); exotic ungulate encephalopathy (nyala, oryx, greater kudu); and the following diseases in man: classic Creutzfeldt-Jakob disease (cCJD), variant Creutzfeldt-Jakob disease (vCJD), Gertsmann-Straeussler-Scheinker (GSS) disease, fatal familial insomnia (FFI) and kuru.

Transmission may be by ingesting infected meat or via the urine, saliva or other body fluids. The incubation period depends on the replication of rogue proteins and may take months if not years. After symptoms become apparent the disease generally progresses fairly rapidly. It reflects the neurodegenerative disease process very much alike for all the known prion diseases.

In particular, there is the formation of amyloid plaques in the central nervous system interposing within the normal tissue structure, spongioform holes and vacuolation of neurons, overabundance of astrocytes and lack of the typical inflammatory response mechanism.

The neuro-degenerative effects include behavioral changes, dementia, loss of motor control, discoordination and ataxia, wasting, convulsions and death. There is no treatment; these diseases are always fatal.

Bovine spongiform encephalopathy (BSE) is a fatal progressive encephalopathy in cattle. The disease is transmitted naturally to felids and many other ungulates, exotic as well as domestic, and can be transmitted artificially to rodents, mink, pigs, sheep and goat as well as monkeys. Cats and other felids are infected by ingesting infected bovine tissues.

The disease has been encountered all over Europe, Canada, the Falkland Islands and Oman. Prion protein, an abnormal form of membrane protein, is the infecting pathogen. Cattle are infected by ingesting contaminated meat and bone meal in their feed. There is no cattle-to-cattle transmission, although, there may be the occasional transplacental transfer. The morbidity is low and only in cattle from 2 years up.

Orally infected cattle show the pathogen in the Peyer’s patches of the small intestine, invading peripheral nerves and the central nervous system. The initial signs of the disease are not obvious and easily missed. Reduced amount of rumination, nose wrinkling and licking, sneezing and snorting, head tossing and grinding of teeth suggest neurological problems. This observation is supported by increase in excitability and exaggerated reflexes, frenzy, head shyness and kicking as well as exaggerated responses to sensatory stimuli. Left undisturbed, the animal will just stand there, not moving, head down, staring into nothing. When moving, the gait is

Filoviridae

Molecular biology

VIRION

Filamentous 790 nm long for Marburg virus and 970 nm long for Ebolavirus. Diameter is about 80nm.

REPLICATIONNUCLEUS1. Virus attaches to sialic acid receptor though HA protein and is endocytosed in the host cell.2. Endosome acidification induces fusion of virus membrane with the vesicle membrane;

encapsidated RNA segments migrate to the nucleus.3. Transcription of genomic segments by the viral polymerase produces mRNAs.4. Genomic (-)RNA5. High level of M1 protein induces genomes segments export from nucleus by NEP protein.6. Virus assembly and budding occurs at the plasma membrane.

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uncoordinated, ataxic, overshooting its aim and running into things. The animal may fall down, develop tremors, muscle twitches and spasms and generalized progressive paresis. There is weight loss and lowered milk production. Euthanasia is advisable before things go too far.

Pathologically there will be bilateral symmetric spongiosis of gray matter, neutropil, vacuolation and loss of nerve cells, increased number of astrocytes and the deposition of amyloid plaque. There are no signs of inflammation. The long incubation period in cattle suggests the possible presence of prions in the meat from an infected animal. Creutzfeldt-Jakob disease in man is thought to be caused by the consumption of infected meat.

Disinfection must consider the protein nature of prions. Prions are resistant to proteases, ionizing radiation, heat (even at boiling temperatures), and formalin. Strong acidic detergents, bleach, and caustic soda are possible disinfectants. Recovery of infectivity after partial denaturation is possible.

Scrapie represents the transmissible spongiform encephalopathy in sheep and goats. It exists worldwide except for Australia and New Zealand, where it has been eliminated through a compulsory eradication program. In the United States, scrapie disease costs sheep producers $10 million to $20 million per year due to lost market share, sales abroad and increased production costs.

In 2001, the U.S. Department of Agriculture initiated an accelerated scrapie eradication program based on:

■ Identification of pre-clinical infected sheep through live-animal testing and active slaughter surveillance.

■ Effective tracing of infected animals to their flock/herd of origin, made possible as a result of new identification requirements.

■ Providing effective cleanup strategies that will allow producers to stay in business, preserve breeding stock, and remain economically viable. The USDA Animal and Plant Health Inspection Service (APHIS) will do this by providing the following to exposed and infected flocks/herds that participate in cleanup plans:

□ Indemnity for high-risk, suspect, and scrapie-positive sheep and goats, which owners agree to destroy.

□ Scrapie live-animal testing. □ Genetic testing. □ Testing of exposed animals that have

been sold out of infected and source flocks/herds.

For identification and tracking, the following tagging requirements were recommended:

■ All sheep/goats sexually intact regardless of age and wethers 18 months of age and older upon change of ownership, or cull animals 18 months or older moving to slaughter.

■ All sheep sexually intact regardless of age and wethers 18 months of age and older for show or exhibition.

■ All breeding sheep regardless of age. ■ All goats sexually intact regardless of age

and wethers 18 months of age and older for show or exhibition that do not have a registration tattoo with registration or a tattoo that is illegible.

■ All commercial goats sexually intact regardless of age and wethers 18 months of age and older not in slaughter channels.

The disease is thought to be passed on through careless movement of animals between herds, ingestion of feed contaminated with infected brain matter, placental tissue and afterbirth. Wild ruminants and domestic and feral cats that have ingested infected tissue are potential carriers.

Because the disease seems to be transmitted in genetically related members of a flock, a genetic predisposition for susceptibility has been hypothesized. Suffolk and Cheviot sheep are among the most susceptible breeds.

There have been reports of the spontaneous development of the disease. In view of the long incubation periods observed, this is difficult to verify. Moreover, prions may be spread with the urine and last for decades in the environment. The infectious protein seems to pass through broken skin and the intestinal tract. Peyer’s patches of the small intestine seem to be the first to gather them.

The clinical picture is much like other transmissible spongiform encephalopathies, including excitability, exaggerated reflex reactions to sudden movements or noises, tremors of head and neck, ataxic gait, inability to control leg movement, discoordinated trotting or hopping and convulsions. Other symptoms include changed drinking and urination patterns, such as drinking and voiding more frequently in smaller amounts; and dry, brittle fleece and loss of areas of fleece. There may be skin itching, pruritus, raw areas of skin caused by persistent rubbing on fixed objects, emaciation and weakness. Some sheep may die without noticeable symptoms.

Microscopic changes in neurons are those expected with spongiform degeneration of the central nervous system, including vacuolation and amyloid deposits in and around the neuropil and the walls of blood vessels in the area.

There is no evidence of transmission to man. Direct contact with goats and grazing on the same pastures can transmit the disease to goats, but the disease incidence is low.

There are two variants of Creutzfeldt-Jakob disease: the classic Creutzfeldt-Jakob disease (cCJD), which is not related to bovine spongiform encephalopathy, and the variant Creutzfeldt-Jakob disease (vCJD), which is caused by BSE.

The variant version (vCJD) has been contracted through ingestion of contaminated beef. Because the pathogen can lie dormant for years, anyone having consumed meat from the same infected beef could come down with the disease even years after consumption.

So it is impossible to judge or estimate how many more will die from an outbreak in 1986 in the United Kingdom. In that outbreak, more than 179,000 cattle were infected and more than 4 million were slaughtered during the course of an eradication program. Nearly a half-million BSE-infected animals entered the food chain at the time. The human toll since 1986 was 10 during the first 10 years to 1996, increasing to 140 by 2004 and to 166 by 2009. Of course, there is also the

Clinical and pathologic characteristics distinguishing classic CJD from variant CJD

Characteristic Classic CJD Variant CJD

Median age at death 68 years 28 years

Median duration of illness 4-5 months 13-14 months

Clinical signs and symptoms Dementia; early neurologic signs

Prominent psychiatric/behavioral symptoms; painful dyesthesiasis; delayed neurologic signs

Periodic sharp waves on electroencephalogram

Often present Often absent

Pulvinar sign on MRI* Not reported Present in >75 percent of cases

Presence of florid plaques on neuropathology

Rare or absent Present in large numbers

Immunohitochemical analysis of brain tissue

Variable accumulation Marked accumulation of protease-resistance prion protein

Presence of agent in lymphoid tissue Not readily detected Readily detected

Increased glycoform ratio on immunoblot analysis of protease-resistance prion protein

Not reported Marked accumulation of protease-resistance prion protein

Source: Adapted from Belay E., Schonberger L. Variant Creutzfeldt-Jakob Disease and Bovine Spongiform Encephalopathy. Clin Lab Med 2002;22:849-62.

*An abnormal signal in the posterior thalami on T2- and diffusion-weighted images and fluid-attenuated inversion recovery sequences on brain magnetic resonance imaging (MRI); in the appropriate clinical context, this signal is highly specific for vCJD.

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possibility that prions derived from spontaneous formation or from other sources, including blood transfusions and organ transplants.

The clinical illness consists of depression and schizophrenic psychoses, lack of coordination, unsteadiness, difficulty walking, reflexive movements, inability to move and muteness.

The disease process for vCJD varies from the classic CJD. Onset is earlier, and it includes behavioral changes, loss of sensation and peripheral acuity, and an inability to coordinate muscle activity, in contrast to classical CJD with its changes in brain wave activity, mental activity and thinking ability.

Each disease also has a particular genetic profile of the prion protein gene. The median age at death for vCJD patients is 28 years, compared with 68 years for patients with classic CJD. The median duration of illness for vCJD is 14 months, compared to five months for classic CJD.

Amplification and identification Lymphocytic choriomeningitis symptoms in the early stage of the disease are minimal and somewhat influenza-like. The first suggestion of meningitis is a stiff neck, altered mental state, ataxia, incontinence or fetal malformation, hydrocephalus, microcephalus and choroid retinitis. Virus isolation requires a sample of cerebrospinal fluid via lumbar puncture (spinal tap) and blood for laboratory investigation.

Isolate, identify, look for antibody in blood and cerebrospinal fluid, look for increased leucocyte count and lymphocytic infiltration. Diagnostics for man and mouse are essentially the same, with the exception that you can use the mouse brain for virus isolation.

Lassa virus: The initial symptoms, one to three weeks after exposure, are fever, swelling of face, muscle fatigue, mucosal bleeding and conjunctivitis, and low counts of white blood cells and thrombocytes. There likely will be raised levels of aspartate aminotransferase.

Whitewater Arroyo virus and the Tamiami virus belong to the same family of viruses, the Arenaviridae. They are likely to require similar procedures.

Yellow fever: Three to six days after mosquito exposure comes a first phase of the disease: mild fever, chills, headache, nausea, vomiting, and some back pain, subsiding quickly (three to four days); and followed by a second phase in which recurrent fever, jaundice, abdominal pain, bleeding from mouth, nose and eyes, and vomiting blood are seen.

West Nile virus: Three phases, (1) asymptomatic, (2) West Nile fever (mild), (3) neuroinvasive (meningitis, encephalitis). Early symptoms lasting seven to 10 days: fever, headache, chills and sweats, nausea, vomiting, diarrhea, weakness, joint pain, persistent lymphadenopathy, fatigue and drowsiness. In addition to the usual symptoms for meningitis and encephalitis, there may be movement

disorders, anterior myelitis of spinal cord, multifocal chorioretinitis, Guillain-Barré syndrome and inflammation of visceral organs. All members of the flaviviridae family should require similar testing.

La Cross virus-induced symptoms include headache, lethargy, nausea and vomiting. In children less than 16 years of age, there is occasionally encephalitis with its concomitant effects, including seizures, paralysis and coma. Virus can be isolated in cell cultures, and antibody can be measured by virus neutralization in the same system. Because the viruses of this family are antigenically related, it may be necessary for a battery of cross tests to clearly identify the pathogenic culprit.

Hantavirus: Although this virus is a member of the family of Bunyaviridae, its mode of infection is different as it is carried by rodents and infects via aerolized rodent debris, urine, saliva and droppings.

We recognize two types of disease syndrome appearing two to four weeks after exposure:

■ Renal, which includes a: □ Febrile phase (influenza-like, i.e. fever,

chills and sweats, malaise, abdominal and back pain, nausea and vomiting) lasting maybe a week.

□ Hypotensive phase (thrombocytopenia, tachycardia and hypoxemia) lasting two days.

Recent exposure: Rodents, rodent infested areas, dusty old houses, sheds; source of protein

Differential diagnoses: Encephalitis, Mosquito-borne diseases, mumps

Procedure Blood/serum CSF Biopsy

Isolation CF, IFA, ELISA, WBC, AST CF, IA, ELISA WBC PCR, IFA, ELISA, MAP,

Serology IgM/IgG-ELISA’s

Recent exposure: Rodents, rodent infested areas, dusty old houses, sheds; source of protein

Differential diagnoses: Malaria, marburg or ebola virus

Procedure Blood/serum Urine CSF Biopsy )*

Isolation CF, IFA, ELISA, WBC MAP CF, IA, ELISA WBC MAP, PCR, ELISA

Serology ELISA

)* Not advisable in patients prone to bleeding

Recent exposure: Travel to mosquito regions during mosquito season

Differential diagnoses: malaria, dengue fever, typhoid, leptospirosis, tick-borne relapsing fever, typhus, Q fever, severe viral hepatitis, Rift Valley fever, Crimean-Congo haemorrhagic fever, Lassa, Marburg and ebola fever, other viral hemorrhagic fevers

Procedure Blood/serum Urine CSF Stool Biopsy )*

Isolation rt-PCR, ELISA, cell culture rt-PCR, ELISA

Serology IgM/IgG-ELISA’s

)* Not advisable in patient prone to bleeding

Recent exposure: Travel to mosquito regions during mosquito season

Differential diagnoses: malaria, dengue fever, other viral hemorrhagic fevers

Procedure Blood/serum CSF Biopsy

Isolation Cell culture, FCMIA, rt-PCR Cell culture, FCMIA, rt-PCR

Cell culture, FCMIA, rt-PCR

Serology IgM/IgG-ELISA, IHC, FCMIA

IgM/IgG-ELISA, IHC

)* rt-PCR and IHC: dDetect the virus/antigen in mosquitoes, bird tissue

Recent exposure: Travel to mosquito regions during mosquito season

Differential diagnoses: malaria, dengue fever, other viral hemorrhagic fevers

Procedure Blood/serum CSF Biopsy

Isolation Cell culture, FC MIA, rt-PCR Cell culture, FCMIA, rt-PCR Cell culture, FCMIA, rt-PCR

Serology IgM/IgG-ELISA, IHC, FCMIA

IgM/IgG-ELISA, IHC

)* rt-PCR and IHC: detect the virus/antigen in mosquitoes

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□ Oliguric phase (renal failure and proteinuria) for three to seven days.

□ Diuretic phase (excessive urine production), which may last for weeks.

□ Convalescent phase (improvement and recovery).

■ Cardiopulmonary, resulting from the inhalation of infectious virus, which brings tachycardia, tachypnea and cardiovascular shock, leading to hospitalization and, often, death.

Smallpox virus (as paraphrased from the CDC)

Seven to 17 days after exposure:1. Prodromal phase, sudden onset of high fever,

malaise, head and body aches, vomiting (lasting two to four days).

2. Early rash phase, small red spots on mucosa of mouth and tongue, developing into sores, especially mouth and throat (four days).

3. Generalization of rash over entire body, fever dropping, feeling better (one day).

4. Three days after onset of rash: rash turns to raised bumps (one day).

5. Fourth day after onset of rash: bumps fill with thick, opaque liquid, depression in center of bump (an important characteristic).

6. Fever reappearing and high until scabbing of bumps.

7. Pustular rash: Bumps turn into sharply raised, round, firm pustules (five days duration).

8. Pustules and scabs: Crusting and scabbing of most sores (five days).

9. Resolving scabs: Scabs falling off, pitted scars (six days).

Guarneri bodies are detected in epithelial cells of lesions, brick-shaped virions in electron microscopy. Chickenpox, by contrast, exhibits varying size and progress of pustules and does not appear on palms and soles.

Pseudocowpox virus: Small, red blistering papules on teats and udder, turning to pustules that will scab quickly with ring-like or horseshoe-shaped appearance within seven to 12 days, which heal within six weeks or so. Nursing calves will develop lesions on mouth and muzzle.

Lyssavirus: Changes in behavior, avoidance and shyness by a pet that is usually friendly and approachable; no fear of humans in a wild animal; restlessness, agitation, excitability and unexpected mood changes; aggressiveness; overabundant drooling; behavior patterns of night animals during the day; and gnawing on and craving unedibles.

The approach is to sacrifice the animal, examine its brain, cortex, spinal bulb by virus isolation through passage in mice and cell culture, direct immunofluorescence.

In man, the incubation period from bite to first symptom is two to three months, but occasionally within days to more than a year. They include fever, confusion, sore throat and coughing, pain and numbness at site of bite, itching; and abdominal pain. The symptoms progress to anxiety and weakness, restlessness and extreme excitability, drooling, convulsions and muscle spasms, loss of feeling and muscle function, numbness and tingling,

difficulty swallowing. Death may occur any time (from two days to five years).

Attempt virus isolation from saliva or cerebro spinal fluid.

Vesicular stomatitis virus: After the incubation in cattle of two to eight days, initial signs of the disease include fever, hypersalivation, ulcerating vesicles around mouth and muzzle, sloughing of tongue epithelium, ulcerating lesions on teats and mastitis, loss of appetite, coronitis and lameness. The virus is zoonotic and may cause influenza-like disease in people working in close contact with infected cattle.

Eastern equine encephalitis: Region and season supporting a clinical picture of arbovirus encephalitis are suggestive of this type of

pathogen. However, this picture is alike for many other causes and requires additional testing. In man, the clinical picture and travel background are important considerations.

Newcastle disease virus: Two to 12 days after exposure there may be respiratory symptoms making the birds gasp, sneeze, cough and produce rattling or railing breathing sounds; progressing to tremors, paralysis of wings, legs, neck, clonic spasms, circling and complete paralysis; depression; green watery diarrhea; swelling of the head and neck. Other signs include reduced or stopped egg production and misshapen eggs.

Young birds are more susceptible. The virus can produce conjunctivitis in poultry workers and veterinarians.

Recent exposure: Rodents, rodent-infested areas, dusty old houses, sheds; source of protein

Differential diagnoses: leptospirosis, Legionnaire’s disease, mycoplasma, Q fever, chlamydia, septicemic plague, tularemia, coccidioidomycosis, histoplasmosis. Goodpasture’s syndrome

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation IHC, rt-PCR

Serology IgM/IgG-ELISA, RIBA, Western blot, cell culture plaque neutralization, Goose RBC HAI

Recent exposure: Direct contact

Differential dagnoses: Granulomatous dermatitis

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation PCR, RFLP, ELISA, EM

Serology ELISA

Recent exposure: Exchange of saliva, bite

Differential diagnoses: Arbovirus encephalitis, post viral encephalitis, Guillain-Barré

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation rt-PCR, MIT rt-PCR, MIT, TC, mice

rt-PCR, IF, dFA, MIT, EM, IHC, RFFIT, mice

Serology IF, virus neutralization in TC, in mice, dFA, FCMIA

Recent exposure: Moving infected cattle, black fly, sand fly regions

Differential diagnoses: Foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation TC, PCR

Serology ELISA, CF test, Virus neutralization

Recent exposure: Mosquito region, mosquito season

Differential diagnoses: Cranial trauma, rabies, hepatoencephalopathy, leukoencephalomalacia, protozoal encephalomyelitis, equine herpesvirus 1, verminous meningoencephalomyelitis, botulism, meningitis.

Procedure Blood/serum CSF Biopsy

Isolation WBC, TC, rt-PCR, IHC, ELISA

TC, rt-PCR, IHC, ELISA

Serology IgM/IgG-ELISA, PRNT, HI, CF

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Nipah virus encephalitis: Symptoms include fever; respiratory problems; a severe, barking cough; and encephalitis. In man, after incubation of three to 14 days, there is encephalitis, fever, headache, drowsiness, mental confusion and disorientation, and seizures leading to coma in a day or two. Hendra virus is member of the same family, and diagnostic tests are similar.

Encephalomyocarditis virus: Symptoms include fever, loss of appetite, listlessness, breathing difficulties, discoordinated movement, trembling, paralysis, cardiac insufficiency, edema of the lungs, frothy exudates from respiratory system, and drowning in respiratory exudates. Other signs are quick death in suckling swine, stillbirths, and fetus mummification.

In man, occasionally, there is fever, central nervous system complications and often death through myocarditis.

Porcine teschovirus: One to four weeks after exposure to the more virulent serotype 1 strain, there is discoordinated movement (ataxia), fever, listlessness, anorexia, sometimes seizures, involuntary eye movements, opisthotonus and coma. Progressive paraplegia advances to quadriplegia and death within three to four days of the appearance of symptoms. Less virulent strains will produce ataxia and paresis in young pigs with frequent recovery.

Cercopithecine herpesvirus 1: Asymptomatic in the monkey, it may take two days to five weeks to show symptoms in man, which include vesicular rash at site of contact, occasionally itching, tingling, numbness, and regional swollen lymph nodes; there may be malaise, fever, chills and muscle pain, followed by peripheral and central nervous system complications. Symptoms that spread along the peripheral nervous system towards the spinal cord and brain include paresthesia, meningismus, persistent headache, confusion, double vision, ascending paralysis, respiratory failure and coma.

Rotavirus: After a two-day incubation, the child will have a temperature and begin vomiting; after another two to three days, it will produce non-bloody diarrhea with 10 to 20 bowel movements a day. Combined with ongoing vomiting, dehydration can become life-threatening.

Coltivirus: Symptoms that appear three to five days after tick bite include fever, headache, malaise, intestinal disease for two to three days that subsides and re-appears with conjunctivitis, spotty rash on body and limbs, lymhadenopathy, stiffness of the neck, photophobia and retro-orbital pain, and reduced consciousness. Other signs: encephalitis hepatitis, myopericarditis and meningitis.

Marburgvirus: An incubation period of five to seven days may range from three to 10 days and then is followed by sudden onset of fever, chills, malaise, extreme exhaustion and weight loss. Five days later, a maculopapular rash appears and hemorrhaging starts. Hemorrhaging will get worse, coming from the nose, mouth, eyes, ears, and rectum. Other symptoms include headache, sore throat and cough, joint and muscle

Recent exposure: Movement of birds

Differential diagnoses: Avian influenza, fowl cholera, laryngotracheitis, fowl pox (diphtheritic form), psittacosis, mycoplasmosis, infectious bronchitis, Pacheco’s parrot disease (psittacine birds)

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation HA, day-old chicks, IC, PCR, TC-CEF,

Serology HAI, ELISA

Recent exposure: Movement of infected pigs

Differential diagnoses: Japanese encephalitis, arbovirus encephalitis

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation rt-PCR rt-PCR rt-PCR, IHC, TC

Serology IgM/IgG-ELISA, PRNT

IgM/IgG-ELISA, PRNT

Recent exposure: Rodents, movement of new animals into facility

Differential diagnoses: Viral encephalitis, FMDV, septic infarction, vitamin E/selenium deficiency

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation rt-PCR rt-PCR rt-PCR

Serology NT, ELISA, HAI

Recent exposure: Direct and indirect contact, fecal-oral route of infection

Differential diagnoses: Classical swine fever, African swine fever, pseudorabies, rabies, edema disease, water deprivation and salt intoxication, toxic and nutritional neuropathies

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation rt-PCR, TC TC, rt-PCR rt-PCR rt-PCR, TC

Serology ELISA,NT, CF (AB rise)

Recent exposure: Bite, scratch, or mucosal contact with body fluid from monkey, MK cell culture

Differential diagnoses: Rabies, herpes simplex encephalitis, arthropod-associated meningoencephalitis

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation rt-PCR rt-PCR

Serology ELISA, Western blot, NT

Recent exposure: Day care centers, homes for the elderly and family homes. Food handlers can contaminate foods that will not be subsequently cooked

Differential diagnoses: Bacterial gastroenteritis, norovirus, adenovirus and astrovirus

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation rt-PCR, EIA, EM , PAGE rt-PCR, EIA, EM, PAGE

Serology EIA

Recent exposure: Tick attachment; tick region, season

Differential diagnoses: Rocky Mountain spotted fever

Procedure Blood/serum CSF Biopsy

Isolation Baby mice IC, FA in RKC, rt-PCR

Baby mice IC, BHK21, rt-PCR Baby mice IC, BHK21, rt-PCR

Serology ELISA, Western blot

Recent exposure: Contact with infected person or primate, secretions, body fluids, excretions, semen

Differential diagnoses: Malaria, typhoid fever, viral hemorrhagic fevers (dengue, lassa)

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation rt-PCR, ELISA EM, rt-PCR, IHC

Serology IgM/IgG-ELISA WBC, AST/ALT

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pain, inflammation of intestine and diarrhea (sometimes bloody), chest and abdominal pain, internal organs, interstitial pulmonary edema, red eyes, eyelid and eye membrane issues, icterus and renal failure. About one out of four infected individuals will die.

Influenza A virus: After an incubation period of less than one week, the low pathogenic avian influenza strain produces respiratory disease, swollen infraorbital sinuses, discharge from eyes and nose, congestion and inflammation of trachea and lungs, reduction in egg production, misshaped eggs, inflammation and exudation from oviduct.

HP (highly pathogenic) strain can produce death before any noticeable symptoms; in the peracute phase of the disease or in the acute phase, you will see cyanosis, swelling of the head, comb and wattle; discoloration of legs; ecchymosis; subcutaneous hemorrhages and bloody discharges; green diarrhea, muscular discoordination, opisthotonus, torticollis and other CNS complications.

Bovine spongiforme encephalitis: The incubation period ranges from months to years. The early signs of the disease are behavioral, including reduced rumination intervals, nose licking, snorting, wrinkling, head rubbing on hard object, head tossing, teeth grinding, exaggerated responses to normal noises and touches, drowsiness, distress, ataxic gait and discoordination, muscle tremors, and fasciculations.

Scrapie: Incubation is indeterminate. Symptoms include behavioral changes, excitability, nervousness, aggressiveness, and excessive responses to normal noise and touch; tremors of the head and neck; convulsions; loss of muscular coordination; an ataxic and hopping gait; reduced but more frequent water uptake and voidance; and dry, brittle, or lost fleece.

Virus and antigen identificationOnce the general symptomatology of a disease is determined, laboratory tests must confirm the presumptive diagnosis and identify the pathogen causing it. The classical procedure has been and in many places still is the inoculation and if necessary, repeated passage in preferably young, often-newborn laboratory animals such as mice, hamsters, embryonated eggs.

The clinical, pathological and immunological changes produced may identify the isolate or at least suggest further steps at identification.

Traces of the virus or its antigen can be marked by immunological means as, for instance, specific antibody that has been labeled with some kind of indicator substance, the fluoresces in UV light (fluorescent assays) or that colorizes colorless substrates by enzyme action (enzyme linked antibody assays). Immunohistochemistry (ICH) takes advantage of this characteristic.

Of course, the antibody stimulated by the test inoculum can be screened against various known antigens and viruses, like in the mouse antibody production test (MAP). Embryonated eggs exhibit

distinctive foci in the chorioallantoic membrane after the inoculation with certain viruses, which then can be neutralized and again specifically identify the agent. The allantoic fluid of embryonated eggs may develop hemagglutinating agents that can be inhibited by specific antibody.

Laboratory confirmation, generally, involves isolating the agent, identifying it and determining its effect on the diseased body, which includes the symptoms observed and the body’s defense response.

The following procedures describe the classical concept of pathogen isolation, amplification and identification. The microscopic recognition of a specific pathogenic organism is not always easy or clear-cut, although there are pictorial representations of them to be found everywhere on the Web (the Google image gallery, Wikipedia or similar sites and the image library of the CDC at (http://www.dpd.cdc.gov/DPDx/html/Image_Library.htm).

Recent exposure: Unknown, movement of birds between flocks

Differential diagnoses: Infectious bronchitis; infectious laryngotracheitis; lentogenic Newcastle disease and paramyxoviruses; mycoplasmosis; infectious coryza; ornithobacteriosis; turkey coryza; and the respiratory form of fowl cholera, aspergillosis. Also seen are velogenic Newcastle disease, peracute septicemic fowl cholera, heat exhaustion, and severe water deprivation.

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation HA CE, HA, rt-PCR

Serology AGID, ELISA, HAI, CENT

Recent exposure: Possibly from feed containing bone meal, and meat by-products from infected cattle

Differential diagnoses: Rabies, encephalitic listerosis, hypomagnesemia, lead poisoning, downer cow syndrome, nervous ketosis, intracranial abscess or tumor, CNS lesions, trauma to spinal cord

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation IHC, EM

Serology ELISA, Western blot

Recent exposure: Possibly from feed containing bone meal, and meat byproducts from infected cattle

Differential diagnoses: Borna disease, Coenurus cerebralis, botulism, rabies, ectoparasites, Aujeszky’s disease (pseudorabies), dermatophilosis, allergic dermatitis, photodermatitis

Procedure Blood/serum Urine CSF Stool Biopsy

Isolation IHC, EM

Serology ELISA, Western blot

TriageType of animal Race, sex, size and weight, age

Central nervous system

Normal, obtunded, stuporous and comatose; attentiveness, bright and alert; gait, posture, balance; pupils constricted, dilated, equal size, or responsive to light; breathing pattern, seizures, convulsions, ataxia, circling; and response to pain stimulus.

Circulatory Color of mucous membranes, gums, conjunctiva, capillary refill time; pulse strength and rate, heart rate and regularity and skin turgor; temperature of extremities, and reduced urine production.

Agility, weakness, signs of blood loss; tachycardia, hypovolemia, bradycardia.

Respiratory Coughing, shortness of breath, stance of animal; open mouth, flaring nostrils, chest wall motion ; cyanosis, hypoxemia.

Gastrointestinal Nausea, vomiting, diarrhea, loss of appetite, weight loss; stool appearance (firm, watery), frequency; distended abdomen.

Renal Urination, frequency, color.

Systemic Fever, loss of appetite, weakness, debilitation, depression; bleeding from orifices, blood in stool, urine; yellow mucosae: icterus (liver), hemolysis: pale/white: blood loss, anemia, shock; brick red: sepsis, polycythemia, hyperthermia; blue: hypoxia.

Present medication

Poison, abnormal intake, chemicals around house.

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Sometimes it is not easy to locate the pathogen among all the debris or distinguish it from similar looking organisms. In that case, specific antibody can be tagged with a fluorescent dye, such as Fluorescein isothiocyanate or Rhodamide B isothiocyanate to seek out and identify the antigen by means of fluorescent microscopy i. e. the direct fluorescent assay (dFA). Fluorescent-labeled antibody conjugates are readily available commercially.

If a specific antibody to a particular antigen is not available in the fluorescent dye conjugated form, fluorescein-labeled antibody to the serum of the species of animal that had produced the pathogen-specific antibody could be used as well with the indirect fluorescent assay (iFA).

Instead of linking the antibody with a fluorochrome, the enzyme immune assay (EIA), also called enzyme-linked immuno-sorbent assay (ELISA), achieves similar results by conjugating the antibody with an enzyme, such as horseradish peroxidase, that will act upon chromogenic substrates such as TMB (3,3’,5,5’-tetramethylbenzidine), DAB (3,3’-Diaminobenzidine) or ABTS (2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid).

The enzyme-conjugated antibody will enhance and visualize the antigen that retains the antibody by its action on the chromogenic substrate. Varieties of such reagents are commercially available singly or in kit form. Another way to detect certain viruses is to utilize their

characteristic to attach to erythrocytes of certain species of birds or mammal.

Direct hemagglutination test (HA): Some viruses, among them adenoviruses, parvoviruses, togaviruses, some coronaviruses, picornaviruses, orthomyxo- and paramyxoviruses agglutinate erythrocytes by binding to specific receptor sites on the surface of the red blood cells. It is a quick and simple screening test for these viruses.

Quite often, the sample to be tested requires virus enrichment before hemagglutination is possible. This can be achieved by passage in 9-day-old embryonated eggs or in cell culture. The allantoic fluid or culture medium harvests are mixed with a suspension of erythrocytes to check agglutination.

Isolation and amplificationCollect sample From blood, stool, urine, cerebrospinal fluid, suspicious tissue (biopsy), pharyngeal swabs, tracheal lavage, amniotic fluid, aborted

fetus;

Inoculate culture Indicator animals (embryonated eggs, mice, hamsters etc), cell cultures; substrates, growth conditions and procedures dependent on suspected pathogen

Harvest product Morphology of pathogen, antigenicity, DNA content, infectivity for other indicator substrates

Store harvest Aliquots: unadulterated (-70 degrees C), prepared for eventual use. If transport to lab will be in more than three days, it is preferred to keep the sample on ice rather than frozen!

Sample collection/manipulation must be carried out under conditions precluding contamination from other sources, such as equipment, hands, chemicals involved in sample collection and preservation, airborne contaminants, dust, from hair or orifices of technician.

Isolation Agent Source/culture

Lymphocytic choriomeningitis Mouse IC, primary rabbit kidney cells, L cells

Lassa virus Vero cells, L cells

Yellow fever virus Newborn Swiss mice IC, vero, LLC MK-2, BHK, arthropod cell lines.

West Nile virus Vero cells

Lacrosse virus Suckling mice, BHK-21, vero cells

Hantavirus Bank vole, vero cells, MAP

Smallpox virus Chick embryo CA membrane, vero cells

Pseudocowpox virus Chick embryo CA membrane, vero cells

Lyssa virus Mouse inoculation test, vero cells

Vesicular stomatitis virus Weanling mice IC, baby hamster kidney cells

Eastern equine encephalitis virus Vero cells, AG-capture ELISA, A549, MRC-5 cell cultures

Newcastle disease virus Chick embryo allantoic cavity

Nipahvirus Hamster IP, IN, vero cells

Encephalomyocarditis virus Krebs II mouse ascites tumor cells, vero cells

Porcine teschovirus Swine primary kidney cells, swine testicular cells

Cercopithecine herpesvirus 1 Vero cells

Rotavirus BSC-1

Coltivirus C6/36 cells (Aedes albopictus monolayer cell cultures)

Marburgvirus IHC, rt-PCR (Virus isolation requires highest level biohazard equipment)

Influenza A virus Chick embryo HA, MDCK cells

Bovine spongiforme encephalitis RIII and C57Bl mice

Scrapie Mice, hamsters

* For identification, specific antibody or batteries of specific antibody are used to neutralize and thereby identify the virus effects in the indicator cultures. Other antigen/antibody tests include HAI, ELISA, CF

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When agglutination occurs, a test to identify the agglutinating agent requires incubation with a battery of sera containing specific antibody. The inhibiting antibody, obviously, is identifying the culprit. It is recommended that all test sera be pretreated with the test erythrocytes to remove non-specific agglutinins. This process also provides for the determination of the amount of virus in the original isolate.

Progressive ten-fold dilutions of the isolate sample are injected, and the highest dilution still infecting 50 percent of the inoculated eggs or cell cultures provide a measure of the amount of virus present (expressed in ID50). Enrichment steps would not be necessary in virus or vaccine producing facilities dealing with the production of large quantities of the virus. Here the measure of hemagglutination would be part of the production control process.

The product sample is diluted out by adding a given amount of sample to the same amount of diluent, mixing and passing on the same amount of the half dilution to the next container and so forth, then adding a red blood cell suspension and finding the highest dilution that still will agglutinate those cells. This same test can be performed in tubes and microtiter plates.

If a virus has been detected in cell cultures, it usually has produced some kind of effect to make it noticeable. It may be in the form of focal cell lysis, or the cells may pile up or change their appearance in other ways, producing vacuoles inside the cells (foamy virus).

In the plaque reduction neutralization test (PRNT), specific antibody will block this cytopathic effect and by this very action identify the virus producing the CPE.

Primary cell cultures are usually more susceptible to virus infection and will provide cytopathic effects at higher dilutions, that is, with fewer infectious units of the virus than continuous cell lines. However, their availability is more limited, they have to be produced fresh every time, and they are subject to the variability of the source, the technique employed to produce them and the possibility of contamination, viral or otherwise, from the donor animal.

Continuous cell lines, by contrast, are highly uniform and reproducible. They come out of the freezer and can be used anytime.

The appearance of cytopathic effect (CPE) is usually fairly characteristic for a given type of virus. The definitive confirmation of virus identity is achieved by determining which of a battery of sera containing specific antibody will neutralize its cytopathic effect in the cell culture.

Another enrichment procedure, the mouse antibody production test, involves laboratory animals. It can be used to find unknown suspected infectious agents in established cell culture lines and biopsy samples. The sample is inoculated twice into sero-negative mice, serum is collected three weeks later and examined for antibody stimulated by the viral contaminant. One example is Lymphocytice choriomeningitis

Direct fluorescent assay (dFA)Step Material Process

1 Infected matter Fix on microscopic slide or flat-bottomed microtiter plate.

2 Diluent containing BSA Rinse repeatedly; BSA to block uncovered plastic area.

3 Pathogen-specific AB fluorescent dye labelled

Add to infected matter and incubate for required time and temperature.

4 Test diluent Rinse repeatedly: remove unattached antibody.

5 UV microscope Read; along with positive and negative control samples.

Indirect fluorescent assay (iFA)Step Material Process

1 Infected matter Fix on microscopic slide or flat-bottomed microtiter plate.

2 Diluent containing BSA Rinse repeatedly; BSA to block uncovered plastic area.

3 Pathogen-specific antibody from given animal-species

Add to infected matter, incubate for required time and temperature.

4 Test diluent Rinse repeatedly: remove unattached antibody.

5 AB to given animal-species labeled with fluorescent dye

Add and incubate for required time and temperature.

6 Test diluent Rinse: remove unattached fluorescein labeled AB.

7 UV microscope Read, along with positive and negative control samples.

Enzyme-linked immuno-sorbent assay (ELISA)Step Material Process

1 Capture AB for AG sought

Coat flat-bottomed polystyrene microtiter plate with capture AB.

2 Incubator Allow attachment to surface (several hours, overnight).

3 Diluent with BSA Rinse, remove unattached AB; BSA blocks uncovered plastic areas.

4 AG (?) sample matter Add to AB coated surface, incubate as instructed to allow attachment.

5 Diluent Rinse repeatedly to remove unattached sample matter.

6 Enzyme labeled AB to AG Add, allow to react according to instructions.

7 Diluents Rinse repeatedly to remove unattached enzyme labeled AB.

8 Chromogenic substrate Add, incubate for limited time, stop enzyme action, read signals, optical density, in spectrophotometer.

Sandwich enzyme-linked immuno-sorbent assay (sELISA)Step Material Process

1 Capture AB for AG that is sought Coat flat-bottomed polystyrene microtiter plate with capture AB

2 Incubator Allow attachment to surface (several hours, overnight)

3 Diluent with BSA Rinse, remove unattached AB; BSA blocks uncovered plastic areas

4 AG (?) sample matter Add to AB-coated surface, incubate as instructed

5 Diluent Rinse repeatedly to remove unattached sample matter

6 AB from given animal-species Add, allow to react according to instructions

7 Diluents Rinse repeatedly to remove unattached AB

8 Enzyme-labeled AB specific to given animal species AB

Add, allow to react according to instructions to measure specific AB retained by pathogen; none retained if negative

9 Diluents Rinse repeatedly to remove unattached enzyme-labeled AB

10 Chromogenic substrate Incubate for limited time, stop enzyme action, read signals, optical density, in spectrophotometer

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virus, often a contaminant in mouse-derived materials and the cause of human infection.

However, these are being pushed aside by the more and more widely recognized polymerase chain reaction test (PCR) which not only isolates but also amplifies, and by the very same means, genetically identifies the pathogen in less time and more specifically than the standard techniques here described.

While the above assay systems base pathogen identification on morphology and antigenic coat components of the pathogenic agent, the PCR test looks for matching DNA sections. The polymerase chain reaction is based on the duplication of a selected short section of DNA and re-duplication from its product exponentially to billions of identical copies within a very short period of time.

This selective amplification makes it a very efficient and highly sensitive procedure for the identification of minute traces of DNA. It is used for cloning and gene sequencing, analysis of gene function, diagnosis of hereditary diseases, genetic fingerprinting and paternity, the identification of DNA traces left at crime scenes, a hair, a drop of blood and, of course, of traces of pathogenic matter found in the course of an infectious disease.

It is found in stool, urine, blood and other body fluids, infected tissue, lymph nodes, skin ulcerations et alia.

The test itself requires a starter DNA sequence (target: template containing the target region); deoxynucleoside triphosphates (dNTPs: DNA building blocks); a pair of primers, complementary to the target DNA; taq DNA polymerase (temperature optimum of about 70 degrees C); buffer solution, including the divalent cation Mg² and monovalent cation potassium.

As the primer is finding and connecting with its complementary segment on the target DNA, the polymerase will commence synthesizing that section of DNA with the dNTPs provided. Messenger RNA (mRNA) can be processed as well by using reverse transcriptase to convert mRNA into complementary DNA (cDNA) and then proceeding as with the DNA PCR.

In the past few years, the PCR has become a standard in many laboratories and equipment, and most essential reagents have become commercially available.

Real-time PCR is an improvement as it permits real-time observation of the process of polymerization. All that is required is a real-time machine tied into a computer with real-time software able to read nucleic acid stains. Cybre green is a nucleic acid stain that binds to the double-stranded DNA product of polymerization and produces a bright green. It does less so or not at all to single-stranded DNA.

Isolation and identification of the pathogenic agent are crucial for early treatment and prognosis of the disease it caused. However, the development of an effective host immune response and its persistence in the long term must be verified to measure effectiveness of such treatment and protection from reinfection.

Antibody assayHemagglutination inhibition test (HI): While antibody in the hemagglutination process described above is used to identify a

Hemagglutination inhibition testStep Material Process

1 Isolate passage product Add to test tubes, diluted or undiluted

2 Known antibody Add to sample tube shake and incubate

3 Indicator RBC (0.4 percent) Add to test tube, shake and incubate

4 White background Read agglutination

* Make sure to include negative control AB and negative control sample in test! 0.01 M phosphate-buffered saline is one of the recommended diluents for HA

Polymerase chain reaction (PCR)Process step Materials Procedures

Sampling (1) Target or template DNA in stool, CSF, infected tissue, their exudates, urine

Is found by specific primers, identifying key for a specific gene section of a specific pathogen, seeking out and attaching only to the matching DNA region for assembly and polymerisation.

Initialization (2) Opposing primers (3’, 5’)(3) Taq DNA polymerase(4) dNTP’s and(5) buffer(6) Divalent cations (Mg2+)(7) Monovalent cations (K)(8) Thermal cycler

Target/template DNA, dNTP’s (in excess), primers (excess), Taq DNA polymerase, buffer + Mg² K; DNA thermal cycler; combine reagents in test tube (0.2 to 0.5 ml size) 10μl-200μl working volume; place in thermal cycler, heat to 94 degrees C to 96 degrees C for 5 minutes (1-9 min).

Denaturation Step 1 94 degrees C to 98 degrees C for 20 to 30 seconds: melt complementary hydrogen bonds between double strands of DNA, separate into two single-stranded complementary molecules.

Annealing Step 2 50 degrees C to 65 degrees C for 20 to 40 seconds. Anneale single-stranded primer to complementary single-stranded DNA target; temperature/time is critical to limit non-specific annealing (background noise). Polymerase cum primer-template hybrid will commence DNA synthesis.

Elongation Step 3 72 degrees C DNA polymerase copies new DNA strand from template; will polymerize 1,000 bases per minute.

Repeat 1-2-3 Recycle steps 1, 2, and 3 30 to 40 times, with each time a reduplication of product: 230-40 .

Final elongation

70 degrees C to 74 degrees C for 5 to 15 minutes after last cycle to allow full extension of single-stranded DNA molecules.

Hold till use 4 degrees C to 15 degrees C.

Agarose gel electrophoresis

Confirm end result: Separate PCR products by size; read using ethidium bromide or cybre green stain.

dNTP’s: Deoxynucleoside triphosphates (DNA building blocks)Opposing primers: must match specific section of single-stranded denatured DNA (double-stranded DNA separated by heat denaturation)Limiting factors: dNTP’s and primers available for use

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hemagglutinating virus, the HI test here is used to identify and measure the amount of specific antibody as expression of past experience with an infecting agent, vaccine efficacy in the field, and endemic or epidemic background studies.

In this case, two-fold dilutions of the test sera are combined with a minimal effective hemagglutinating dose, say two to four HA units of the virus or viral antigen and the highest dilution inhibiting hemagglutination would represent the HI antibody titer.

The indirect hemagglutination test, also called the passive hemagglutination test, consists of tanned or formalinized erythrocytes coated with an antigen and then agglutinated by the presence of antibody to that antigen.

In the latex agglutination test, erythrocytes are replaced by latex particles as carriers of the test antigen.

The equipment used is more a matter of convenience, efficiency and economics, use of microscopic slide, test tubes, microtiter plates produce similar results.

The complement fixation test can identify and measure antigen or antibody. The presence of both in a sample will bind and use up a defined amount of complement in the test and thus compete with the indicator system (sheep red blood cells and anti-sheep red blood cell antibody) necessary to visualize the presence of complement.

If either antigen or antibody is absent from the test tube, the complement is available to activate and visualize sheep red blood cell lysis. The test is fairly complex and requires rigorous control over all components of the system. It is being or has been already replaced by the more efficient and quicker fluorescent and enzyme linked immune assay systems.

The above-mentioned immunoassays applied to the identification of pathogens can just as easily be modified to permit antibody assays and to measure antibody response. Most labeled antigens and antibody are commercially available.

Although the old standby of hemagglutination inhibition and complement fixation tests are still being used occasionally, the much more sophisticated techniques of enzyme and

fluorescein-linked antibody assays have become the gold standard.

There are many ways of performing the enzyme-linked immunosorbant assay. The ELISA can be used to identify and measure antigen, as mentioned above, or to detect and measure antibody levels induced by acute or recent or long-passed illness to help diagnosis and course of action.

Antibody determination is required in the field-testing of vaccines and the establishment of endemic or epidemic baselines for infectious diseases in probable target populations.

There are many ways by which the ELISA for antibody is performed in different laboratories, but they all are based on the same basic premise and require the same basic four components:

■ The antigen to which one wants to find the antibody.

■ The test serum (or other body fluids, such as CSF, ascites, organ extracts) in question, to be tested for the presence of antibody to that antigen (vaccine virus, disease producing agent), and:

□ A negative control. □ A positive control of known titer.

■ The indicator antibody, produced in a mammalian species other than the test serum source, against IgA, IgM, IgG of serum from the test serum source species. This indicator antibody is conjugated with an enzyme such as horseradish peroxidase or alkaline phosphatase.

■ A clear uncolored chromagen substrate that will be converted to color by the action of the enzyme tagged onto the indicator antibody and can be read by the eye or by means of the optical density in a colorimetric instrument.

These materials are then employed in different ways: indirect ELISA, sandwich ELISA, competitive ELISA, MAC-ELISA, EIM-ELISA. In the ELISA screen, 96 samples, including the known negative and known positive controls, provide a yes or no answer to the question of antibody presence.

This is essentially the same test as the one above, except that it is seeking out antibody molecules instead of antigen.

In the sandwich ELISA, already described above, the antigen is sandwiched between a capture antibody on the bottom of the well and the test sample antibody, which if positive, will be retained by the antigen and its presence measured by the enzyme-labeled indicator antibody. Evidently, the antigen capture antibody and the test antibody must not come from the same animal species or the enzyme labeled indicator antibody would react to both.

The same test can also be used to measure the amount of antibody present, by diluting test samples and determining the highest dilution still retaining the indicator antibody.

The competitive ELISA test consists of a measured amount of known antibody combined with the test antigen sample, allowed to interact and then added to the capture AG on the bottom of the plate. The more antibody tied up by the

Indirect enzyme-linked immuno-sorbent assay (indirect ELISA)Step Material Process

1 Specific antigen Coat bottoms of polystyrene microtiter plate with AG.

2 Incubator Allow attachment to surface (several hours, overnight).

3 Diluent with BSA Rinse, remove unattached AG; BSA blocks uncovered plastic.

4 Test AB sample Add to AG coated surface, incubate as instructed.

5 Diluent Rinse repeatedly to remove unattached test AB sample.

6 Enzyme labeled AB to test AB Add, allow to react according to instructions.

7 Diluents Rinse repeatedly to remove unattached enzyme labeled AB.

8 Chromogenic substrate Add, incubate for limited time, stop enzyme action, read signals, optical density, in spectrophotometer.

* Make sure to include positive and negative control samples with each plate (Step 4)Enzyme labeled Ab, produced in an animal species other than the species from which the test AB sample came. Usually, enzyme labeled AB is directed against IgA, IgM, and IgG from serum of the species being tested

Competitive enzyme-linked immuno-sorbent assay (competitive ELISA)Step Material Process

1 Specific antigen Coat bottoms of polystyrene microtiter plate with AG.

2 Incubator Allow attachment to surface (several hours, overnight).

3 Diluent with BSA Rinse, remove unattached AG; BSA to block uncovered plastic.

4 Known AB/sample AG mix Add to AG coated surface, incubate as instructed.

5 Diluent Rinse, remove unattached AB/AG mix, the more AG in the mix the more AB is tied up and will not attach to the capture AG.

6 Enzyme labeled AB to test AB Add, allow to react according to instructions.

7 Diluents Rinse repeatedly to remove unattached enzyme labeled AB.

8 Chromogenic substrate Add, incubate for limited time, stop enzyme action, read signals, optical density, in spectrophotometer.

* Make sure to include positive and negative control samples with each plate (Step 4).

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test antigen, the less will be available for the capture antigen.

The advantage of this test is that even in a raw and not very clean sample, antigen, if present, can selectively attach to the antibody and tie it up in a measurable way. The MAC-ELISA (M-antibody-capture-ELISA) is based on the use of anti-IgM antibody at the bottom of the microtiter plate. Because IgM is one of the early antibody types

to be found in the acute disease (along with IgA), it is of diagnostic value. Functioning like the enzyme-linked antibody, the enzyme tag can be replaced by a fluorescein cyanate tag and visualized by UV microscopy.

The fluorescent covalent microsphere immuno assay (FCMIA) is a variation and further development of the same theme. Microbeads, identifiable and countable through flow

cytometry, can substitute for the microtiter well bottom and carry a variety, up to 100, of different analytes through the fluorescent assay system. The microspheres are then passed through a flow cytometer to be identified and counted.

A far simpler test than the ones mentioned above is reported to be the AuBioDOT assay, based on the same principle as the ELISA IgM capture system (MAC-ELISA). Looking for the presence

Indirect enzyme-linked immuno-sorbent assay (MAC-ELISA)Step Material Process

1 IgM AB (e.g. Goat anti-human) Coat bottoms of polystyrene microtiter plate with anti-IgM AB.

2 Incubator Allow attachment to surface (several hours, overnight)

3 Diluent with BSA Rinse, remove unattached AB; BSA blocks uncovered plastic.

4 Test serum sample Add to anti-IgM AB coated surface, incubate as instructed.

5 Diluent Rinse repeatedly to remove unattached test AB sample.

6 Virus or Antigen Add, incubate overnight.

7 Diluent Rinse repeatedly to remove unattached test AB sample.

8 Enzyme labeled AB to Test IgG Add, allow to react according to instructions.

9 Diluents Rinse repeatedly to remove unattached enzyme labeled AB.

10 Chromogenic substrate Add, incubate for limited time, stop enzyme action, read signals, optical density, in spectrophotometer.

* Make sure to include positive and negative control samples with each plate (Step 4).

Enzyme labeled Ab, produced in an animal species other than the species from which the test AB sample came.

Indirect enzyme linked immuno-sorbent assay (EIM-ELISA)Step Material Process

1 IgM AB (e.g. goat antihuman) Coat bottoms of polystyrene microtiter plate with anti-IgG AB.

2 Incubator Allow attachment to surface (several hours, overnight).

3 Diluent with BSA Rinse, remove unattached AB; BSA blocks uncovered plastic.

4 Virus or antigen Add to anti-IgG AB-coated surface, incubate as instructed.

5 Diluent Rinse repeatedly to remove unattached test virus or antigen.

6 Test serum samples Two-fold dilution steps, add to plate, incubate as instructed.

7 Diluent Rinse repeatedly to remove unattached test AB sample dilution.

8 Anti virus/antigen IgG Add limited dose to determine inhibition by serum dilution.

9 Diluents Rinse repeatedly to remove unattached IgG.

10 Enzyme labeled AB to test IgG Add, allow to react according to instructions.

11 Diluents Rinse repeatedly to remove unattached enzyme labeled AB.

12 Chromogenic substrate Add, incubate for limited time, stop enzyme action, read signals, optical density, in spectrophotometer.

* Make sure to include positive and negative control samples with each plate (Step 4)Enzyme-labeled Ab, produced in an animal species other than the species from which the test AB sample came.

Indirect Fluorescent Assay (iFA)Step Material Process

1 Specific antigen Attach to well bottoms of microtiter plates.

2 Diluent containing BSA Rinse repeatedly; BSA blocks non-specific attachment to free plastic area.

3 Test AB dilutions Add to wells; incubate for required time and temperature.

4 Diluent Rinse repeatedly: remove unattached antibody.

5 Fluorescein-conjugated AB to test AB donor animal

Fluorescein-conjugated AB will attach to test AB retained by Ag in bottom of well; incubate for required time and temperature.

6 Diluent Rinse repeatedly: remove unattached antibody.

7 UV microscope Read; along with positive and negative control samples.

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Fluorescent covalent microsphere immuno assay (FCMIA)Step Material Process

1 Carboxylated microspheres One of a possible 100, washed and prepared according to instructions.

2 Antigen (virus) Add, incubate 2 hrs at rt degrees.

3 PBS + 0.05 percent Tween20 Wash twice.

4 PBS + 1 percent BSA Store at 4 degrees C.

5 Hemocytometer To determine microsphere concentration: 100 spheres/μl.

6 PBS + 5 percent dried skim milk, 0.05 percent sodium azide [pH 7.4]

Blocking buffer to minimize non-specific binding of carrier spheres; incubate microsphere usage suspension (100 spheres/μl) for 1 hr at 37 degrees C.

7 1.2μm millipore filter microtiter plate 50 μl aliquots (5,000 beads) placed in each well and washed 3 times with 200 μl of wash buffer; buffer removal via filter by vacuum manifold.

8 Test serum 50 μl in blocking buffer + 1 percent tween20

Add to well, incubate for 30 minutes at 37 degrees C, shaking.

9 Wash buffer Wash 3 times 200 μl of wash buffer; buffer removal via filter by vacuum.

10 Detection antibody 50 μl antihuman IgG-red algae-phycoerythrin, incubate for 30 minutes at 37 degrees C, shaking.

11 Wash buffer Wash 3 times 200 μl of wash buffer, buffer removal via filter by vacuum and resuspend in 100 μl of wash buffer, shake vigorously.

12 Luminex 100 flow analyzer Analize as recommended by supplier.

* Make sure to include positive and negative control samples with each plate (Step 8)

AuBioDOTStep Material Process

1 White opaque polyethylene strips* Coat with goat IgG against human IgM, 3 hrs at 37 degrees C.

2 Test serum samples, controls Add, incubate 15 min at rt degrees.

3 PBS-0.05 percent Tween 20 Rinse 3 times (after three distilled water rinses).

4 Virus or antigen Add measured effective amount, incubate 15 min at rt degrees.

5 PBS-0.05 percent Tween 20 Rinse 3 times (after three distilled water rinses).

6 Colloidal gold conjugated AB Add 1 OD (540 nm), incubate 15 min at rt degrees.

7 PBS-0.05 percent Tween 20 Rinse 3 times (after three distilled water rinses).

8 Silver enhancer solution Read after 10 minutes.

* Greiner, Frickenhausen, Germany* * Make sure to include positive and negative control samples with each plate (Step 2)

Western blotStep Material Process

1 Virus or antigen May require lysis of cells with RIPA buffer, NP-40, Triton X-100.

2 SDS-PAGE Sort by size/molecular weight.

3 Copper stain (0.3 M CuCl2) To visualize fractions and control SDS-PAGE process.

4 0.1- 0.25 M tris/0.25 M EDTA pH 8.0 To destain; add 1 time tris-glycine buffer + methanol 20 percent for transfer.

5 SDS-PAGE fractions Apply electrical field to transfer onto nitrocellulose blotting paper.

6 Ponceau red (0.2 percent in TBST) Visualize fractions and control effective transfer, destain with TBST.

7 The blot: Blocking Rinse with TBST plus nonfat milk protein or BSA to block sticky surface.

8 Specific primar antibody in TBST Add, incubate overnight at 4 degrees C (may keep blocking agent in TBST or not).

9 TBST Rinse repeatedly 5 minutes or more each time to remove unattached AB.

10 Enzyme-linked secondary antibody Add, incubate, shake at rt degrees for 1-2 hours (no blocking agent in TBST).

11 TBST Rinse to remove unattached secondary antibody.

12 Add luminal substrate, ECL, ECL+ Read enhanced chemiluminescence, photograph (CCD camera), scan.

TBST: Tris buffered saline plus 0.1 percent Tween20.Step 10: Horseradish peroxidase-conjugated secondary antibody directed at primary antigen-specific antibody.ECL: Enhanced chemiluminescence.

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of IgM in a human serum nonhuman IgG (goat or otherwise) active against human IgM is affixed to a carrier surface to detect the IgM of an early or acute infection.

Western blotting, also called protein immune blot, is a procedure that allows the detection and identification of proteins, viruses and antigens by the antibody to which they will bind. Antibody directed at most known pathogens and their antigens as well as at any kind of antibody are commercially available and allow today even the smallest laboratory to run sophisticated diagnostic tests.

In short, sample material is separated according to molecule size by means of SDS-PAGE, (sodium dodecyl sulfate polyacrylamide gel electrophoresis); the separation products are transferred to nitrocellulose blotting paper (the blot); and identified by its specific antibody.References and recommended reading1. Abed, Y., and G. Boivin. 2008. New Saffold cardiovirus in

three children in Canada. Emerg. Infect. Dis. 14:834-8362. Acharya R, Fry E, Stuart D, Fox G, Rowlands D, et al.

(1989) The three-dimensional structure of foot-and-mouth disease virus at 2.9 Å resolution. Nature (London) 337: 709–716

3. Adami C, Pritchard AE, Knauf T, Luo M, Lipton HL. A determinant for central nervous system persistence localized in the capsid of Theiler’s murine encephalomyelitis virus by using recombinant viruses. J Virol. 1998;72:1662–5

4. Aguilar PV, Robich RM, Turell MJ, O’Guinn ML, Klein TA, Huaman A, et al. Endemic eastern equine encephalitis in the Amazon region of Peru. Am J Trop Med Hyg 2007;76(2):293-8

5. Alexander DJ (2007) An overview of the epidemiology of avian influenza. Vaccine 25: 5637–5644

6. Alper T, Cramp WA, Haig DA, Clarke MC (May 1967). Does the agent of scrapie replicate without nucleic acid?. Nature 214 (5090): 764–6

7. Al-Sunaidi M, Williams CH, Hughes PJ, Schnurr DP, Stanway G. Analysis of a new human parechovirus allows the definition of parechovirus types and the identification of RNA structural domains. J Virol. 2007;81:1013–21

8. Al-Tikriti SK, Al-Ani F, Jurji FJ, Tantawi H, Al-Moslih M, Al-Janabi N, Mahmud MI, Al-Bana A, Habib H, Al-Munthri H, Al-Janabi S, AL-Jawahry K, Yonan M, Hassan F, Simpson DI. Congo/Crimean haemorrhagic fever in Iraq. Bull World Health Organ. 1981;59(1):85-90.

9. Aubert, C., and M. Brahic. 1995. Early infection of the central nervous system by the GDVII and DA strains of Theiler’s virus. J. Virol. 69:3197-3200.

10. Balkhy HH, Schreiber JR. Severe La Crosse encephalitis with significant neurologic sequelae. Pediatr Infect Dis J 2000;19(1):77-80.

11. Bamborough P, Wille H, Telling GC, Yehiely F, Prusiner SB, Cohen FE (1996). Prion protein structure and scrapie replication: theoretical, spectroscopic, and genetic investigations. Cold Spring Harbor Symposium on Quantitative Biology 61: 495–509

12. Bangari, D. S., Pogranichniy, R. M., Gillespie, T., Stevenson, G. W. (2010). Genotyping of Porcine teschovirus from nervous tissue of pigs with and without polioencephalomyelitis in Indiana. jvdi 22: 594-597

13. Barquet N, Domingo P (15 October 1997). Smallpox: the triumph over the most terrible of the ministers of death. Ann. Intern. Med. 127 (8 Pt 1): 635–42

14. Barton LL, Budd SC, Morfitt WS, Peters CJ, Ksiazek TG, Schindler RF, et al. Congenital lymphocytic choriomeningitis virus infection in twins. Pediatr Infect Dis Journal 1993; 12: 942-6.

15. Barragán F, Retana OG, Naranjo EJ. The rodent trade of Tzeltzal Indians of Oxchuc, Chiapas, Mexico. Hum Ecol. 2007;36:769–73.

16. Barrett AD, Higgs S (2007). Yellow fever: a disease that has yet to be conquered. Annu. Rev. Entomol. 52: 209–29.

17. Barton LL, Mets MB, Beauchamp CL. Lymphocytic choriomeningitis virus: emerging fetal teratogen. Am J Obstet Gynecol 2002;187: 1715—6

18. Barton LL, Budd SC, Morfitt WS, Peters CJ, Ksiazek

TG, Schindler RF, et al. Congenital lymphocytic choriomeningitis virus infection in twins. Pediatr Infect Dis J 1993; 12:942-6.

19. Bauer, K. Foot and mouth disease as zoonosis. Arch. Virol. Supp. 1997; 13:95-97.

20. Baumgarte S, de Souza Luna LK, Grywna K, Panning M, Drexler JF, Karsten C, et al. Prevalence, types, and RNA concentrations of human parechoviruses, including a sixth parechovirus type, in stool samples from patients with acute enteritis. J Clin Microbiol. 2008;46:242–8

21. Beekes M, Baldauf E, Diringer H (1996). Sequential appearance and accumulation of pathognomonic markers in the central nervous system of hamsters orally infected with scrapie. Journal of General Virology 77: 1925–1934

22. Bell JC, Stojdl D; Lichty; Knowles; Marius; Atkins; Sonenberg Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nature Medicine 2000; 6 (7): 782–789

23. Belnap DM, Filman DJ, Trus BL, Cheng N, Booy FP, et al. (2000) Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus. J Virol 74: 1342–1354

24. Benenson, A.S. (Ed.). Control of Communicable Diseases Manual.1995. American Public Health Assoc.; Washington, D.C.

25. Bennett SG, Milazzo ML, Webb JP Jr, Fulhorst CF. Arenavirus antibody in rodents indigenous to coastal southern California. Am J Trop Med Hyg. 2000;62:626–30.

26. Berge T, editor. International catalogue of arboviruses and certain other viruses of the world (supplement). Tamiami (TAM), strain W-10777. Am J Trop Med Hyg 1970;19:1157-8.

27. Bishop RF, Davidson GP, Holmes IH, Ruck BJ (1973). Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet 2 (7841): 1281–3

28. Bolton DC, Rudelli RD, Currie JR, Bendheim PE (1991). Copurification of sp33-37 and scrapie agent from hamster brain prior to detectable histopathology and clinical-disease. Journal of General Virology 72: 2905–2913

29. Borson, N. D., C. Paul, X. Lin, W. K. Nevala, M. A. Strausbauch, M. Rodriguez, and P. J. Wettstein. 1997. Brain-infiltrating cytolytic T lymphocytes specific for Theiler’s virus recognize H2Db molecules complexed with a viral VP2 peptide lacking a consensus anchor residue. J. Virol. 71:5244-5250

30. Bowen MD, Peters CJ, Nichol ST. The phylogeny of New World (Tacaribe complex) arenaviruses. Virology 1996;219:285-90.

31. Bowen MD, Rollin PE, Ksiazek TG, et al. (August 2000). Genetic diversity among Lassa virus strains. J. Virol. 74 (15): 6992–7004

32. Brabec M, Baraville G, Blaas D, Fuchs R (2003) Conformational changes, plasma membrane penetration and infection by Human Rhinovirus Type 2: Role of receptors and low pH. J Virol 77: 5370–5377

33. Brahic, M., J. F. Bureau, and T. Michiels. 2005. The genetics of the persistent infection and demyelinating disease caused by Theiler’s virus. Annu. Rev. Microbiol. 59:279-298.

34. Brault AC, Powers AM, Chavez CL, Lopez RN, Cachon MF, Gutierrez LF, et al. Genetic and antigenic diversity among eastern equine encephalitis viruses from North, Central, and South America. Am J Trop Med Hyg 1999;61(4):579-86.

35. Buxton D, Reid HW. Experimental infection of red grouse with louping-ill virus (flavivirus group). II. Neuropathology. J Comp Pathol. 1975;85(2):231-5.

36. Cajimat MNB, Milazzo ML, Hess BD, Rood MP, Fulhorst CF. Principal host relationships and evolutionary history of the North American arenaviruses. Virology. 2007;367:235–43.

37. Cajimat MNB, Milazzo ML, Borchert JN, Abbott KD, Bradley RD, Fulhorst CF. Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the Mexican woodrat (Neotoma mexicana). Virus Res. 2008;133:211–7.

38. Calisher CH, Tzianabos T, Lord RD, Coleman PH. Tamiami virus, a new member of the Tacaribe group. Am J Trop Med Hyg 1970;19:520-6.

39. Callan RJ, Van Metre DC. Viral diseases of the ruminant nervous system. Vet Clin North Am Food Anim Pract. 2004;20(2):327-62.

40. Carlson CS, O’Sullivan MG, Jayo MJ, Anderson DK, Harber ES, Jerome WG, et al. Fatal disseminated cercopithecine herpesvirus 1 (herpes B infection in cynomolgus monkeys (Macaca fascicularis). Vet Pathol

1997;34:405–14 41. Casals J. Antigenic similarity between the virus causing

Crimean hemorrhagic fever and Congo virus. Proc Soc Exp Biol Med. 1969 May;131(1):233-6.

42. Chamorro, M., C. Aubert, and M. Brahic. 1986. Demyelinating lesions due to Theiler’s virus are associated with ongoing central nervous system infection. J. Virol. 57:992-997

43. Chard, L. S., Kaku, Y., Jones, B., Nayak, A., Belsham, G. J. (2006). Functional Analyses of RNA Structures Shared between the Internal Ribosome Entry Sites of Hepatitis C Virus and the Picornavirus Porcine Teschovirus 1 Talfan. J. Virol. 80: 1271-1279

44. Charrel RN, Attoui H, Butenko AM, Clegg JC, Deubel V, Frolova TV, Gould EA, Gritsun TS, Heinz FX, Labuda M, Lashkevich VA, Loktev V, Lundkvist A, Lvov DV, Mandl CW, Niedrig M, Papa A, Petrov VS, Plyusnin A, Randolph S, Süss J, Zlobin VI, de Lamballerie X. Tick-borne virus diseases of human interest in Europe. Clin Microbiol Infect. 2004;10(12):1040-55.

45. CDC. Surveillance for human West Nile virus disease---United States, 1999--2008. Surveillance Summaries, April 2, 2010.MMWR 2010;59(No. SS-2)

46. CDC. Hantavirus pulmonary syndrome---United States: updated recommendations for risk reduction. MMWR 2002;51(No. RR-9).

47. CDC. Lymphocytic choriomeningitis virus infection in organ transplant recipients---Massachusetts, Rhode Island, 2005. MMWR 2005;54:537--9.

48. Centers for Disease Control and Prevention (CDC) (January 2009). Possible congenital infection with La Crosse encephalitis virus--West Virginia, 2006-2007. MMWR Morb. Mortal. Wkly. Rep. 58 (1): 4–7

49. Centers for Disease Control and Prevention. Fatal illnesses associated with a New World arenavirus-California, 1999-2000. MMWR Morb Mortal Wkly Rep 2000;49:709-11.

50. Centers for Disease Control and Prevention (CDC) (2007). Household transmission of vaccinia virus from contact with a military smallpox vaccinee—Illinois and Indiana, 2007. Morbidity and Mortality Weekly Report 56 (19): 478–81

51. Centers for Disease Control and Prevention Fatal Cercopithecine herpesvirus 1 (B virus) infection following a mucocutaneous exposure and interim recommendations for worker protection. MMWR Morb Mortal Wkly Rep 1998;47:1073–6, 1083

52. Chang PC, Hsieh ML, Shien JH, Graham DA, Lee MS, et al. (2001) Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks. J Gen Virol 82: 2157–2168

53. Chen, H. H., W. P. Kong, L. Zhang, P. L. Ward, and R. P. Roos. 1995. A picornaviral protein synthesized out of frame with the polyprotein plays a key role in a virus-induced immune-mediated demyelinating disease. Nat. Med. 1:927-931

54. Childs JE, Peters CJ. Ecology and epidemiology of arenaviruses and their hosts. In: Salvato MS, editor. The Arenaviridae. New York: Plenum Press; 1993. p. 331-84.

55. Childs JE, Glass GE, Ksiazek TG, et al. Human-rodent contact and infection with lymphocytic choriomeningitis and Seoul viruses in an inner-city population. Am J Trop Med Hyg 1991;44:117--21.

56. Chua KB, Goh KJ, Wong KT, et al. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 1999; 3541257-9

57. Cohen JI, Davenport DS, Stewart JA, Deitchman S, Hilliard JK, Chapman LE, et al.Recommendations for prevention of and therapy for exposure to B virus (Cercopithecine herpesvirus-1). Clin Infect Dis 2002;35:1191–203

58. Colchester AC, Colchester NT (2005). The origin of bovine spongiform encephalopathy: the human prion disease hypothesis. Lancet 366 (9488): 856–61

59. Colchester, A; Colchester, N (28 January 2006). Origin of bovine spongiform encephalopathy — Authors’ reply. The Lancet (Elsevier) 367 (9507): 298–299

60. Collinge J, Whitfield J, McKintosh E (June 2006). Kuru in the 21st century--an acquired human prion disease with very long incubation periods. Lancet 367 (9528): 2068–74

61. Cranwell MP, Josephson M, Willoughby K, Marriott L. Louping ill in an alpaca. Vet Rec. 2008;162(1):28.

62. Cros, J; Palese P (September 2003). Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses. Virus Res 95 (1–2): 3–12

63. Crotty S, Cameron C, Andino R (2002). Ribavirin’s antiviral mechanism of action: lethal mutagenesis?. J. Mol.

Page 32 Elite

Med. 80 (2): 86–9564. Crowcroft NS, Morgan D, Brown D. Viral haemorrhagic

fevers in Europe--effective control requires a co-ordinated response. Euro Surveill. 2002 Mar;7(3):31-2

65. Cuevas JM, Sanjuan R, Moya A, Elena SF (2005) Mode of selection and experimental evolution of antiviral drugs resistance in vesicular stomatitis virus. Infect Genet Evol 5: 55–65

66. Dacheux L, Reynes J-M, Buchy P, et al. (2008). A reliable diagnosis of human rabies based on analysis of skin biopsy specimens. Clin Infect Dis 47 (11): 1410–17

67. Daddario-DiCaprio KM, Geisbert TW, Ströher U, et al. (2006). Postexposure protection against Marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment. Lancet 367 (9520): 1399–404

68. Daniels, J. B., A. M. Pappenheimer, and S. Richardson. 1952. Observations on encephalomyelitis of mice (DA strain). J. Exp. Med. 96:517-530

69. Das SC, Nayak D, Zhou Y, Pattnaik AK (2006) Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells. J Virol 80: 6368–6377

70. Davies MV, Chang HW, Jacobs BL, Kaufman RJ (1 March 1993). The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms. J. Virol. 67 (3): 1688–92

71. Davis, B. S., G-J. J. Chang, B. Cropp, J. T. Roehrig, D. Martin, C. J. Mitchell, R. Bowen, and M. L. Bunning. 2001. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays. J. Virol. 75:4040-4047

72. Day JF, Stark LM. Transmission patterns of St. Louis encephalitis and eastern equine encephalitis viruses in Florida: 1978-1993. J Med Entomol 1996;33(1):132-9

73. Deibel R, Woodall JP, Decher WJ, Schryver GD. Lymphocytic choriomeningitis virus in man: serologic evidence of association with pet hamsters. JAMA 1975;232:501--4.

74. Delhaye, S., V. van Pesch, and T. Michiels. 2004. The leader protein of Theiler’s virus interferes with nucleocytoplasmic trafficking of cellular proteins. J. Virol. 78:4357-4362

75. de los Reyes EC, McJunkin JE, Glauser TA, Tomsho M, O’Neal J. Periodic lateralized epileptiform discharges in La Crosse encephalitis, a worrisome subgroup: clinical presentation, electroencephalogram (EEG) patterns, and long-term neurologic outcome. J Child Neurol 2008;23:167-72

76. Dennehy PH (2000). Transmission of rotavirus and other enteric pathogens in the home. Pediatr. Infect. Dis. J. 19 (10 Suppl): S103–5.

77. Deresiewicz RL, Thaler SJ, Hsu L, Zamani AA. Clinical and neuroradiographic manifestations of eastern equine encephalitis. N Engl J Med 1997;336(26):1867-74.

78. Descoteaux, J. P., and S. Mihok. 1986. Serologic study on the prevalence of murine viruses in a population of wild meadow voles (Microtus pennsylvanicus). J. Wildl. Dis. 22:314-319

79. de Serres G, Skowronski DM, Mimault P, et al. (2009). Bats in the bedroom, bats in the belfry: Reanalysis of the rationale for rabies postexposure prophylaxis. Clin Infect Dis 48 (11): 1493–1499.

80. Despond O, Tucci M, Decaluwe H, Grégoire MC, S Teitelbaum J, Turgeon N (March 2002). Rabies in a nine-year-old child: The myth of the bite. Can J Infect Dis 13 (2): 121–5

81. Dietl CA, Wernle JA, Pett SB, et al. Extracorporeal membrane oxygenation support improves survival of patients with severe hantavirus cardiopulmonary syndrome. J Thorac Cardiovasc Surg 2008;135:579--84.

82. Doherty PC, Reid HW. Experimental louping-ill in sheep and lambs. II. Neuropathology. J Comp Pathol. 1971;81(3):331-7.

83. Dvorak, C. M., D. J. Hall, M. Riddle, A. Pranter, J. Dillman, M. Deibel, and A. C. Palmenberg. 2001. Leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation. Virology 290:261-271

84. Ergönül Ö, Celikbas A, Dokuzoguz B, et al. (2004). The chacteristics of Crimean-Congo hemorrhagic fever in a recent outbreak in Turkey and the impact of oral ribavirin therapy. Clin Infect Dis 39: 285–9

85. Erwin PC, Jones TF, Gerhardt RR, Halford SK, Smith

AB, Patterson LE, et al. La Crosse encephalitis in Eastern Tennessee: clinical, environmental, and entomological characteristics from a blinded cohort study. Am J Epidemiol 2002;155:1060-5.

86. Feltz, E. T., B. Mandel, and E. Racker. 1953. Susceptibility of mice free of Theiler’s virus (TO strain) to infection with TO virus. J. Exp. Med. 98:427-432.

87. Finke S, Conzelmann KK (August 2005). Replication strategies of rabies virus. Virus Res. 111 (2): 120–31

88. Fischer TK, Viboud C, Parashar U, et al. (2007). Hospitalizations and deaths from diarrhea and rotavirus among children <5 years of age in the United States, 1993–2003. J. Infect. Dis. 195 (8): 1117–25

89. Fisher-Hoch SP, McCormick JB (2004). Lassa fever vaccine. Expert review of vaccines 3 (2): 189–97

90. Fontenille, D; Diallo, M; Mondo, M; Ndiaye, M; Thonnon, J (1997). First evidence of natural vertical transmission of yellow fever virus in Aedes aegypti, its epidemic vector Transactions of the Royal Society of Tropical Medicine and Hygiene 91 (5): 533–5

91. Fouchier R, Schneeberger P, Rozendaal F, Broekman J, Kemink S, Munster V, Kuiken T, Rimmelzwaan G, Schutten M, Van Doornum G, Koch G, Bosman A, Koopmans M, Osterhaus A (2004). Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome.Proc Natl Acad Sci USA 101 (5): 1356–61

92. Frame JD, Baldwin JM, Gocke DJ, Troup JM (1 July 1970). Lassa fever, a new virus disease of man from West Africa. I. Clinical description and pathological findings. Am. J. Trop. Med. Hyg. 19 (4): 670–6

93. Fry EE, Newman JW, Curry S (2005) Structure of Foot-and-mouth disease virus serotype A10 61alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation. J Gen Virol 86: 1909–1920

94. Fuentes-Marins, R., A. R. Rodriguez, S. S. Kalter, A. Hellman, and R. A. Crandell. 1963. Isolation of enteroviruses from the normal baboon (Papio doguera). J. Bacteriol. 85:1045-1050

95. Fulhorst CF, Bowen MD, Ksiazek TG, Rollin PE, Nichol ST, Kosoy MY, et al. Isolation and characterization of Whitewater Arroyo virus, a novel North American arenavirus. Virology 1996;224:114-20.

96. Fulhorst CF, Bennett SG, Milazzo ML, Murray HL Jr, Webb JP Jr, Cajimat MNB, et al. Bear Canyon virus: an arenavirus naturally associated with Peromyscus californicus (California mouse). Emerg Infect Dis. 2002;8:717–21.

97. Fulhorst CF, Charrel RN, Weaver SC, Ksiazek TG, Bradley RD, Milazzo ML, et al. Geographical range and genetic diversity of Whitewater Arroyo virus (family Arenaviridae) in the southwestern United States. Emerg Infect Dis. 2001;7:403–7.

98. Fulhorst CF, Milazzo ML, Bradley RD, Peppers LL. Experimental infection of Neotoma albigula (Muridae) with Whitewater Arroyo virus (Arenaviridae). Am J Trop Med Hyg. 2001;65:147–51.

99. Gentsch, J. R., R. I. Glass, P. Woods, V. Gouvea, M. Gorziglia, J. Flores, B. K. Das, and M. K. Bhan. 1992. Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30:1365-1373

100. Gerety, S. J., W. J. Karpus, A. R. Cubbon, R. G. Goswami, M. K. Rundell, J. D. Peterson, and S. D. Miller. 1994. Class II-restricted T cell responses in Theiler’s murine encephalomyelitis virus-induced demyelinating disease. V. Mapping of a dominant immunopathologic VP2 T cell epitope in susceptible SJL/J mice. J. Immunol. 152:908-918

101. Gerhard, W., A. Taylor, Z. Wroblewska, M. Sandberg-Wollheim, and H. Koprowski. 1981. Analysis of a predominant immunoglobulin population in the cerebrospinal fluid of a multiple sclerosis patient by means of an anti-idiotypic hybridoma antibody. Proc. Natl. Acad. Sci. USA 78:3225-3229

102. Ghedin E, Sengamalay N, Shumway M, Zaborsky J, Feldblyum T, Subbu V, Spiro D, Sitz J, Koo H, Bolotov P, Dernovoy D, Tatusova T, Bao Y, St George K, Taylor J, Lipman D, Fraser C, Taubenberger J, Salzberg S (Oct 20 2005). Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution.Nature 437 (7062): 1162–6

103. Gilbert L, Jones LD, Laurenson MK, Gould EA, Reid HW, Hudson PJ. Ticks need not bite their red grouse hosts to infect them with louping ill virus. Proc Biol Sci. 2004;271 Suppl 4:S202-5.

104. Goldfarb, L. G., and D. C. Gajdusek. 1992. Viliuisk encephalomyelitis in the Iakut people of Siberia. Brain 115:961-978

105. Gonzalez JP, Georges AJ, Kiley MP, Meunier DM, Peters CJ, McCormick JB. Evolutionary biology of a Lassa virus complex. Med Microbiol Immunol 1986;175:157-9

106. Gould EA, de Lamballerie X, Zanotto PM, Holmes EC (2003). Origins, evolution, and vector/host coadaptations within the genus Flavivirus. Advances in Virus Research 59: 277–314

107. Gould EA, Solomon T. Pathogenic flaviviruses. Lancet. 2008;371:500-9

108. Grard G, Moureau G, Charrel RN, Lemasson JJ, Gonzalez JP, Gallian P, Gritsun TS, Holmes EC, Gould EA, de Lamballerie X. Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy. Virology. 2007;361(1):80-92.

109. Gregg MB. Recent outbreaks of lymphocytic choriomeningitis in the United States of America. Bull World Health Organ 1975;52:549--53

110. Greve GM, Forte CP, Marlor CW, Meyer AM, Hoover-Litty H, et al. (1991) Mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of ICAM-1. J Virol 65: 6015–6023

111. Griffith JS (September 1967). Self-replication and scrapie. Nature 215 (5105): 1043–4

112. Hachiya M, Osborne M, Stinson C, Werner BG. Human eastern equine encephalitis in Massachusetts: predictive indicators from mosquitoes collected at 10 long-term trap sites, 1979-2004. Am J Trop Med Hyg 2007;76(2):285-92.

113. Hallin GW, Simpson SQ, Crowell RE, et al. Cardiopulmonary manifestations of hantavirus pulmonary syndrome. Crit Care Med 1996;24:252--8.

114. Hardin SG, Erwin PC, Patterson L, New D, Graber C, Halford SK. Clinical comparisons of La Crosse encephalitis and enteroviral central nervous system infections in a pediatric population: 2001 surveillance in East Tennessee. Am J Infect Control 2003;31:508-10.

115. Hartley CA, Ficorilli N, Dynon K, Drummer HE, Huang JA, et al. (2001) Equine rhinitis A virus: structural proteins and immune response. J Gen Virol 82: 1725–1728

116. Hayes EB, Komar N, Nasci RS, Montgomery SP, O’Leary DR, Campbell GL. Epidemiology and transmission dynamics of West Nile virus disease. Emerg Infect Dis 2005;11:1167—73

117. Heberling, R. L., and F. S. Cheever. 1965. Some characteristics of the simian enteroviruses. Am. J. Epidemiol. 81:106-123

118. Hertzler S, Luo M, Lipton HL (2000) Mutation of predicted virion pit residues alters binding of Theiler’s murine encephalomyelitis virus to BHK-21 cells. J Virol 74: 1994–2004

119. Hevey, M.; Negley, D.; Pushko, P.; Smith, J.; Schmaljohn, A. (Nov 1998). Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates. Virology 251 (1): 28–37

120. Hewat EA, Neumann E, Blaas D (2002) The concerted conformational changes during Human Rhinovirus 2 uncoating. Molecular Cell 10: 317–326.

121. Hoehne M, Schreier E. Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3´-minor groove binder-DNA probe. BMC Infect Dis. 2006;6:69

122. Hoffert, W. R., M. E. Bates, and F. S. Cheever. 1958. Study of enteric viruses of simian origin. Am. J. Hyg. 68:15-30

123. Huff J, Barry P (2003). B-virus (Cercopithecine herpesvirus 1) infection in humans and macaques: potential for zoonotic disease. Emerg Infect Dis 9 (2): 246–50.

124. Huggins JW (1989). Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug. Rev Infect Dis 11 Suppl 4: S750–61

125. Huhn, G. D., J. J. Sejvar, S. P. Montgomery, and M. S. Dworkin. 2003. West Nile virus in the United States: an update on an emerging infectious disease. Am. Fam. Physician 68:653-660

126. Hull, R. N. 1968. The simian viruses. Virol. Monogr. 2:1-66

127. Hull, R. N., J. R. Minner, and C. C. Mascoli. 1958. New viral agents recovered from tissue cultures of monkey kidney cells. III. Recovery of additional agents both from cultures of monkey tissues and directly from tissues and excreta. Am. J. Hyg. 68:31-44

128. Huygelen C (1996). Jenner’s cowpox vaccine in light of current vaccinology (in Dutch; Flemish). Verh. K. Acad. Geneeskd. Belg. 58 (5): 479–536

129. Hyde J, Nettleton P, Marriott L, Willoughby K. Louping ill in horses. Vet Rec. 2007;160(15):532.

130. International Committee on Taxonomy of Viruses

Elite Page 33

Universal Virus Database [ICTVdB] Management. 00.026.0.01. Flavivirus. In: Büchen-Osmond C, editor. ICTVdB - The universal virus database, version 4 [online]. New York: Columbia University; 2006. Available at: http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB. Accessed 17 Aug 2009.

131. Jahrling PB, Peters CJ. Lymphocytic choriomeningitis virus. A neglected pathogen of man [editorial]. Arch Pathol Lab Med 1992;116:486-8.

132. Jendroska K, Heinzel FP, Torchia M, Stowring L, Kretzschmar HA, Kon A, Stern A, Prusiner SB, DeArmond SJ (1991). Proteinase-resistant prion protein accumulation in syrian-hamster brain correlates with regional pathology and scrapie infectivity. Neurology41 (9): 1482–1490

133. Jennings WL, Lewis AL, Sather GE, Pierce LV, Bond JO. Tamiami virus in the Tampa Bay area. Am J Trop Med Hyg 1970;19:527-36.

134. Jimenez-Clavero, M. A., Fernandez, C., Ortiz, J. A., Pro, J., Carbonell, G., Tarazona, J. V., Roblas, N., Ley, V. (2003). Teschoviruses as Indicators of Porcine Fecal Contamination of Surface Water. Appl. Environ. Microbiol. 69: 6311-6315

135. Johnson KM, Webb PA, Justines G. Biology of Tacaribe-complex viruses. In: Lehman-Grube F, editor. Lymphocytic choriomeningitis virus and other arenaviruses. Berlin: Springer-Verlag; 1973. p. 241-58.

136. Johnson ED, Johnson BK, Silverstein D, Tukei P, Geisbert TW, Sanchez AN, et al. Characterization of a new Marburg virus isolated from a 1987 fatal case in Kenya. Arch Virol Suppl. 1996;11:101–14

137. Jones SM, Feldmann H, Stroher U et al. (2005). Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nature Med 11 (7): 786–90

138. Jones LD, Gaunt M, Hails RS, Laurenson K, Hudson PJ, Reid H, Henbest P, Gould EA. Transmission of louping ill virus between infected and uninfected ticks co-feeding on mountain hares. Med Vet Entomol. 1997;11(2):172-6.

139. Jones MS, Lukashov VV, Ganac RD, Schnurr DP. Discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin. J Clin Microbiol. 2007;45:2144–50

140. Jones-Engel, L., G. Engel, M. A. Schillaci, A. Rompis, A. Putra, K. G. Suaryana, A. Fuentes, B. Beer, S. Hicks, R. White, B. Wilson, and J. S. Allan. 2005. Primate-to-human retroviral transmission in Asia. Emerg. Infect. Dis. 11:1028-1035

141. Jones TF, Craig AS, Nasci RS, Patterson LE, Erwin PC, Gerhardt RR, et al. Newly recognized focus of La Crosse encephalitis in Tennessee. Clin Infect Dis 1999;28:93-7.

142. Jones M, Peden AH, Prowse CV (September 2007). In vitro amplification and detection of variant Creutzfeldt–Jakob disease PrPSc. J. Pathol. 213 (1): 21–6

143. Jordan Lite (2008-10-08). Medical Mystery: Only One Person Has Survived Rabies without Vaccine--But How?. Scientific American. pp. 4. Retrieved 2010-01-30

144. Kaku, Y., Chard, L. S., Inoue, T., Belsham, G. J. (2002). Unique Characteristics of a Picornavirus Internal Ribosome Entry Site from the Porcine Teschovirus-1 Talfan. J. Virol. 76: 11721-11728

145. Kallio-Kokko H, Uzcategui N, Vapalahti O, Vaheri A. Viral zoonoses in Europe. FEMS Microbiol Rev. 2005;29(5):1051-77.

146. Kalter, S. S. 1982. Enteric viruses of nonhuman primates. Vet. Pathol. 19:33-43

147. Kalter, S. S., R. L. Heberling, A. W. Cooke, J. D. Barry, P. Y. Tian, and W. J. Northam. 1997. Viral infections of nonhuman primates. Lab. Anim. Sci. 47:461-467

148. Kanda, N (2001). (Book Review) The Eradication of Smallpox: Edward Jenner and The First and Only Eradication of a Human Infectious Disease. Nature Medicine 7 (1): 15–6

149. Khan AS, Khabbaz RF, Armstrong LR, et al. Hantavirus pulmonary syndrome: the first 100 US cases. J Infect Dis 1996;173:1297--303.

150. Kim S, Boege U, Krishnaswamy S, Minor I, Smith TJ, et al. (1990) Conformational variability of a picornavirus capsid: pH-dependent structural changes of Mengo virus related to its host receptor attachment site and disassembly. Virology 175: 176–190

151. Kirkland PD, Gleeson AB, Hawkes RA, Naim HM, Boughton CR. Human infection with encephalomyocarditis virus in New South Wales. Med J Aust. 1989;151:176, 178

152. Knowles NJ, Dickinson ND, Wilsden G, Carra E, Brocchi E, De Simone F. Molecular analysis of encephalomyocarditis viruses isolated from pigs and rodents in Italy. Virus Res. 1998;57:53–62

153. Kolakofsky D, Pelet T, Garcin D, Hausmann S, Curran J, et al. (1998) Paramyxovirus RNA synthesis and the requirement for hexamer genome length: the rule of six revisited. J Virol 72: 891–899

154. Komar N. West Nile virus: epidemiology and ecology in North America. Adv Virus Res 2003;61:185—234

155. Komar N, Dohm DJ, Turell MJ, Spielman A. Eastern equine encephalitis virus in birds: relative competence of European starlings (Sturnus vulgaris). Am J Trop Med Hyg 1999;60(3):387-91

156. Kosoy MY, Elliot LH, Ksiazek TG, Fulhorst CF, Rollin PE, Childs JE, et al. Prevalence of antibodies to arenaviruses in rodents from the southern and western United States: evidence for an arenavirus associated with the genus Neotoma. Am J Trop Med Hyg 1996;54:570-5.

157. Koster F, Foucar K, Hjelle B, et al. Rapid presumptive diagnosis of hantavirus cardiopulmonary syndrome by peripheral blood smear review. Am J Clin Pathol 2001;116:665--72.

158. Krishnamurthy S, Samal SK (1998) Nucleotide sequences of the trailer, nucleocapsid protein gene and intergenic regions of Newcastle disease virus strain Beaudette C and completion of the entire genome sequence. J Gen Virol 79 ( Pt 10): 2419–2424

159. Lambert AJ, Nasci RS, Cropp BC, Martin DA, Rose BC, Russell BJ, Lanciotti RS. Nucleic acid amplification assays for detection of La Crosse virus RNA. J Clin Microbiol 2005;43:1885-9.

160. LaRue R, Myers S, Brewer L, Shaw DP, Brown C, Seal BS, et al. A wild-type porcine encephalomyocarditis virus containing a short poly(C) tract is pathogenic to mice, pigs, and cynomolgus macaques. J Virol. 2003;77:9136–46

161. Laskowsiki RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst, 26: 283–291.

162. Lee KE, Umapathi T, Tan CB, et al. The neurological manifestations of Nipah virus encephalitis, a novel paramyxovirus. Ann Neurol 1999; 46428-32

163. Letchworth, G.J. et al. Vesicular stomatitis. Vet. J. (BR) 1999; 157: 239-260.

164. Letson GW, Bailey RE, Pearson J, Tsai TF. Eastern equine encephalitis (EEE): a description of the 1989 outbreak, recent epidemiologic trends, and the association of rainfall with EEE occurrence. Am J Trop Med Hyg 1993;49(6):677-85.

165. Li F, Browning GF, Studdert MJ, Crabb BS (1996) Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses. Proc Natl Acad Sci USA 93: 990–995

166. Linhares AC, Gabbay YB, Mascarenhas JD, Freitas RB, Flewett TH, Beards GM (1988). Epidemiology of rotavirus subgroups and serotypes in Belem, Brazil: a three-year study. Ann. Inst. Pasteur Virol. 139 (1): 89–99

167. Linthicum KJ, Bailey CL, Davies FG, Tucker CJ. Detection of Rift Valley fever activity in Kenya by satellite remote sensing imagery. Science 1987;235:1656-9.

168. Lipton HL, Friedmann A, Sethi P, Crowther JR. Characterization of Vilyuisk virus as a picornavirus. J Med Virol. 1983;12:195–203

169. Logan C, O’Leary JJ, O’Sullivan N. Real-time reverse transcription-PCR for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children. J Clin Microbiol. 2006;44:3189–95

170. Logan D, Abu-Ghazaleh R, Blakemore W, Curry S, Jackson T, et al. (1993) Structure of a Major Immunogenic Site on Foot-and-Mouth Disease Virus. Nature (London) 362: 566–568.

171. Macaldowie C, Patterson IA, Nettleton PF, Low H, Buxton D. Louping ill in llamas (Lama glama) in the Hebrides.Vet Rec. 2005;156(13):420-1.

172. Malherbe, H., and R. Harwin. 1963. The cytopathic effects of vervet monkey viruses. S. Africa Med. J. 37:407-411

173. Manuelidis L, Yu ZX, Barquero N, Banquero N, Mullins B (February 2007). Cells infected with scrapie and Creutzfeldt-Jakob disease agents produce intracellular 25-nm virus-like particles. Proceedings of the National Academy of Sciences of the United States of America 104 (6): 1965–70

174. Marriott L, Willoughby K, Chianini F, Dagleish MP, Scholes S, Robinson AC, Gould EA, Nettleton PF. Detection of louping ill virus in clinical specimens from mammals and birds using TaqMan RT-PCR. J Virol Methods. 2006;137(1):21-8.

175. McDonnell G, Burke P (May 2003). The challenge of prion decontamination. Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of

America36 (9): 1152–4176. McJunkin JE, de los Reyes EC, Irazuzta JE, et al. (March

2001). La Crosse encephalitis in children. N. Engl. J. Med. 344 (11): 801–7

177. Meegan JM. Rift Valley fever epizootic in Egypt: description of the epizootic and virological studies. Trans R Soc Trop Med Hyg 1979;73:618-23.

178. Mertz GJ, Hjelle B, Crowley M, Iwamoto G, Tomicic V, Vial PA. Diagnosis and treatment of new world hantavirus infections. Curr Opin Infect Dis 2006;19:437--42.

179. Mertz GJ, Miedzinski L, Goade D. Placebo-controlled, double-blind trial of intravenous ribavirin for the treatment of hantavirus cardiopulmonary syndrome in North America. Clin Infect Dis 2004;39:1307--13.

180. Milazzo ML, Cajimat MNB, Haynie ML, Abbott KD, Bradley RD, Fulhorst CF. Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the white-throated woodrat (Neotoma albigula) in the southwestern United States. Vector Borne Zoonotic Dis. 2008;8:523–40.

181. Monath TP (April 2008). Treatment of yellow fever. Antiviral Res. 78 (1): 116–24

182. Moritz ML, Ayus JC. La Crosse encephalitis in children. N Engl J Med 2001;345:148-9

183. Moseley MH, Marriott L, Nettleton P, Dukes J, Irvine RJ, Mougeot F. Use of real-time RT-PCR to determine the prevalence of louping ill virus in live red grouse chicks. Vet Rec. 2007;161(19):660-1.

184. Mostashari F, Bunning ML, Kitsutani PT, et al. Epidemic West Nile encephalitis, New York, 1999: results of a household-based seroepidemiological survey. Lancet 2001;358:261--4.

185. Muir P, Kammerer U, Korn K, Mulders MN, Poyry T, Weissbrich B, et al. Molecular typing of enteroviruses: current status and future requirements. The European Union Concerted Action on Virus Meningitis and Encephalitis. Clin Microbiol Rev. 1998;11:202–27

186. Murray, K, P Selleck, P Hooper, et al. A morbillivirus that caused fatal disease in horses and humans. Science 1995, 268:94-7

187. Nakaya T, Cros J, Park MS, Nakaya Y, Zheng H, et al. (2001) Recombinant Newcastle disease virus as a vaccine vector. J Virol 75: 11868–11873

188. Nasci RS, Moore CG, Biggerstaff BJ, Panella NA, Liu HQ, Karabatsos N, et al. La Crosse encephalitis virus habitat associations in Nicholas County, West Virginia. J Med Entomol 2000;37:559-70.

189. Nelson CB, Pomeroy BS, Schrall K, Park WE, Lindeman RJ. (1952). An outbreak of conjunctivitis due to Newcastle disease virus (NDV) occurring in poultry workers. Am J Public Health 42(6):672–8.

190. Niimi Y, Iwasaki Y, Umemura T (December 2008). MM2-cortical-type sporadic Creutzfeldt–Jakob disease with early stage cerebral cortical pathology presenting with a rapidly progressive clinical course. Neuropathology 28 (6): 645–51

191. Nix, W. A., M. S. Oberste, and M. A. Pallansch. 2006. Sensitive, semi-nested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J. Clin. Microbiol. 44:2698-2704

192. Nobusawa E, Sato K (April 2006). Comparison of the mutation rates of human influenza A and B viruses. J Virol 80 (7): 3675–8

193. Oberste, M. S., K. Maher, and M. A. Pallansch. 2007. Complete genome sequences for nine simian enteroviruses. J. Gen. Virol.88:3360-3372

194. Oberste, M. S., and M. A. Pallansch. 2005. Enterovirus molecular detection and typing. Rev. Med. Microbiol. 16:163-171.

195. Ogbu O, Ajuluchukwu E, Uneke CJ (2007). Lassa fever in West African sub-region: an overview. Journal of vector borne diseases 44 (1): 1–11

196. Ohsawa K, Black DH, Sato H, Eberle R Sequence and genetic arrangement of the u(s) region of the monkey B virus (cercopithecine herpesvirus 1) genome and comparison with the u(s) regions of other primate herpesviruses. J Virol 2002;76:1516–20

197. Okorie TG. Comparative studies on the vector capacity of the different stages of Amblyomma variegatum Fabricius and Hyalomma rufipes Koch for Congo virus, after intracoelomic inoculation. Vet Parasitol. 1991 Mar;38(2-3):215-23

198. Oldstone, Michael B. A. (2000). Viruses, Plagues, and History (1st ed.). Oxford University Press

199. Özduman K, Wollman G; Piepmeier; van den Pol (2008). Systemic Vesicular Stomatitis Virus Selectively Destroys

Page 34 Elite

Multifocal Glioma and Metastatic Carcinoma in Brain. The Journal of Neuroscience 28 (8): 1882–1893.

200. Pan KM, Baldwin M, Nguyen J, et al. (December 1993).Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proceedings of the National Academy of Sciences of the United States of America 90 (23): 10962–6

201. Park MS, Steel J, Garcia-Sastre A, Swayne D, Palese P (2006) Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease. Proc Natl Acad Sci U S A 103: 8203–8208

202. Paton NI, Leo YS, Zaki SR, et al. Outbreak of Nipah-virus infection among abattoir workers in Singapore. Lancet 1999; 3541253-6

203. Patton JT, Vasquez-Del Carpio R, Spencer E (2004). Replication and transcription of the rotavirus genome.Curr. Pharm. Des. 10 (30): 3769–77

204. Peters CJ, Buchmeier M, Rollin PE, Ksiazek TG. Arenaviruses. In: Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, et al., editors. Fields virology. 3rd ed. Philadelphia: Lippincott-Raven Publishers; 1996. p. 1521-51.

205. Peters CJ. Marburg and Ebola - Arming ourselves against the deadly filoviruses. New England Journal of Medicine. 2005;352:2571

206. Pfeifer A, Eigenbrod S, Al-Khadra S (December 2006). Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice. The Journal of Clinical Investigation 116 (12): 3204–10

207. Pisarev, A. V., Chard, L. S., Kaku, Y., Johns, H. L., Shatsky, I. N., Belsham, G. J. (2004). Functional and Structural Similarities between the Internal Ribosome Entry Sites of Hepatitis C Virus and Porcine Teschovirus, a Picornavirus.J. Virol. 78: 4487-4497

208. Porter, F. W., Y. A. Bochkov, A. J. Albee, C. Wiese, and A. C. Palmenberg. 2006. A picornavirus protein interacts with Ran-GTPase and disrupts nucleocytoplasmic transport. Proc. Natl. Acad. Sci. USA 103:12417-12422

209. Quaresma JA, Barros VL, Pagliari C, Fernandes ER, Guedes F, Takakura CF, Andrade HF Jr, Vasconcelos PF, Duarte MI (2006). Revisiting the liver in human yellow fever: virus-induced apoptosis in hepatocytes associated with TGF-beta, TNF-alpha and NK cells activity. Virology 345 (1): 22–30

210. Ramasamy I, M Law, S Collins, F Brook (April 2003). Organ distribution of prion proteins in variant Creutzfeldt-Jakob disease. The Lancet Infectious Diseases 3 (4): 214–222

211. Ramos MM, Overturf GD, Crowley MR, Rosenberg RB, Hjelle B. Infection with Sin Nombre hantavirus: clinical presentation and outcome in children and adolescents. Pediatrics 2001;108:e27.

212. Reece JF, Chawla SK. (2006). Control of rabies in Jaipur, India, by the sterilization and vaccination of neighbourhood dogs.. Vet Rec 159 (12): 379–83

213. Reid HW. Experimental infection of red grouse with louping-ill virus (flavivirus group). I. The viraemia and antibody response. J Comp Pathol. 1975;85(2):223-9.

214. Reid HW, Doherty PC. Experimental louping-ill in sheep and lambs. I. Viraemia and the antibody response. J Comp Pathol. 1971;81(2):291-8.

215. Reid HW, Duncan JS, Phillips JD, Moss R, Watson A. Studies of louping-ill virus (Flavivirus group) in wild red grouse (Lagopus lagopus scoticus).J Hyg (Lond). 1978;81(2):321-9.

216. Reid HW, Moss R, Pow I, Buxton D. The response of three grouse species (Tetrao urogallus, Lagopus mutus, Lagopus lagopus) to louping-ill virus. J Comp Pathol. 1980;90(2):257-63.

217. Reimann CA, Hayes EB, DiGuiseppi C, et al. Epidemiology of neuroinvasive arboviral disease in the United States, 1999--2007. Am J Trop Med Hyg 2008;79:974—9

218. Rheingans RD, Heylen J, Giaquinto C (2006). Economics of rotavirus gastroenteritis and vaccination in Europe: what makes sense?. Pediatr. Infect. Dis. J. 25 (1 Suppl): S48–55

219. Riffel N, Harlos K, Iourin O, Rao Z, Kingsman A, et al. (2002) Atomic resolution structure of Moloney Murine Leukemia Virus matrix protein and its relationship to other retroviral matrix proteins. Structure 10: 1627–1636.

220. Robinson HL, Hunt LA, Webster RG (1993) Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 11: 957–960

221. Rodriguez M, Roos RP. Pathogenesis of early and late disease in mice infected with Theiler’s virus, using intratypic recombinant GDVII/DA viruses. J

Virol.1992;66:217–25.222. Rozengurt N, Sanchez S. A spontaneous outbreak

of Theiler’s encephalomyelitis in a colony of severe combined immunodeficient mice in the UK. Lab Anim. 1993;27:229–34

223. Salvato MS, Clegg JCS, Buchmeier MJ, Charrel RN, Gonzalez J-P, Lukashevich IS, et al. Family Arenaviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus taxonomy: eighth report of the International Committee on Taxonomy of Viruses. Amsterdam: Elsevier Academic Press; 2005:725–33.

224. Sampath A, Padmanabhan R (January 2009). Molecular targets for flavivirus drug discovery. Antiviral Research 81 (1): 6–15.

225. Sbrana E, Xiao SY, Guzman H, Ye M, Travassos da Rosa AP, Tesh RB (2004). Efficacy of post-exposure treatment of yellow fever with ribavirin in a hamster model of the disease. Am J Trop Med Hyg 71 (3): 306–12

226. Sinclair R, Boone SA, Greenberg D, Keim P, Gerba CP. Persistence of category A select agents in the environment. Appl Environ Microbiol. 2008;74(3):555-63.

227. Simpson DI, Knight EM, Courtois G, Williams MC, Weinbren MP, Kibukamusoke JW.Congo virus: a hitherto undescribed virus occurring in Africa. I. Human isolations--clinical notes.East Afr Med J. 1967 Feb;44(2):86-92

228. Smith DH, Johnson BK, Isaacson M, Swanepoel R, Johnson KM, Killey M, et al. Marburg-virus disease in Kenya. Lancet. 1982;1:816–20

229. Smith GL, Vanderplasschen A, Law M (1 December 2002). The formation and function of extracellular enveloped Vaccinia virus. J. Gen. Virol. 83 (Pt 12): 2915–31.

230. Smyth M, Pettitt T, Symonds A and Martin J (2003) Identification of the Pocket Factors in a Picornavirus.Archives Virol 148: 1225-33

231. Smyth, M.S. and Martin, JHJ (2002) Picornavirus Uncoating. J Clin Path; Mol Path 55: 214-219

232. Smyth MS, Symonds A, Brazinova S and Martin JHJ (2002) Bovine Enterovirus as an Oncolytic Virus: Foetal Calf Serum Facilitates its infection of Human Cells. Int. J. Mol. Med. 10, 49-53

233. Smyth, M.S. & Martin, J.H.J. (2001)Structural, biochemical and electrostatic basis of serotype specificity in bovine enteroviruses. Archives Virol. 146, 347-355

234. Stallknecht D.E. et al. Potential for contact and mechanical vector transmission of vesicular stomatitis virus New Jersey in pigs. A.J.V.R.1999; 60:43-48.

235. Stanway G, Joki-Korpela P, Hyypia T. Human parechoviruses—biology and clinical significance. Rev Med Virol. 2000;10:57–69

236. St Jeor S. Three-week incubation period for hantavirus infection. Pediatr Infect Dis J 2004;23:974--5.

237. Steinhardt E, Israeli C, Lambert RA (September 1913). Studies on the cultivation of the virus of vaccinia. J. Inf Dis 13: 294–300

238. Stoner GD, Williams B, Kniazeff A, Shimkin MB. Effect of neuraminidase pretreatment on the susceptibility of normal and transformed mammalian cells to bovine enterovirus 261. Nature. 1973 245:319-20

239. Studdert MJ, Gleeson LJ (1978) Isolation and characterisation of an equine rhinovirus. Zentbl Vetmed B 25: 225–237

240. Subbiah M, Xiao S, Collins PL, Samal SK (2008) Complete sequence of the genome of avian paramyxovirus type 2 (strain Yucaipa) and comparison with other paramyxoviruses. Virus Res 137: 40–48

241. Swanepoel R, Smit SB, Rollin PE, Formenty P, Leman PA, Kemp A, et al. International Scientific and Technical Committee for Marburg Hemorrhagic Fever Control in the Democratic Republic of Congo. Studies of reservoir hosts for Marburg virus. Emerg Infect Dis. 2007;13:1847–51

242. Tardei, G., S. Ruta, V. Chitu, C. Rossi, T. F. Tsai, and C. Cernescu. 2000. Evaluation of immunoglobulin M (IgM) and IgG enzyme immunoassays in serologic diagnosis of West Nile virus infections. J. Clin. Microbiol. 38:2232-2239

243. Tellier R (November 2006). Review of aerosol transmission of influenza A virus. Emerging Infect. Dis. 12 (11): 1657–62

244. Timoney PJ.Susceptibility of the horse to experimental inoculation with louping ill virus. J Comp Pathol. 1980;90(1):73-86.

245. Tognotti E. (June 2010). The eradication of smallpox, a success story for modern medicine and public health: What lessons for the future? (PDF). J Infect Dev Ctries4 (5): 264–266

246. Tolle MA (April 2009). Mosquito-borne diseases. Curr Probl Pediatr Adolesc Health Care 39 (4): 97–140

247. Tolonen N, Doglio L, Schleich S, Krijnse Locker J (1 July 2001). Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei. Mol. Biol. Cell 12 (7): 2031–46.

248. Toltzis P, Huang AS (1986) Effect of ribavirin on macromolecular synthesis in vesicular stomatitis virus-infected cells. AntimicrobAgents Chemother 29: 1010–1016

249. Toltzis P, O’Connell K, Patterson JL (1988) Effect of phosphorylated ribavirin on vesicular stomatitis virus transcription. AntimicrobAgents Chemother 32: 492–497

250. Tschampa, Henriette J.; M; F; P; S; U (1 May 2003). Thalamic Involvement in Sporadic Creutzfeldt–Jakob Disease: A Diffusion-Weighted MR Imaging Study. American Journal of Neuroradiology (American Society of Neuroradiology) 24 (5): 908–915

251. Tuthill TJ, Bubeck D, Rowlands DJ, Hogle JM (2006) Characterization of early steps in the poliovirus infection process: receptor-decorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles. J Virol 80: 172–180

252. Valleron AJ, Boelle PY, Will R, Cesbron JY (November 2001). Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom. Science 294(5547): 1726–8

253. Varasso A, Drummer HE, Huang JA, Stevenson RA, Ficorilli N, et al. (2001) Sequence conservation and antigenic variation of the structural proteins of equine rhinitis A virus. J Virol 75: 10550–10556

254. Vázquez, S., O. Váldes, M. Pupo, I. Delgado, M. Alvarez, J. L. Pelegrino, and M. G. Guzmán. 2003. MAC-ELISA and ELISA inhibition methods for detection of antibodies after yellow fever vaccination. J. Virol. Methods 110:179-184

255. Velázquez FR, Matson DO, Calva JJ, Guerrero L, Morrow AL, Carter-Campbell S, Glass RI, Estes MK, Pickering LK, Ruiz-Palacios GM (1996). Rotavirus infections in infants as protection against subsequent infections. N. Engl. J. Med. 335 (14): 1022–8

256. Verstrepen WA, Kuhn S, Kockx MM, Van De Vyvere ME, Mertens AH. Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single-tube real-time reverse transcription-PCR assay. J Clin Microbiol. 2001;39:4093–6

257. Vladimirtsev VA, Nikitina RS, Renwick N, Ivanova AA, Danilova AP, Platonov FA, et al. Family clustering of Viliuisk encephalomyelitis in traditional and new geographic regions. Emerg Infect Dis. 2007;13:1321–6

258. Wang, Y., X. Tu, C. Humphrey, H. McClure, X. Jiang, C. Qin, R. I. Glass, and B. Jiang. 2007. Detection of viral agents in fecal specimens of monkeys with diarrhea. J. Med. Primatol. 36:101-107.

259. Watts DM, Ussery MA, Nash D, Peters CJ. (1989). Inhibition of Crimean-Congo hemorrhagic fever viral infectivity yields in vitro by ribavirin. Am J Trop Med Hyg.41: 581–5.

260. Weaver SC, Salas RA, de Manzione N, Fulhorst CF, Duno G, Utrera A, et al. Guanarito virus (Arenaviridae) isolates from endemic and outlying localities in Venezuela: sequence comparisons among and within strains isolated from Venezuelan hemorrhagic fever patients and rodents. Virology 2000;266:189-95.

261. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56: 152–179

262. Weigler BJ, Hird DW, Hilliard JK, Lerche NW, Roberts JA, Scott LM Epidemiology of cercopithecine herpesvirus 1 (B virus) infection and shedding in a large breeding cohort of rhesus macaques. J Infect Dis 1993;167:257–63

263. Will RG, Ironside JW, Zeidler M (April 1996). A new variant of Creutzfeldt–Jakob disease in the UK. Lancet 347 (9006): 921–5

264. Wong, S. J., V. L. Demarest, R. H. Boyle, T. Wang, M. Ledizet, K. Kar, L. D. Kramer, E. Fikrig, and R. A. Koski. 2004. Detection of human anti-flavivirus antibodies with a West Nile virus recombinant antigen microsphere immunoassay. J. Clin. Microbiol. 42:65-72

265. Woodall JP, Williams MC, Simpson DI. Congo virus: a hitherto undescribed virus occurring in Africa. II. Identification studies. East Afr Med J. 1967 Feb;44(2):93-8.

266. Wutz G, Auer H, Nowotny N, Grosse B, Skern T, et al. (1996) Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses. J Gen Virol 77: 1719–1730

267. Wurtz R, Paleologos N. La Crosse encephalitis presenting like herpes simplex encephalitis in an immunocompromised adult. Clin Infect Dis 2000;31:1113-4

Elite Page 35

268. Xiao S, Paldurai A, Nayak B, Subbiah M, Collins PL, et al. (2009) Complete genome sequence of avian paramyxovirus type 7 (strain Tennessee) and comparison with other paramyxoviruses. Virus Res 145: 80–91

269. Young, Geoffrey S.; G; F; M; H; L; L; W et al. (June–July 2005). Diffusion-Weighted and Fluid-Attenuated Inversion Recovery Imaging in Creutzfeldt–Jakob Disease: High Sensitivity and Specificity for Diagnosis. American Journal of Neuroradiology (American Society of Neuroradiology) 26 (6): 1551–1562

270. Zell, R., Seitz, S., Henke, A., Munder, T., Wutzler, P. (2005). Linkage map of protein-protein interactions of Porcine teschovirus. J. Gen. Virol. 86: 2763-2768

271. Zhang Z, Mitchell DK, Afflerbach C, Jakab F, Walter J, Zhang YJ, et al. Quantitation of human astrovirus by real-time reverse-transcription-polymerase chain reaction to examine correlation with clinical illness. J Virol Methods. 2006;134:190–6.

272. Zoecklein LJ, Pavelko KD, Gamez J, Papke L, McGavern DB, Ure DR, et al. Direct comparison of demyelinating disease induced by the Daniel’s strain and BeAn strain of Theiler’s murine encephalomyelitis virus. Brain Pathol. 2003;13:291–308

INFECTIOUS DISEASES DIAGNOSTICS (B) SUBCELLULAR

PATHOGENSFinal Examination Questions

Choose True or False for questions 1-15 then complete your test online at

www.elitecme.com.

1. Facilities for laboratory workers concerned with early diagnostic processes for emerging human diseases that are zoonoses should have positive internal air pressure.

True False

2. Meningitis caused by lymphocytic choriomeningitis is usually fatal.

True False

3. Birds appear as intermediate hosts for the St. Louis encephalitis virus.

True False

4. Infection with one of the four dengue virus strains will cross-protect again each other.

True False

5. Dengue fever and dengue hemorrhagic fever are a major cause of disease and death in the tropics and subtropics.

True False

6. Hantavirus is transmitted through inhalation of dust from droppings and secretions of infected rodents or through broken skin.

True False

7. People exposed to Rift Valley fever who experience a fever for more than 48 hours should urgently seek medical advice.

True False

8. Mosquito eggs can survive for years in a dry environment, and heavy rainfalls will allow those eggs to hatch and produce an active and highly infectious population of mosquito vectors.

True False

9. Once Rift Valley virus has appeared in a herd, live virus vaccines should be given to bring it to a quick end.

True False

10. Rhabdoviridae infect only mammals.

True False

11. Vesicular stomatitis virus involves two serotypes, Indiana and New Jersey, and both are zoonotic.

True False

12. The primary reservoirs of alphaviruses are horses and man.

True False

13. There are many alphavirus species active in the United States.

True False

14. Man is the only host for rubella virus.

True False

15. Exotic birds infected with Newcastle disease have been shown to shed the virus for over a year.

True False

16. Bovine enteroviruses as a rule do not produce a disease but are found in the feces of cattle in large numbers.

True False

17. Pigs can spread the encephalomyocarditis virus through bodily secretions and excretions.

True False

18. Foot-and-mouth disease can only be passed on by direct contact.

True False

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19. Equine rhinitis A virus infection in a horse cannot be transmitted to man.

True False

20. Frequent stomping of feet (dancing disease), lameness and walking on knees are symptoms of blue tongue disease virus infection.

True False

21. Highly pathogenic avian influenza (HPAI) is highly contagious, involves multiple organs, and has high mortality rates.

True False

22. Transmission of prions may be by ingesting infected meat or via the urine, saliva and other body fluids.

True False

23. Prions can be easily treated and cured.

True False

24. Prions cannot survive in the open environment.

True False

25. Because the vCJD pathogen can lie dormant, it can produce illness in man many years after the consumption of meat from an infected animal.

True False