adenoviruses. adenovirus- classification - dna viruses first isolated from adenoidal tissue in 1953...
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ADENOVIRUS- ClassificationADENOVIRUS- Classification
- DNA viruses first isolated from adenoidal tissue in 1953
- approximately 100 serotypes have been recognized, at least 47 of which infect humans.
•Subdivided into 6 subgroups based on hemagglutination (A-F)•Human pathogens belong to 49 serotypes•Common serotypes:- 1-8, 11, 21, 35, 37, 40•Enteric Adenoviruses belong to subgroup F
- Molecular biology research : splicing……
- Gene therapy : cystic fibrosis………..
Disease Patient Population
Respiratory Diseases
Febrile, undifferentiated upper respiratory tract infection
Infants, young children
Pharyngoconjunctival fever Children, adults
Acute respiratory disease Military recruits (serotype 4, 7)
Pertussis-like syndrome Infants, young children
Pneumonia Infants, young children; military recruits; immunocompromised patients
Other Diseases
Acute hemorrhagic cystitis Children; bone marrow transplant recipients
Epidemic keratoconjunctivitis Any age; renal transplant recipients
Gastroenteritis Infants, young children
Hepatitis Liver transplant recipients; other immunocompromised patients
Meningoencephalitis Children; immunocompromised patients
Table 52-1. Illnesses Associated with Adenoviruses
ADENOVIRUS - StructureADENOVIRUS - Structure
• Non-enveloped DNA virus• Icosadeltahedrons (20 면체 )• 70-90 nm in size • Linear ds DNA genome (36kb) with a
terminal protein (molecular mass, 55 kDa)
- capsid comprises 240 capsomeres, which consist of hexons and pentons. -12 pentons : have a penton base and a fiber. - fiber : viral attachment proteins can act as a hemagglutinin. -penton base and fiber are : toxic to cells, carry type-specific antigens.
BOX 52-1. Unique Features of Adenovirus
•Naked icosadeltahedral capsid has fibers (viral attachment proteins) at vertices. •Linear double-stranded genome has 5' terminal proteins. •Synthesis of viral DNA polymerase activates switch from early to late genes. •Virus encodes proteins to promote messenger RNA and DNA synthesis, including its own DNA polymerase. •Human adenoviruses are grouped A through F by DNA homologies and by serotype (more than 42 types). •Serotype is mainly a result of differences in the penton base and fiber protein, which determine the nature of tissue tropism and disease. •Virus causes lytic, persistent, and latent infections in humans, and some strains can immortalize certain animal cells.
Simplified genome map of adenovirus type 2
-Transcription : both strand- Early transcription – early protein : E1A, E1B, E2A, E2B, E3, E4-Late transcription –late protein- 11 polypeptide : 9 – structural protein (capsid) 2 – core (DNA-binding protein)
Gene Number
M.W (kDa2) Function
E1A* Activates viral gene transcription
Binds cellular growth suppressor: p105RB promotes transformation
Deregulates cell growth
Inhibits activation of interferon response elements
E1B Binds cellular growth suppressor: p53 promotes transformation
Blocks apoptosis
E2 Activates some promoters
Terminal protein on DNA
DNA polymerase
E3 Prevents tumor necrosis factor-α (TFN-α) inflammation
E4 Limits viral cytopathologic effect
VA RNAs Inhibit interferon response
Capsid
II 120 Contains family antigen and some serotyping antigens
III 85 Penton base protein
Toxic to tissue culture cells
IV 62 Fiber
Responsible for attachment and hemagglutination; contains some serotyping antigens
VI 24 Hexon-associated proteins
VIII 13 Penton-associated proteins
IX 12
IIIa 66
Core
V 48 Core protein 1: DNA-binding protein
VII 18 Core protein 2: DNA-binding protein
Entry and replicationEntry and replication• Fiber protein determines target cell specificity and attachment • Viral DNA enters host cell nucleus• Virus replicates in nucleus
- Fiber receptor : Ig superfamily Coxsackie B viruses 도 이용 - Coxsackie adenovirus receptor - MHC I 도 이용- Penton base interact with v integrin - receptor-mediated endocytosis (clathrin-coated vesicles)- Capsid delivers the DNA genome to the nucleus
- Early transcription - Replication - Late gene transcription : capsid proteins in cytoplasm nucleus viral assembly
Pathogenesis and ImmunityPathogenesis and Immunity
• viral fiber proteins determine the target cell specificity. • toxic activity of the penton base protein can result in inhibition of
cellular mRNA transport and protein synthesis, cell rounding, and tissue damage.
-Lytic ( 용해감염 ) : mucoepithelial cells ( 점막상피세포 ) -Latent ( 잠복감염 ) : lymphoid and adenoid cells-Transforming ( 형질전환 ) : hamster, not human
BOX 53-2. Disease Mechanisms of Adenoviruses
•Virus is spread by aerosol, close contact, or fecal-oral means to establish
pharyngeal infection. Fingers spread virus to eyes. •Virus infects mucoepithelial cells in the respiratory tract, gastrointestinal tract,
and conjunctiva or cornea, causing cell damage directly. •Disease is determined by the tissue tropism of the specific group or serotype of the
virus strain. •Virus persists in lymphoid tissue (e.g., tonsils, adenoids, Peyer's patches). •Antibody is important for prophylaxis and resolution.
The histologic hallmark of adenovirus infection is a dense, central intranuclear inclusion within an infected epithelial cell that consists of viral DNA and protein (Figure 52-3).
These inclusions may resemble those seen in cells infected with cytomegalovirus, but adenovirus does not cause cellular enlargement (cytomegaly).
Mononuclear cell infiltrates and epithelial cell necrosis are seen at the site of infection.
ADENOVIRAL INCLUSION BODIESADENOVIRAL INCLUSION BODIES
Histologic appearance of adenovirus-infected cells. Inefficient assembly of virions yields dark basophilic nuclear inclusion bodies containing DNA, proteins, and capsids
• Viremia : immunocompromised
patients • latent and persist in lymphoid and
other tissue, such as adenoids, tonsils, and Peyer's patches
- Antibody : important for resolving lytic adenovirus infections and protects the person from reinfection with the same serotype
- Cell-mediated immunity is important in limiting virus outgrowth, as borne out by the fact that immunosuppressed people suffer more serious and recurrent disease.
- host defenses evasion (1) encode small virus-associated RNAs (VA RNA) : prevent the activation of the
interferon-induced protein kinase R mediated inhibition of viral protein synthesis. (2) viral E3 and E1A proteins block apoptosis induced by cellular responses to the
virus or by T cell or cytokine (e.g., TNF-α) actions. (3) adenoviruses can inhibit CD8(+) cytotoxic T-cell action by preventing proper
expression of MHC I molecules and therefore antigen presentation.
Epidemiology
BOX 53-3. Epidemiology of Adenoviruses
Disease/Viral Factors •Capsid virus is resistant to inactivation by gastrointestinal tract and drying. •Disease symptoms may resemble those of other respiratory virus infections. •Virus may cause asymptomatic shedding.
Transmission •Direct contact via respiratory droplets and fecal matter, on hands, on fomites (e.g.,
towels, contaminated medical instruments), close contact, and inadequately
chlorinated swimming pools.
Who Is at Risk? •Children younger than 14 years of age. •People in crowded areas (e.g., daycare centers, military training camps, swimming
clubs).
Geography/Season •Virus is found worldwide. •There is no seasonal incidence.
Modes of Control •Live vaccine for serotypes 4 and 7 is available for military use.
Adenoviruses primarily infect children and less commonly infect adults. Disease from reactivated virus occurs in immunocompromised children and adults.
BOX 52-4. Clinical Summaries
•Pharyngoconjunctival fever (
인두결막염 ) : A 7-year-old student
develops sudden onset of red eyes, sore
throat, and a fever of 38.9°C (102°F).
Several children in the local elementary
school have similar symptoms. •Gastroenteritis: An infant has diarrhea
and is vomiting. Adenovirus serotype 41
was identified by polymerase chain
reaction analysis of stool for
epidemiologic reasons.
Clinical syndromes
ACUTE FEBRILE PHARYNGITIS ( 급성 열성 인두염 )AND PHARYNGOCONJUNCTIVAL FEVER ( 인두 결막염 )
- pharyngitis, which is often accompanied by conjunctivitis (pinkeye) and pharyngoconjunctival fever.
- young children- mild, flulike symptoms (including nasal congestion, cough, coryza, malaise, fever, chills,
myalgia, and headache) that may last 3 to 5 days. - Pharyngoconjunctival fever occurs more often in outbreaks involving older children.
ACUTE RESPIRATORY DISEASE ( 급성 호흡기 질환 ) -fever, cough, pharyngitis, and cervical adenitis.-adenovirus serotypes 4 and 7. -military recruits stimulated the development and use of a vaccine for these serotypes.
OTHER RESPIRATORY TRACT DISEASES - coldlike symptoms, laryngitis, croup, and bronchiolitis. - pertussis-like illness, viral pneumonia.
CONJUNCTIVITIS AND EPIDEMIC KERATOCONJUNCTIVITIS ( 유행성 결막염 )- follicular conjunctivitis : (Figure 52-6). - Swimming pool conjunctivitis - Epidemic keratoconjunctivitis may be an occupational hazard- for industrial workers.
GASTROENTERITIS AND DIARRHEA - major cause of acute viral gastroenteritis; - Adenovirus serotypes 40 to 42 have been grouped as enteric adenoviruses (group F)
Laboratory Diagnosis - Immunoassays, including fluorescent antibody and enzyme-linked immunosorbent assays- genome assays, PCR to detect, type, -must be used for enteric adenovirus serotypes 40 to 42, which do not grow readily in available cell cultures.
PreventionPrevention• Good handwashing• Contact precautions• Chlorination of water• Disinfection or sterilization of
ophthalmologic equipment• Use of single dose vials• Oral vaccine- restricted use
• Used as VECTORS to transfer desired genetic material into cells• Viral genome is relatively easily manipulated in vitro • Efficient expression of inserted DNA in recipient cell
Gene therapy
Gene Therapy
The transfer of selected genes into a host with the hope of ameliorating or curing a disease state
Human many diseases absence or inappropriate presence of a protein
Isolate and produce these natural proteins genetic engineering and recombinant technology
Protein deliverySustained drug delivery
Gene therapyUltimate method of protein delivery
Body’s cells Small factories
produce a therapeutic protein for a specific disease over a prolonged period
Gene Transfer Technology
Viral Vectors for Gene Transfer Retroviral Vectors HIV MMLV Adenoviral Vector Adeno-Associatedviral Vector Herpes Simplex Viral Vector
Nonviral Techniques Naked DNA Liposome Molecular conjugates
Antisense Technology Non-catalytic antisense Catalytic antisense molecules Ribozymes: hammerhead or hairpin
Retrovirus Vectors Advantages DisadvantagesHigh transduction efficiency Requires dividing cells for infectivityInsert size up to 8kB Low titers(106-107)Integrates into host genome resulting Integration is randomin sustained expression of vectorExtremely well studied system In vivo delivery remains poor.Vector proteins not expressed in host Effective only when infecting helper cell lines
Adenovirus vectors
Advantages DisadvantagesHigh transduction efficiency Expression is transientInsert size up to 8kb (viral DNA does not integrate) Viral proteins can be expressed in High viral titer (1010-1013) host following vector administrationInfects both replicating and In vivo delivery hampered by hostdifferentiated cells immune response
Herpes simplex virus
Advantages DisadvantagesLarge insert size System currently under developmentCould provide long-term CNS Current vectors provide transient expressiongene expressionHigh titer Low transduction efficiency
-Lack of association with disease
-The ability to latently infect a high fraction of
exposed cells
-A minimal number of viral antigens to induce a host
immune response
-The possible ability to latently infect
non-dividing cells
-The possible advantage of site-specific integration
-The ability to latently infect a broad range of
human cell types
AAV : Useful for a vector for gene therapy
Delivery System Requirements A practical gene delivery system must meet five demanding requirements. It must be:
Efficient •Capable of achieving the duration of expression of protein from the gene required by the applicable medical situation •Flexible with respect to the tissues it can deliver to •Able to handle a wide range of therapeutic genes •Able to demonstrate a dose-response relationship
•Efficient delivery of genes to both dividing and non-dividing target cells •Absence of viral genes that may be responsible for causing an undesirable immune response •In vivo administration to patients •High levels of gene expression •Excellent stability allowing AAV vectors to be manufactured, stored and handled like more traditional pharmaceutical products.
AAV Virus Particles
Structure of AAV Virus Genomic DNA
Structure of AAV Vector DNA
AAV Vector Delivery System