Bovine ephemeral fever and rhabdoviruses endemic to Australia

PJ WALKER and DH CYBINSKI CSIRO, Division of Tropical Animal Production, Long Pocket Laboratories

Private Bag No. 3, PO Indooroopilly, Queensland 4068

Introduction Several decades of investigation of arthropod-borne diseases

in Australia have seen the isolation and identification of a large number of endemic viruses which infect man, livestock and native animals. Some are important pathogenic agents which have costly social or economic consequences. Many others, while not presently known to cause disease, can confuse serological diagnosis of related pathogenic infections, or may have the potential for pathogenic effects which are not cur- rently recognised. In this sense, the endemic rhabdoviruses are typical of many other Australian arboviruses. The group includes bovine ephemeral fever virus (BEF), which is a path- ogen of considerable economic importance, and several viruses of livestock which appear to cause mild or asymptomatic infections. The importance of these viruses lies in their possible pathogenic potential and in the implications of their known serological links to bovine ephemeral fever and rabies viruses (Calisher et a1 1989). This brief review will examine the en- demic Australian rhabdoviruses, focusing on recent studies of the structural and antigenic properties of the group.

Bovine Ephemeral Fever The Disease in Australia

Bovine ephemeral fever is an acute and often severe infection of cattle and water buffaloes which occurs in many tropical and sub-tropical regions of the world. The disease appears to have first entered Australia from islands to the north more than 50 years ago. The first confirmed outbreak occurred in the Humbert River District of the Northern Territory in Feb- ruary 1936. A major epidemic followed and, by the summer of 1937, a wave of infection had swept through eastern Aus- tralia reaching into northern Victoria (Seddon 1938). Similar sweeping epidemics occurred in 1955-56, 1%7-68, 1970-71, 1972-74 and 1974-75, infecting large numbers of cattle at a significant cost to industry (St George et a1 1977). After 1976, this epidemic pattern began to subside. The disease occurred in successive years of high and low prevalence, moving more slowly southward and infecting mainly younger animals Wren el a1 1983). In recent years, sweeping epidemics have not been seen. The disease now appears to have become endemic over a wide area of eastern Australia, emerging as localised out- breaks, mainly in the summer and autumn months Wren et 01 1987). However, successive years of low disease prevalence followed by a summer of high rainfall could interrupt the pattern of endemic foci and provide the conditions necessary to sustain an epidemic involving the majority of cattle in Australia.

Clinical Signs As the name suggests, bovine ephemeral fever is character-

ised by rapid onset of clinical signs and an equally rapid recovery. There are usually two febrile phases, each lasting 1 to 2 days (TD St George 1988). The first phase is usually mild and is accompanied by a severe decline in milk production in lactating cows. More severe clinical signs evident in the second febrile phase typically include muscle stiffness, lameness, depression, nasal and ocular discharge, salivation, anorexia, ruminal stasis and recumbency. Some cattle develop severe pneumonia, emphysema or long term paralysis. Mortality oc- curs mainly in heavy, well conditioned animals and lactating cows but the overall mortality rate rarely exceeds 1% (St George 1986).

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Effects on Production A ephemeral fever is a seasonal disease, the scale of eco-

nomic loss is determined largely by climatic conditions and the availability of susceptible cattle. Although mortality is relatively low, large epidemics are of major economic con- sequence to the dairy and beef industries. Less extensive ep- idemics and scattered outbreaks can cause significant losses at the district and producer level.

The most significant production losses result from reduced milk yield in dairy cows and loss of condition in beef cattle. Milk yield declines rapidly from early in clinical infection and commonly remains low for several weeks. Some cattle fail to return to pre-infection yields until the next lactation (Theo- doridis et a1 1973; Davis et al 1984). Other losses in dairy herds include reduced milk and cream quality, high mortality in best milkers and breeding stock and late term abortions in infected cows (TD St George personal communication). In beef herds, average weight loss following BEF infection has been estimated at 10 kg (GM Murphy and TD St George, personal communication). Meat quality is also reduced and mortality is often high in stud cattle (TD St George personal communication).

The overall economic impact of ephemeral fever in Australia is often underestimated because of the relatively low mortality and characteristic rapid recovery from clinical disease. How- ever, in 1986 it was estimated that, in terms of reduced milk yield in dairy herds, weight loss in beef animals and overall mortality, a major BEF epidemic would cost in excess of $100 million. More limited outbreaks could cause losses totalling $3 million in the affected areas (St George 1986). These figures indicate that ephemeral fever is of major economic importance in Australia and that effective control of the disease would be a significant benefit to the cattle industry.

Bovine Ephemeral Fever Virus The agent of ephemeral fever infection is an arthropod-

borne rhabdovirus which has antigenic and structural links to rabies virus (Calisher et a1 1989; Walker et a1 1989). The virus appears to exist as a single serotype but considerable anitgenic variation has been detected between strains. The structure of BEF is similar to that of rabies virus. By electron microscopy, the virus displays cone or bullet-shaped morphology with clear surface projections through a viral envelope, and a precisely coiled nucleocapsid (It0 et 01 1968; Murphy et al 1972). In molecular terms, the virion structure consists of a 42s single- stranded RNA genome, 5 structural proteins and a lipid en- velope (Della-Porta and Brown 1979; Walker et a1 1989). By analogy with rabies virus the virion proteins have been assigned as the large polymerase protein L (180K), membrane glyco- protein G (81K). nucleoprotein N (53K), polymerase-associated protein M, (43K) and matrix protein M, (29K) (Walker ef a1 1989). The nucleoprotein and the matrix protein of BEF virus are known to be phosphorylated.

In BEF-infected BHK-21 cells, 6 viral proteins can be de- tected from 12 h post-infection, followed by a rapid inhibition of cellular protein synthesis (Walker et 01 1989). The L, N, M, and M, proteins correspond to those detected in virions. The G protein exists in 2 forms which appear to be synthesised independently. Each contains complex N-linked carbohydrates which are added to 67K and 71K polypeptide precursors to form the 81K virion G protein and a 90K glycoprotein (GNs) which appears to remain cell-associated. Little is yet known of the function of the cell associated glycoprotien or of i

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structural relationship to the virion G protein. However, there is recent evidence that a similar glycoprotein may also exist in rabies virus infected cells (Tordo and Poch 1988).

The 81K virion G protein appears to be the target for type- specific neutralising antibody. The G protein forms the pro- jections on the virion surface which can be removed by treat- ment with trypsin or non-ionic detergents (Della-Porta and Brown 1979; Walker et al 1989). Four neutralisation sites have been detected on the G protein and monoclonal antibodies directed at these sites have been useful in defining antigenic variation between BEF strains (D H Cybinski, unpublished).

Control by Vaccination Since 1985, a live attenuated BEF vaccine has been available

in Australia. The vaccine consists of cell culture-adapted BEF virus prepared in the saponin adjuvant Quil A and is given in 2 doses at an interval of approximately one month (Van- selow ef a1 1985). The vaccine has proven relatively effective in experimental trials and incidents of vaccine failure in the field have been uncommon. In 1988-89, approximately 150,000 doses of the vaccine were used in dairy and beef herds in endemic areas of Queensland and New South Wales. Vacci- nation has not been sufficiently widespread to have influenced the epidemiological pattern. Questions remain about the ef- fectiveness of the vaccine against all circulating BEF strains and its performance in the face of a major BEF epidemic is untested. However, it is clear that the costs of BEF to rural industry could be effectively contained by vaccination.

Other Australian Rhabdoviruses Thirteen other rhabdoviruses have been isolated from insect

and vertebrate hosts in Australia. None is yet known to cause clinical disease but there is serological evidence that many infect cattle and buffaloes. A detailed study of antigenic relationships between a large assembly of rhabdoviruses has recently shown that the Austrlaian viruses constitute 3 major serogroups (BEF, rabies and Tibrogargan) and several un- grouped viruses (Calisher et a1 1989). Complex antigenic re- lationships were detected between serogroups and between grouped and ungrouped viruses. All Australian rhabdoviruses other than Almpiwar and Kununurra were shown to be in- directly related to rabies virus and, as such, fall in the Lys- savirus genus.

BEF Serogroup The bovine ephemeral fever serogroup can be defined as

BEF, Berrimah and Kimberley viruses from Australia, Malakal virus from Africa and Puchong virus from Malaysia (Calisher et al 1989). Berrimah and Kimberley viruses were isolated from healthy sentinel cattle in northern Australia (Gard et a1 1983; Cybinski and Zakrzewski 1983). Berrimah virus is closely related to BEF with which it shares one of the major G protein neutralisation sites (DH Cybinski, unpublished). Cross-reac- tions between BEF and Berrimah viruses in serum neutralis- ation tests have presented difficulties in assessing the preva- lence of Berrimah virus infection in Australia and it is still uncertain if Berrirnah can cause clinical ephemeral fever (Cy- binski 1987).

Kimberley is a more distantly related virus which has a distribution in cattle in northern and eastern Australia which is similar to that of BEF (Liehne ef a1 1981; Cybinski and Zakrzewski 1983). Antibody to Kimberley virus has also been detected in cattle in Papua-New Guinea, Malaysia, the South Pacific and China. There is no evidence that Kimberley in- fection causes clinical disease. In Australia, most young sus- ceptible cattle in endemic areas develop subclinical infections each year. Previous infection with Kimberley does not protect against BEF and animals with evidence of sequential infections with both viruses are common (St George et al 1984). The structural proteins of Berrimah and Kimberley viruses resemble those of BEF with some differences in the size of the G and M, proteins (Walker et a1 1989; DL Doolan and PJ Walker, unpublished).

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Malakal and Puchong viruses were both isolated from Ma- sonia ungormk mosquitoes in Sudan and western Malaysia (Karabatsos 1985). Their host range and prevalence is not known and further work should be conducted to determine if the viruses cause clinical disease.

Rabies Serogroup Adelaide River virus was isolated from a healthy sentinel

steer near Darwin in 1981 (Gard et a1 1984). Recent serological evidence has indicated that the virus is a Lyssavirus with direct serological links to rabies, Duvenhague, Mokola and Obod- hiang viruses (Calisher et a1 1989). The virus also cross-reacts by immunofluorescence with BEF and Kimberley viruses (Gard et a1 1984) and appears to link the BEF and rabies serogroups. Antibody to Adelaide River virus has also been detected in cattle in China, Papua New Guinea and New Caledonia (DH Cybinski unpublished). There is also evidence of infection in buffaloes and pigs. In Australia, the distribution appears similar to that of BEF and Kimberley viruses (Gard et a1 1984) suggesting that, unlike most other rabies serogroup viruses, Adelaide River is transmitted by biting insects. There is no evidence that Adelaide River virus causes clinical disease but it is known that Kotonkon, another rabies serogroup arbov- irus, causes clinical ephemeral fever in cattle (Kemp et a1 1973, Tomori et a1 1973).

The existence in Australian cattle of a virus with close links to rabies virus is a matter for concern. In the event of a rabies outbreak, the presence of cross-reactive Adelaide River anti- body in feral and domestic animals could confuse serological diagnosis and epidemiological studies of the disease. Further work should be conducted to define more clearly the rela- tionship between Adelaide River virus and exotic rhabdovi- ruses and to determine if other similar viruses are endemic in Australia.

Tibrogargan Serogroup The Tibrogargan serogroup can be defined as Tibogargan,

Coastal Plains and Ngaingan viruses from Australia and Biv- ens Arm and Sweetwater Branch viruses from Florida (Cy- binski and Gard 1986; Gibbs et a1 1989; Calisher et al 1989). All Tibrogargan serogroup viruses appear to infect cattle but there has been no indication that the viruses are associated with clinical disease. Antibody to Tibrogargan serogroup vi- ruses has also been detected in buffaloes, deer, horses and dogs. Low level serological cross-reactions have been detected between Tibrogargan and BEF serogroup viruses (Calisher et a1 1989).

Ungrouped Rhabdoviruses Other Australian rhabdoviruses remain ungrouped but most

have been linked indirectly to rabies or BEF by immunofluo- escence (DH Cybinski unpublished data; Calisher et a1 1989). Charleville virus is related to rabies through Kolongo and Obodhiang viruses; Oakvale virus cross-reacts with Kimberley virus; Parry’s Creek and Wongabel viruses appear to be linked to BEF and Tibrogargan serogroup viruses; and Humpty Doo virus is related to the Tibrogargan serogroup. Almpiwar virus is antigenicly unrelated to other known rhabdoviruses. Ku- nunurra virus has been described as a rhabdovirus on mor- phological grounds (Liehne et al 1981) but serological data has failed to support this classification @H Cybinski unpub- lished data).

Future Research I t is now more than 50 years since the first major epidemic

of ephemeral fever occurred in Australia yet many questions still remain about the agent, the infection and the disease. The site of replication of the virus in infected cattle remains unclear and this has prevented the development of rapid di- agnostic tests. The changing epidemiology of the disease is not understood. The regular sweeping epidemics of the late 1960s and early 1970s have subsided but the factors which sustain the current endemic pattern are unclear. The virus

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appears to persist in sub-tropical and temperate regions be- tween seasons of high vector prevalence but the overwintering mechanism is unknown. Variations in the pathogenicity of BEF strains and the role of some related viruses in clinical disease have not been defined. I t is not known if new exotic strains are responsible for major epidemics or if all endemic strains are effectively targeted by the current vaccine. Molec- ular studies of BEF strains from different outbreaks, epidemics and geographic locations should resolve many of these im- portant questions.

Despite the availability of an attenuated BEF vaccine, little is known about the mechanism of immunity to BEF. Neu- tralising antibody has been used as a measure of protective immunity but there is evidence that animals with high levels of neutralising antibody may not resist challenge (Della-Porta and Snowdon 1979). Reported cases of vaccine failure and the high cost and limited shelf life of available vaccines indicate that better vaccination strategies are necessary. Progress in identifying protective antigens and in defining the role of cell- mediated immunity in long term protection should lead to the development of a more stable and inexpensive vaccine that will provide reliable lifelong protection. Through effective vaccination and a thorough understanding of the disease, the economic impact of BEF in Australia will be minimised.

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