Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus.

The nucleotide sequence of the bovine ephemeral fever virus (BEFV) genome has been determined from the 3' terminus to the end of the nucleoprotein (N) gene. The 3' leader sequence comprises 50 nucleotides and shares a common terminal three nucleotides (3'-UGC-) and a downstream U-rich domain with vesicular stomatitis virus (VSV) and rabies virus. The N gene comprises 1328 nucleotides from the transcription initiation consensus sequence (AACAGG) to the conserved transcription termination-poly(A) sequence [CATG(A)7] and encodes a polypeptide of 431 amino acids with an estimated M(r) of 49,159 and a pI of 5.4. The deduced amino acid sequence of the BEFV N protein is similar to those of other mammalian rhabdoviruses and is more closely related in sequence to vesiculoviruses (VSV Indiana and New Jersey, Piry, Chandipura) than to lyssaviruses (rabies and Mokola). An almost full-length clone, 1301 bp in length, of the BEFV N gene and clones derived from 5'-terminal (559 bp) and 3'-terminal (742 bp) fragments were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. A panel of 12 BEFV N protein-specific monoclonal antibodies was shown to react in immunoblots with fusion proteins containing the almost full-length N protein and the C-terminal fragment, but not the N-terminal fragment. Two of these antibodies also reacted with baculovirus-expressed rabies virus N protein. Polyclonal mouse ascitic fluids derived from BEFV, rabies virus and several other related viruses were also shown to cross-react in immunoblots with purified preparations of rabies virus and BEFV N proteins.

Studies on the pathogenesis of bovine ephemeral fever in experimental cattle. III. Virological and biochemical data.

In an attempt to define the nature of the response of cattle to ephemeral fever infection, a number of indicators of inflammation were monitored during clinical disease. The total Ca, Zn, Fe, Cu, glucose and phosphate in plasma, together with blood ammonia, were assayed relative to changes in the rectal temperature. CaT levels fluctuated markedly and hypocalcaemia occurred in 4 of 8 cattle. Plasma Zn and Fe values fell while plasma Cu levels rose markedly in all cattle. Mean levels of serum NH3 of 20-30 mumol l-1 rose to a peak value of 56 mumoll-1. Plasma glucose levels rose to a peak of 4.6 +/- 0.5 mMl-1 and the plasma phosphate levels fell from 2.4 +/- 0.1 mMl-1 to 1.17 +/- 0.2 mMl-1 during fever. Values of pCO2 fell from a mean of 46.9 +/- 3.6 mmHg to 36.4 +/- 3.1 mmHg and coincided with a rise in pH. Virus was isolated 73 h (+/- 23) after inoculation and persisted until 130 h (+/- 21). The common role of these parameters in generalised inflammation and ephemeral fever is discussed.

Proteins of bovine ephemeral fever virus.

The proteins of bovine ephemeral fever virus (BEFV) were examined in purified virions and in infected BHK-21 cells. Five structural proteins were named L (180K), G (81K), N (52K), M1 (43K) and M2 (29K). The 81K G protein incorporated [3H]glucosamine, was removed from virions by treatment with Triton X-100 and bound monoclonal antibodies which were both neutralizing and protective. Treatment of virions with Triton X-100 and 0.2 to 1.0 M-NaCl progressively released L, M1 and M2. The N protein remained associated with nucleocapsids in up to 2.5 M-NaCl. The glycoprotein (G), nucleoprotein (N) and matrix protein (M2) were phosphorylated. In BEFV-infected BHK-21 cells, five virus-induced proteins were detected from 12 h post-infection. The L, N, M1 and M2 proteins corresponded to those detected in virions whereas the G protein existed in two forms. In tunicamycin-treated cells these occurred as 67K and 71K non-glycosylated precursors. In the absence of tunicamycin, 77K and 79K glycosylated forms were further modified to produce the 81K virion G protein and a 90K cell-associated form. Five viral proteins were also detected in cells infected with the closely related Berrimah virus; the Berrimah virus G protein was also present in two forms.

Establishment and characterization of monoclonal antibodies against bovine ephemeral fever virus.

We established fourteen monoclonal antibodies (MAbs) reactive to bovine ephemeral fever virus YHL strain, and characterized six representatives including three IgG1s (YG3/4, YG5/8, and YG6/7) and three IgMs (YM4/9, YM2/6, and YM6/8). Among them, YG3/4 and YM4/9 gave especially strong reactivities to the virus. YM4/9 reacted specifically with a 43K antigen of the virus, corresponding to the matrix protein 1. The other MAbs reacted most strongly with the 43K antigen, but also reacted with unknown 23K and 21K antigens. By a simultaneous two-site method using YM4/9 and YG3/4, it was possible to detect 10(4.10)TCID50/ml of the virus, in the presence of serum.

Mapping of antigenic sites on the bovine ephemeral fever virus glycoprotein using monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against the G, M2 and N proteins of bovine ephemeral fever virus (BEFV) and 29 were selected for further study. Thirteen neutralizing MAbs were assigned to one conformation-independent and at least two conformation-dependent antigenic sites on the G protein by a competitive binding ELISA. The panel of MAbs were tested by neutralization and immunofluorescence with three strains of BEFV and three BEFV-related viruses. The results indicated that BEFV strains from different sources were not identical and that the M2 protein was the least variable of the proteins investigated. Passive protection studies in mice showed that the correlation between neutralizing titre and resistance to challenge was 0.85 (P less than 0.001).

Bovine ephemeral fever.

Ephemeral fever remains a viral disease of considerable importance to many countries including Australia. The virus has been only partly characterised and still awaits final classification. Although BEF virus was first thought to contain 6 structural proteins there is increasing evidence to suggest that it contains the 5 proteins characteristic of the Rhabdoviridae. Although BEF is thought to be arthropod borne, the vector has yet to be identified but it is clear from the distribution of BEF that more than one vector is capable of transmitting the disease. Despite rigorous investigation of the clinical signs and the pathology of ephemeral fever, little progress has been made on the pathogenesis of the disease. This has been partly due to the difficulty of propagating BEF virus in vitro and the inability to define the site of replication. However, there is mounting evidence to suggest that BEF is immunopathologic in nature and that the clinical expression of the disease is influenced by the release of one or more mediators of inflammation. The disease is characterised by a number of haematological and biochemical changes and early and prolonged treatment with phenylbutazone is capable of reversing a number of these changes. The intravenous administration of calcium can now be considered a justifiable addition to the treatment regimen together with prolonged phenylbutazone therapy. The vaccines currently available are prepared from either live attenuated or killed virus and may be less than reliable. There appears to be a need for a reliable, inexpensive, cold-chain independent alternative vaccine.

Epidemiology of bovine ephemeral fever in Australia 1981-1985.

Bovine ephemeral fever is an important viral disease of cattle in Australia. The disease occurred each year, principally in summer and autumn, between 1981 and 1985. Queensland and the northern half of New South Wales were areas of greatest activity with only sporadic cases being reported from the Northern Territory and the northern third of Western Australia. Since 1981, the disease has been endemic in an extensive area of eastern Australia and has tended to occur in widely scattered outbreaks rather than the north-south advancing wave form of the epidemics of 1936-37, 1967-68, 1970-71 and 1972-74. The southernmost outbreaks between 1981 and 1985 were well within the limits of these earlier epidemics. The pattern of disease appears to have become seasonally endemic rather than periodically endemic in the northern two-thirds of eastern Australia. Ephemeral fever was not recorded in Victoria, Tasmania, South Australia or the southern part of Western Australia between 1981 and 1985. The disease was most frequently reported in cattle under 3 years of age, but also occurred in older cattle.

Studies on the pathogenesis of bovine ephemeral fever in sentinel cattle. I. Virology and serology.

Twenty-two sentinel cattle were observed daily during an outbreak of ephemeral fever on a dairy farm in eastern Australia in the summer of 1981-82. Of the 22 cattle, 9 developed clinical ephemeral fever. None developed sub-clinical infection. The pattern of the epidemic was a single index case followed 10 days later by the main epidemic wave which lasted for 7 days. This wave stopped when there were still 14 uninfected susceptible animals remaining in the sentinel group, and when biting flies were very active. Ten isolations of bovine ephemeral fever virus were made in Aedes albopictus tissue cultures from the blood of 5 clinical cases. One hundred and twelve isolations of CSIRO Village virus and one each of Kimberley and Akabane viruses were also made from various members of the sentinel group. There was serological evidence that infections with Tibrogargan, Tinaroo and Aino viruses also occurred in 6 cattle in the observation period. The 13 cattle undergoing a sub-clinical viraemia with CSIRO Village virus, Tibrogargan, Kimberley, Akabane or Aino viruses at the time of the main outbreak, appeared to be temporarily protected against ephemeral fever. However, 9 of the 11 still remaining in the herd were susceptible in a subsequent outbreak of ephemeral fever 2 years later. Evidence is presented that subclinical infections with other arboviruses may limit an ephemeral fever epidemic by providing temporary protection by interference.

Studies on the pathogenesis of bovine ephemeral fever in sentinel cattle. II. Haematological and biochemical data.

Twenty-two non-lactating dairy cattle from a sentinel herd previously described (St. George, 1985) were monitored daily during an outbreak of ephemeral fever. Nine developed clinical ephemeral fever between 25 December 1981 and 30 January 1982. There were no subclinical infections with bovine ephemeral fever virus in the group. There were, however, subclinical infections with CSIRO Village, Akabane, Aino, Tinaroo and Kimberley viruses as described by St. George et al. (1984). Six of the nine affected cattle showed a neutrophilia with a concurrent lymphopaenia on the day of pyrexia; however, the differential white cell profile had begun to change up to 24 h prior to leucocytosis. Serum carboxypeptidase values fell by 24 h following the febrile response. Plasma fibrinogen rose rapidly in all six cows. The peak concentration (15.6 +/- 2.70 g l-1) occurred 3 days after pyrexia with the highest individual increase being from 6.05 to 19.6 g l-1. Plasma fibrinogen levels remained elevated for at least 7 days. Serum calcium fell significantly during Day 1 of the disease, the mean decline being 0.22 +/- 0.08 mmol l-1. The greatest individual fall was from 2.33 to 1.92 mmol l-1. None of the affected cattle showed any compensatory change in serum magnesium. There was no change in the normal values of creatinine, urea, gamma-GT, AST and alkaline phosphatase. Bovine ephemeral fever virus was isolated from only four of the six cases, whereas specific antibody was detected in all cattle 3-4 days after recovery.

Transmission of virus from serosal fluids and demonstration of antigen in neutrophils and mesothelial cells of cattle infected with bovine ephemeral fever virus.

Following intravenous injection of bovine ephemeral fever (BEF) virus 6 cattle were autopsied after clinical disease became evident. Fluid from serosal cavities with serofibrinous inflammatory changes showed large increases in neutrophil numbers. BEF virus was detected for the first time in pericardial, thoracic and abdominal fluids. Virus was also detected in synovial fluids, confirming an earlier report of transmission with a synovial fluid sample. Using a direct fluorescent antibody technique, BEF virus antigen was identified for the first time in synovial, pericardial, thoracic and abdominal fluids, in synovial membranes and epicardium. In synovial membranes and epicardium, specific fluorescence was observed in two cell types, mesothelial cells and neutrophils. In the fluids, fluorescence was restricted to neutrophils, the predominant cell type. Specific fluorescence was observed in blood smears from only one animal although blood samples collected at autopsy from all animals contained infective virus.

Growth of epizootic hemorrhagic disease, akabane, and ephemeral fever viruses in Aedes albopictus cells maintained at various temperatures.

The growth curves of one epizootic hemorrhagic disease (EHD) virus serotype (Reoviridae), two Akabane virus strains (Bunyaviridae) and three bovine ephemeral fever (BEF) group viruses (Rhabdoviridae) were determined in Aedes albopictus cells maintained at 15, 20, 28 and 33 degrees C. Ae albopictus cells supported the growth of all the viruses although not necessarily at all temperatures. Because none of the viruses exhibited cytopathic effect in Ae albopictus cells, growth was assayed in baby hamster kidney 21 (BHK21) cells maintained at 37 degrees C. The temperature at which the Ae albopictus cells were maintained had a marked effect on the growth and yield for each virus studied. EHD virus was heat-stable and grew after 4 days at 28 and 33 degrees C, and after 8 days at 20 degrees C. No growth was recorded up to 12 days at 15 degrees C. The two Akabane viruses were heat-sensitive and exhibited different growth patterns. One strain (B8935) showed no growth at 15 degrees C and only minimal growth at 20, 28 and 33 degrees C. The other strain (CSIRO 16) showed growth after 1-2 days at all temperatures with higher titres reached at 15 and 20 degrees C than at 28 and 33 degrees C. The BEF group viruses grew to approximately the same titres at all temperatures. At the higher temperatures (28 and 33 degrees C) most of BEF group viruses had disappeared within 9 days. In contrast at the lower temperatures (15 and 20 degrees C), there was still virus present 18 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolation and preliminary characterization of a rhabdovirus in Australia related to bovine ephemeral fever virus.

CSIRO 368 virus was isolated from blood collected in the Northern Territory from a healthy cow and electron microscope studies showed that the isolate had rhabdovirus morphology. Fluorescent antibody studies and complement fixation tests related the virus to bovine ephemeral fever (BEF) virus. Neutralization tests in both suckling mice and Vero cells showed that the virus was not BEF virus. Antibodies to CSIRO 368 virus were found in cattle sera from northern and eastern Australia and Papua New Guinea. Antibodies were found in 16 out of 45 buffalo, some of which also had antibodies to BEF virus. In contrast, none of the 419 deer tested had antibodies to CSIRO 368 virus, although 142 of the same deer had antibodies to BEF virus. No antibodies to CSIRO 368 virus were detected in 16 goats, 54 horses, 10 pigs, 31 sheep, 25 kangaroos, or 14 human beings. Both CSIRO 368 and BEF viruses were found to be sensitive to ether and chloroform, but were not affected by the DNA inhibitor 5-bromo-2'-deoxyuridine, showing that they probably had the same type of nucleic acid--namely RNA. CSIRO 368 was also shown to grow to higher titres in BHK21 cells than in Vero cells. Temperature sensitivity studies at -20, 4 and 37 degrees C showed that the presence of foetal calf serum increased the survival time markedly at -20 degrees C, but only slightly at 4 and 37 degrees C. The virus survived the longest at -20 degrees C in the presence of foetal calf serum.