Newcastle disease virus fusion and haemagglutinin-neuraminidase gene motifs as markers for viral lineage.

Reverse transcriptase polymerase chain reaction was used to generate sequence data for 91 Australian Newcastle disease viruses (NDV) isolated from 1932 to 2000 covering the cleavage site of the fusion (F) protein and the C-terminus of the haemagglutinin-neuraminidase (HN) protein. Comparison of sequences at these two sites indicates distinct evolutionary relationships between these viruses. Typically, HN gene relationships revealed by phylogenetic analyses were also maintained in comparisons between F gene cleavage sites; however, the former analyses appeared to give a clearer indication of the lineage of a virus isolate. This data supports and extends earlier observations in that there is no evidence for gene exchange by recombination but that different strains appear to have evolved through synonymous mutations. Inter-relationships, especially between Australian NDV isolates, appear to be associated with lineages having the same C-terminal HN extensions rather than associated with virulence of the virus. A proposed mechanism for this observation is discussed.

Pathotyping isolates of Newcastle disease virus using antipeptide antibodies to pathotype-specific regions of their fusion and hemagglutinin-neuraminidase proteins.

Antipeptide antibodies have been evaluated for their abilities to predict the characteristics of the cleavage motifs of the fusion protein precursors (F0) of 25 isolates of Newcastle disease virus (NDV) with a range of virulences, grouped into 12 sets according to their monoclonal antibody reactivities. A Western blot format was used to show that antisera to synthetic peptides representing sequences at the C-termini of the F2-polypeptides of defined pathotypes of NDV usually distinguish between pathotypes on the basis of their Fo cleavage sequences. However, exceptions were found with three groups of virulent isolates. Protein sequencing and mass spectral analysis of the F2-polypeptide of isolate Texas GB from one of these groups, identified an anomalous cleavage/activation process which removed the amino acids required for recognition by the antisera. This probably also explained the lack of reactivity of the Roakin isolate and low reactivity of the Komarov isolate from this group. The other exceptions involved isolates in groups with cleavage region variations from the usual motif of virulent isolates or isolates with undefined cleavage motifs. Antipeptide antisera were also raised to sections of the 45 residue C-terminal extension the hemagglutinin-neuraminidase precursor (HN0) encoded by the genes of some avirulent isolates. Western blot analysis showed that positive reactions with antibodies to peptides based on sequences between residues 577 and 613 of the HN0 was evidence for the presence of an avirulent isolate but did not exclude the presence of other pathotypes. Antisera designed to target residues 569-577 detected HN0 extensions of 6 residues on isolates known to encode such extensions. These antisera also enabled differentiation of isolates with HN0 extensions of 6 residues from those with no extension, however, it was not possible to determine the virulence of isolates based on reaction with these antisera.

Maturation of immunological reactivity in the fetal lamb infected with Akabane virus.

The development of cell-mediated immunological reactivity was studied in fetal lambs infected with Akabane virus. Examination of hepatic cells from fetuses between 40 and 75 days' gestation that had been infected via the transplacental route revealed inconsistent responses to Akabane, together with a uniform failure to respond to non-specific mitogens which contrasted with the behaviour of control, uninfected lambs. Following direct inoculation of fetal lambs with virus between 50 and 120 days' gestation, specific proliferative responses were observed on the part of the spleen cells from some. Direct challenge of fetal lambs of 4 months' gestation evoked cellular responses in lymph draining from the site of virus inoculation similar to those produced by challenge of adult sheep. The proliferative response of lymph-borne cells was substantially better if live, rather than inactivated, virus had been used.

Transmission of Akabane virus from the ewe to the early fetus (32 to 53 days).

The role of the placental junction in AKA virus infection in the ewe was examined during the time when the chorionic villi were first becoming firmly attached to the maternal caruncles. The studies were made over 21 days covering the period between 32 and 53 days of pregnancy. Viral tropism in the fetal membranes and tissues of the fetuses was identified by virus isolation and immuno-fluorescence studies. Areas of virus replication were noted from 24 h post-inoculation in the fetal membranes and persisted in these tissues throughout the experiment. Viral antigen was first detected in the fetus from day 5 post-inoculation by virus isolation and immuno-fluorescence. From this time on, viral activity increased in specific areas of the fetus, notably in the brain and, to a lesser extent, the skeletal muscles. Gross pathological changes occurred in the fetuses between day 14 and day 21 post-inoculation (46 to 53 days gestation). Despite the relatively high titres of AKA virus present in the placental tissues and the developmental changes occurring in the fetus due to the virus, the placental junction continued to carry out its physiological function of maintaining pregnancy.

Biochemical and serological comparisons of Australian bunyaviruses belonging to the Simbu serogroup.

Comparative analysis of the structural and possible non-structural proteins of seven Simbu serogroup bunyaviruses isolated in Australia revealed them all to be similar in size to those of Bunyamwera virus, the prototype of the Bunyavirus genus. The molecular weights of the structural proteins for these bunyaviruses (Akabane, Aino, Tinaroo, Douglas, Peaton, Facey's Paddock and Thimiri viruses) were 193K to 205K (L), 103K to 125K (G1), 33K to 37K (G2) and 25K to 26K (N). Analysis of the virion RNA of three viruses (Akabane, Douglas and Facey's Paddock) showed them all to be similar to Bunyamwera virus RNA, apparent Mr values being 2.6 X 10(6) (L), 1.4 X 10(6) to 1.9 X 10(6) (M) and 0.24 X 10(6) to 0.42 X 10(6) (S). Host cell protein synthesis was switched off late during infection, revealing four structural proteins L, G1, G2 and N. Comparative analysis of these protein profiles in infected Vero cells showed each virus, although similar, to be unique and easily identified; this method of comparison was efficient and rapid compared to the difficulty in obtaining adequate amounts of purified virus for analysis. Additionally, for all viruses except Douglas, two to four possible non-structural proteins were identified, with an Mr range from 12K to 30K. The viruses Akabane and Tinaroo, which have previously been shown to cross-react by plaque inhibition virus neutralization tests, were readily distinguished in migration of the G1 glycoprotein and by analysis of plaque reduction virus neutralization data using linear regression analysis of the dose-response curves. Using these same analyses, the differences between Aino and Douglas viruses, also related by plaque inhibition, were even greater. Application of the biochemical analysis of virus-specified proteins and some serological comparisons identified a mixed pool of different viruses in two unknown isolates grouped as Simbu serogroup viruses, and further identified a potential teratogenic strain in one of the two pools.

Akabane disease in sheep.

Perinatal lamb mortality, associated with malformations of the CNS due to Akabane viral infection, occurred in 4 of 9 flocks of ewes lambing on 3 farms between 26 May and 14 November, 1976. Cases were restricted to ewes conceiving prior to the second week of March and lambing between 26 May and 19 July. As judged by seroconversion in sentinel flocks on 2 of the farms, field infection with Akabane virus occurred mainly between mid-February and mid-April. Malformations of the CNS occurred in 42.5%, 51.2%, 100% and 31.0% of the dead lambs examined in the affected flocks respectively. Prevalence in the 4 affected flocks, expressed as the proportion of ewes lambing which delivered at least one malformed foetus, was 6.1%, 8.4%, 88.9% and 5.7% respectively. Lamb mortality due to malformations of the CNS was 7.1%, 5.5%, 92.3% and 5.7% of lambs born. Age-specific prevalence was calculated for 3 of the 4 flocks and 2-year-old ewes accounted for 71.4% and 76.9% of total cases respectively in 2 flocks, whereas in one flock malformations occurred at equivalent frequencies throughout several older age groups. Birthweights of affected lambs were usually significantly lighter than those of unaffected lambs of similar sex and birth-type, and their mean duration of gestation was slightly, and significantly, prolonged. Micrencephaly (88.1% of cases) and hydrocephalus (68.7% of cases) were the outstanding pathological features of the malformations with hydranencephaly, microgyria, porencephaly and attenuation of the spinal cord occurring at much lower frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies with enzyme-linked immunosorbent assays for the serodiagnosis of bluetongue and epizootic haemorrhagic disease of deer.

An ELISA for the detection of serum antibody in sheep, cattle and goats to the viruses of bluetongue (BTV) and epizootic haemorrhagic disease of deer (EHDV) has been developed. Two methods of antigen preparation were analysed for efficacy in the ELISA and inter-group seroreactivity. A freeze-thaw (F/T) antigen appeared to have a narrower specificity than a cytoskeletal preparation from infected cells (P200) which contained all viral proteins. A higher background reactivity was seen when using the P200 antigen, suggesting that a F/T antigen, perhaps as a composite of serotypes, would be of greater value in an ELISA to replace current methods for antibody screening. The effect of multiple infections with unrelated orbiviruses was found to have no effect on the detection of antibody to BTV and EHDV by ELISA. The ELISA was able to demonstrate development and persistence of antibody to BTV in cattle over the course of 120 days.

Experimental infection of bulls and cows with bluetongue virus serotype 20.

Bluetongue virus serotype 20 (BTV20) was inoculated intradermally and subcutaneously in 4 bulls and by the intrauterine route in 8 nulliparous cows after insemination at oestrus. Viraemia was detected intermittently between 8 and 21 days after inoculation. Virus was isolated from tissue samples of 2 cows and a bull after slaughter at 14 days and from one bull at 28 days. Group reactive and type specific antibodies to BTV20 were demonstrated from 17 to 27 days after infection. No antibodies were detected in the animals slaughtered at 14 days. No clinical signs of disease were seen during the experiment and no gross or histopathological changes referable to BTV20 infection were observed post-mortem. Because of the viraemia and the production of detectable serum antibodies, gametes from these cattle would be excluded from export.

Bluetongue virus serotype 20: experimental infection of pregnant heifers.

Three groups of 4 cows at 84 to 95 days, 100 to 160 days, and 170 to 180 days pregnant were inoculated both intradermally and subcutaneously with bluetongue virus serotype 20 (BTV20). Clinical observations and the viraemic and serological responses of the cows were followed for 9 to 17 weeks after inoculation. Viraemia developed in 9 of the 12 cows and was first detected 4 to 9 days after inoculation. Viraemia was detected for 4 to 21 days and in some animals only intermittently. The titre of the viraemia was obtained in 4 cows and ranged from detectable only, to 10(1) to 10(2.8) 50% tissue culture infecting doses per ml. Both serum neutralising and precipitating antibodies were detected in 11 of the 12 cows within 2 to 8 weeks after inoculation. No clinical responses were seen and one cow (516) did not develop a viraemia or produce detectable antibodies to the virus. The cows, calves and foetuses were necropsied following either parturition or slaughter between 200 and 270 days of pregnancy. No virus isolations were made from a wide range of tissues from the cows, calves or foetuses and no immunoglobulins or serum neutralising antibodies were detected in the serums of precolostral calves or foetuses at necropsy. No gross or histopathological lesions were seen in the cows, calves or foetuses, and there was no evidence that BTV20 crossed the bovine placenta or infected the foetus.

Classification of orbiviruses: a need for supergroups of genera.

There has been concern that the present nomenclature system for the members of the Reoviridae family, and particularly the Orbivirus genus, does not represent the actual relationships exhibited between the members. In order to follow the conventions established by the International Committee for the Taxonomy of Viruses (ICTV), it is tentatively proposed that the present Reoviridae genera be upgraded in status to the following sub-families: reovirinae, orbivirinae, Fijivirinae, cypovirinae, rotavirinae, coltivirinae and phytoreovirinae. Below the sub-family level, divisions of genus (equivalent to superserogroup, serocomplex or supergroup), sub-genus (equivalent to serogroup), species (equivalent to serotype or virus), and variant (equivalent to sub-serotype or genotype) could be created. In the orbivirinae this would result in 2 genera of cyanovirus (bluetongue, epizootic hemorrhagic disease, Eubenangee and Palyam sub-genera) and Kemerovovirus (Chenunda, Great Island, Kemerovo and Wad Medani sub-genera) and a number of ungrouped sub-genera (African horsesickness, Changuinola, Corriparta, equine encephalosis, Wallal and Warrego sub-genera and the remaining ungrouped viruses). It is hoped that further biochemical studies shall confirm these groupings at a more fundamental level and eventually a system recognising the double-stranded RNA gene product relationships shall evolve.

Biochemical characterisation of Australian orbiviruses.

There are over 30 antigenically distinct orbiviruses found in Australia, including members in the bluetongue virus (BTV) and epizootic hemorrhagic disease of deer virus (EHDV) serogroups. Genomic RNA profiles were analysed by polyacrylamide gel electrophoresis (PAGE) on both 10% Laemmli and tris-borate-EDTA-(TBE)-urea gels. There was considerably more variation in the RNA profiles in Laemmli gels than was apparent in the TBE-urea gels. Since the latter system separates on molecular size, then presumably migration in the Laemmli gels may depend upon molecular weight (MW) and conformation. Analyses of 35S-methionine labeled proteins in virus-infected cells was carried out by PAGE in 10 to 20% gradient Laemmli gels. Twelve to 15 virus-specific labeled protein bands were observed in cells infected with orbiviruses. A detailed analysis of Australian BTV 1 isolates was made to identify these proteins. In addition to previously reported proteins (P1 to P8A) an additional low MW protein, P9, was observed (approx. MW 12,000). In the EHDV serogroup, 3 viruses (Ibaraki, CSIRo402 and CSIRo439), which were very closely related by virus neutralization tests and in protein-PAGE, were distinct in their migration by RNA-PAGE. Analyses of the individual RNA segments by 1-dimensional T1-ribonuclease oligonucleotide mapping showed minor differences between Ibaraki and the Australian EHDV isolates, suggesting that the 3 viruses have similar 3'-terminal RNA sequences. These studies suggest that Ibaraki (IBA) virus is closely related to but distinct from the Australian isolates.

Immunochemical analyses of Australian bluetongue virus serotypes using monoclonal antibodies.

In order to characterize the immunochemical role of bluetongue virus (BTV)-specified proteins and provide reagents capable of defining the serological relatedness of bluetongue (BT) serotypes and their relationship with other orbiviruses, a panel of 16 IgG monoclonal antibodies was raised to the Australian BTV serotypes, isolate CSIRO156 (BTV 1), CSIRO19 (BTV20) and CSIRO154 (BTV21). Analyses of virus-coded polypeptide specificities of these monoclonals using enzyme-linked immunosorbent assay (ELISA), a radioimmunoprecipitation assay (RIPA), and a virus neutralization assay, revealed the outer coat viral protein P2 to have a major role in the neutralization of both CSIRO156 and CSIRO19. Presumptive evidence for the involvement of the P3 protein in the neutralization of CSIRO19 was also obtained. The virus-specified non-structural protein P6A induced a group reactive immune response to all 3 serotypes. Antigenic relationships between P3 of CSIRO156 and P2 of CSIRO19 were found, and an analysis of the relationships between epitopic regions on P2 and P3 of both viruses revealed several distinct immunogenic sites exist on the P2 protein.

Problems in the interpretation of diagnostic tests due to cross-reactions between orbiviruses and broad serological responses in animals.

Tests presently used for the diagnosis of infections by bluetongue virus (BTV) or related orbiviruses are based on the use of 2 types of serological reactions. Those that are considered group-reactive tests are the agar gel diffusion precipitin (AGDP), complement-fixation (CF) and fluorescent antibody tests and those that are considered type-specific are a wide variety of virus neutralization tests (50% and 80% plaque reduction, plaque inhibition and microtiter neutralization) and cross-protection tests. These tests suffer from problems of standardization between laboratories and of specificity. Group-reactive tests (AGDP and CF) for the BTV serogroup also detect cross-reactions with viruses in the epizootic hemorrhagic disease virus (EHDV), Eubenangee (EUB) and Palyam (PAL) serogroups, with the EHDV cross-reactions being of particular concern. Further, multiple infections of cattle with PAL serogroup members can produce antibodies which will react to BTV and EHDV serogroup antigens in serological tests. Multiple infections of animals with related viruses can produce antibodies which will cross-react with orbiviruses in type-specific, virus neutralization tests to a virus which the animal has not previously been exposed. These observations stress the need to evaluate the tests at present being used, to assess the risks of cross-reactions between related orbiviruses and to develop new tests of defined specificity.

Teratogenicity of Australian Simbu serogroup and some other Bunyaviridae viruses: the embryonated chicken egg as a model.

The use of embryonated chicken eggs as a model for assessing the teratogenic potential of animal viruses was investigated with 12 members of the Bunyaviridae family. Infection of 4-day-old embryonated chicken eggs via the yolk sac with 10 of the viruses resulted in deaths or congenital deformities that were similar to those observed in Akabane virus infections of fetal ruminants and included arthrogryposis, scoliosis, mandible defects, and retarded development. Statistical analysis showed that the viruses fell into three main groupings, namely, those that caused both death and deformities (Akabane, Aino, Tinaroo, and Belmont viruses), those that mainly caused death (Peaton, Thimiri, and Facey's Paddock viruses), and those that required very high doses to cause either death or deformities (Douglas and CSIR0296 viruses). In addition, two viruses (Kowanyama and Mapputta viruses) caused neither death nor deformities. A difference in the pathogenic potential between two Akabane isolates (B8935 and CSIR016) in the embryonated chicken egg model was found to correlate with differences previously observed in experimentally infected sheep; Akabane CSIR016 was the more pathogenic. It is concluded that the embryonated chicken egg model should also be of value in assessing the teratogenic potential of other Bunyaviridae and attenuated vaccine viruses, although it does not assess the ability of the virus to cross the placenta.