Epizootics of sudden death in tammar wallabies (Macropus eugenii) associated with an orbivirus infection.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) occurred at six research facilities and zoological gardens in New South Wales, Australia, in late 1998 and at one Queensland research facility in March 1999. There were 120 confirmed tammar wallaby deaths during this period; however, population censuses indicated that up to 230 tammar wallabies may have died. The majority of animals died without premonitory signs. A small proportion of wallabies exhibited increased respiratory rate, sat with a lowered head shortly before death or were discovered in lateral recumbency, moribund and with muscle fasciculations. Gross postmortem findings consistently included massive pulmonary congestion, mottled hepatic parenchyma and subcutaneous oedema throughout the hindlimbs and inguinal region. Approximately 30% of the animals examined also had extensive haemorrhage within the fascial planes and skeletal muscle of the hindlimb adductors, inguinal region, ventral thorax, dorsal cervical region and perirenal retroperitoneal area. The tissues of affected animals became autolytic within a short period after death. Bacteriological examination of tissues from 14 animals did not provide any significant findings. Toxicological examination of the gastric and colonic contents of four animals did not reveal evidence of brodifacoume or other rodenticides. Viruses from the Eubenangee serogroup of the Orbivirus genus were isolated from the cerebral cortex of nine, and the myocardium of two, tammar wallabies and the liver and intestine of another tammar wallaby. A similar orbivirus was also isolated from the cerebrospinal fluid of another tammar wallaby that died suddenly. The disease agent appears to be a previously unrecognised orbivirus in the Eubenangee serogroup. This is the first report of epizootics of sudden deaths in tammar wallabies apparently associated with an orbivirus infection.

Detection and quantitation of gallid herpesvirus 1 in avian samples by 5' Taq nuclease assay utilizing Minor Groove Binder technology.

A 5' Taq nuclease assay utilizing Minor Groove Binder technology and targeting the thymidine kinase gene of gallid herpesvirus 1 (GaHV-1) was designed and optimized for use in diagnosing avian infectious laryngotracheitis. The assay was specific for GaHV-1 in that it did not react with other avian viral or bacterial pathogens. The detection limit was 1.0x10(-2) median tissue culture infectious dose per reaction or 90 target copies per reaction. Fifteen out of 41 diagnostic samples from sick birds reacted in the assay, five of which produced a typical alphaherpesvirus cytopathic effect (CPE) on chicken kidney (CK) cells. Sequencing, using amplicons generated by a polymerase chain reaction with primers flanking the 5' Taq nuclease amplicon, confirmed the presence of GaHV-1 in six samples (two producing alphaherpesvirus CPE on CK cells, three not producing alphaherpesvirus CPE, and one that was not inoculated onto CK cells). Tracheal swabs taken from 18 healthy broilers did not react in the assay. The ability of the assay to determine viral load in samples was demonstrated. Overall the assay is suitable for the rapid diagnosis of infectious laryngotracheitis.

The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.

Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

OBJECTIVE: To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. DESIGN: A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. RESULTS: The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. CONCLUSION: The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Pulau virus; a new member of the Nelson Bay orthoreovirus species isolated from fruit bats in Malaysia.

After the outbreak of Nipah virus (NiV) in 1998-99, which resulted in 105 human deaths and the culling of more than one million pigs, a search was initiated for the natural host reservoir of NiV on Tioman Island off the east coast of Malaysia. Three different syncytia-forming viruses were isolated from fruit bats on the island. They were Nipah virus, Tioman virus (a novel paramyxovirus related to Menangle virus), and a reovirus, named Pulau virus (PuV), which is the subject of this study. PuV displayed the typical ultra structural morphology of a reovirus and was neutralised by serum against Nelson Bay reovirus (NBV), a reovirus isolated from a fruit bat (Pteropus poliocephalus) in Australia over 30 years ago. PuV was fusogenic and formed large syncytia in Vero cells. Comparison of dsRNA segments between PuV and NBV showed distinct mobility differences for the S1 and S2 segments. Complete sequence analysis of all four S segments revealed a close relationship between PuV and NBV, with nucleotide sequence identity varying from 88% for S3 segment to 56% for the S1 segment. Similarly phylogenetic analysis of deduced protein sequences confirmed that PuV is closely related to NBV. In this paper we discuss the similarities and differences between PuV and NBV which support the classification of PuV as a novel mammalian, fusogenic reovirus within the Nelson Bay orthoreovirus species, in the genus Orthoreovirus, family Reoviridae.

Genetic diversity of bluetongue viruses in south east Asia.

Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.

Regional overview of bluetongue viruses in South-East Asia: viruses, vectors and surveillance.

Structured epidemiological studies based on sentinel herds in Indonesia and Malaysia have provided much information regarding the bluetongue (BT) viruses (BTV) and their likely vectors in South-East Asia. Serotypes 1, 2, 3, 7, 9, 12, 16, 21 and 23 have been isolated. Molecular analyses show all group within the Australasian topotype, with four genotypic sub-groupings identified to date. There are relationships to isolates from both India and Australia. Strains of BTV in South-East Asia do not appear to be highly virulent, since BT disease is not seen in local sheep. Known vector species identified include Culicoides fulvus, C. actoni, C. wadai and C. brevitarsis. C. imicola has not been identified in Malaysian or Indonesian studies. Molecular analyses indicate movement of South-East Asian strains of BTV into northern Australia, and the gradation in observations between India and eastern Australia regarding serotype, genotype, virulence and vector species suggests movement along a conceptual gradient through South-East Asia.

Genetic diversity of bluetongue viruses in Australasia.

The authors have characterised the genetic diversity of the bluetongue virus (BTV) RNA segments 3 and 10 from Indonesia, Malaysia and Australia. Analysis of RNA segment 3, which codes for the core protein VP3, showed conserved sequences in the previously defined Australasian topotype, but which further divided into four distinct clades or genotypes. Certain genotypes appeared to be geographically restricted while others were distributed widely throughout South-East Asia. Ongoing surveillance programmes in Australia have identified the movement of Indonesian genotypes into northern Australia and possible reassortment among them. Similarly, analysis of RNA segment 10, which codes for the non-structural protein NS3/3A, showed they were also conserved and grouped into five clades or genotypes, three Asian and two North American/South African.

Characterisation and monitoring of neutralisation-resistant VP2 phenotypes in BTV-1 isolates from northern Australia collected over a twenty-year period.

Phenotypic profiles of the VP2 protein of isolates of bluetongue virus serotype 1 (BTV-1) collected from Queensland and the Northern Territory, Australia, between 1979 and 1986 were analysed using neutralising monoclonal antibodies (MAbs) raised to the prototype isolate of Australian BTV-1 collected in the Northern Territory in 1979. Two distinct profiles were found. Northern Territory isolates exhibited the prototype profile, yet those from Queensland had a significantly different ('resistant') profile. Nucleotide sequencing of gene segment 2 from both groups of isolates was undertaken. When the nucleotide sequences of isolates from a later period in each State were analysed (1997-2001), all exhibited the 'resistant' profile. Thus, a novel VP2 phenotype, already in existence in Queensland, had supplanted a pre-existing VP2 phenotype in the Northern Territory between the two periods. Furthermore, amino acid differences between the resistant and prototype VP2 proteins were analogous to amino acid substitutions known to be associated with neutralisation resistance. The host immune response may therefore have contributed to selection of the 'resistant' phenotype.

The exceptionally large genome of Hendra virus: support for creation of a new genus within the family Paramyxoviridae.

An outbreak of acute respiratory disease in Hendra, a suburb of Brisbane, Australia, in September 1994 resulted in the deaths of 14 racing horses and a horse trainer. The causative agent was a new member of the family Paramyxoviridae. The virus was originally called Equine morbillivirus but was renamed Hendra virus (HeV) when molecular characterization highlighted differences between it and members of the genus Morbillivirus. Less than 5 years later, the closely related Nipah virus (NiV) emerged in Malaysia, spread rapidly through the pig population, and caused the deaths of over 100 people. We report the characterization of the HeV L gene and protein, the genome termini, and gene boundary sequences, thus completing the HeV genome sequence. In the highly conserved region of the L protein, the HeV sequence GDNE differs from the GDNQ found in almost all other nonsegmented negative-strand (NNS) RNA viruses. HeV has an absolutely conserved intergenic trinucleotide sequence, 3'-GAA-5', and highly conserved transcription initiation and termination sequences similar to those of respiroviruses and morbilliviruses. The large genome size (18,234 nucleotides), the unique complementary genome terminal sequences of HeV, and the limited homology with other members of the Paramyxoviridae suggest that HeV, together with NiV, should be classified in a new genus in this family. The large genome of HeV also fills a gap in the spectrum of genome sizes observed with NNS RNA virus genomes. As such, it provides a further piece in the puzzle of NNS RNA virus evolution.

Development of a polymerase chain reaction to detect Vietnamese isolates of duck virus enteritis.

A polymerase chain reaction (PCR) method for the detection of duck virus enteritis (DVE) virus in tissues of infected and affected ducks, and in cell culture was developed. This required us to obtain specific nucleotide sequence information as we could not find any specific data about the genome of the virus. We found the assay to be highly effective in detecting the virus under experimental conditions and to be easily transferred to laboratories in Vietnam where it is being used in studies on the epidemiology of the disease. We have applied this simple and rapid diagnostic method to the detection of DVE isolates grown in cell culture and tissues from infected birds. The assay was also able to differentiate DVE from other avian herpesviruses, such as Marek's disease, infectious laryngotracheitis virus and goose herpesvirus.

A novel P/V/C gene in a new member of the Paramyxoviridae family, which causes lethal infection in humans, horses, and other animals.

In 1994, a new member of the family Paramyxoviridae isolated from fatal cases of respiratory disease in horses and humans was shown to be distantly related to morbilliviruses and provisionally called equine morbillivirus (K. Murray et al., Science 268:94-97, 1995). To facilitate characterization and classification, the virus was purified, viral proteins were identified, and the P/V/C gene was cloned and sequenced. The coding strategy of the gene is similar to that of Sendai and measles viruses, members of the Paramyxovirus and Morbillivirus genera, respectively, in the subfamily Paramyxovirinae. The P/V/C gene contains four open reading frames, three of which, P, C, and V, have Paramyxovirinae counterparts. The P and C proteins are larger and smaller, respectively, than are cognate proteins in members of the subfamily, and the V protein is made as a result of a single G insertion during transcription. The P/V/C gene has two unique features. (i) A fourth open reading frame is located between those of the C and V proteins and potentially encodes a small basic protein similar to those found in some members of the Rhabdoviridae and Filoviridae families. (ii) There is also a long untranslated 3' sequence, a feature common in Filoviridae members. Sequence comparisons confirm that although the virus is a member of the Paramyxovirinae subfamily, it displays only low levels of homology with paramyxoviruses and morbilliviruses and negligible homologies with rubulaviruses.

EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in the Northern Territory.

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.

Phylogenetic comparison of the serotype-specific VP2 protein of bluetongue and related orbiviruses.

Regions of the VP2 gene from various bluetongue virus serotypes were sequenced and phylogenetic comparisons were performed. The sequences were characteristic for each BTV serotype and isolates of the same serotype could be grouped geographically, mimicking the topotyping characteristics of BTV VP3 gene sequences. PCR amplification and sequence analysis were used to show the close relationship between Caribbean BTV isolates and South African BTV isolates of the same serotype. Similarly, Australian BTV isolates showed a close genetic relationship with Asian BTV isolates of the same serotype. A multiple amino acid sequence alignment of fifteen BTV serotypes and other orbiviruses over a proposed major neutralization site showed this region (317 335 aa.) was highly variable and nucleotide sequences showed that BTV serotypes could be grouped into nucleotypes, or related serotypes, in broad agreement with the inter-relationships postulated by Erasmus (1990), using plaque-reduction tests.

Complete nucleotide sequence of RNA segment 3 of bluetongue virus serotype 2 (Ona-A). Phylogenetic analyses reveal the probable origin and relationship with other orbiviruses.

The nucleotide sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 2 (Ona-A) from North America was determined to be 2772 nucleotides containing a single large open reading frame of 2703 nucleotides (901 amino acid). The predicted VP3 protein exhibited general physiochemical properties (including hydropathy profiles) which were very similar to those previously deduced for other BTV VP3 proteins. Partial genome segment 3 sequences, obtained by polymerase chain reaction (PCR) sequencing, of BTV isolates from the Caribbean were compared to those from North America, South Africa, India, Indonesia, Malaysia and Australia, as well as other orbiviruses, to determine the phylogenetic relationships amongst them. Three major BTV topotypes (Gould, A.R. (1987) Virus Res. 7, 169-183) were observed which had nucleotide sequences that differed by approximately 20%. At the molecular level, geographic separation had resulted in significant divergence in the BTV genome segment 3 sequences, consistent with the evolution of distinct viral populations. The close phylogenetic relationship between the BTV serotype 2 (Ona-A strain) from Florida and the BTV serotypes 1, 6 and 12 from Jamaica and Honduras, indicated that the presence of BTV serotype 2 in North America was probably due to an exotic incursion from the Caribbean region as previously proposed by Sellers and Maaroof ((1989) Can. J. Vet. Res. 53, 100-102) based on trajectory analysis. Conversely, nucleotide sequence analysis of Caribbean BTV serotype 17 isolates suggested they arose from incursions which originated in the USA, possibly from a BTV population distinct from those circulating in Wyoming.

Major core protein VP7 of Australian bluetongue virus serotype 15: sequence and antigenicity divergence from other BTV serotypes.

Full-length cDNA of the RNA genome segment coding for the major core protein VP7 of Australian bluetongue virus serotype 15 (BTV-15) has been isolated by reverse transcription-PCR cloning. Comparative analysis indicated that the BTV-15 VP7 sequence had diverged significantly from that of other members of the BTV serogroup. At the amino acid level, BTV-15 VP7 exhibited sequence identities of 80 to 84% with VP7 molecules of other serotypes, significantly lower than the sequence identities of between 93 and 100% observed among other serotypes characterized to date. This was consistent with previous observations that there were significant immunological differences between BTV-15 and other BTV serotypes and that monoclonal antibodies raised against BTV-1 VP7 failed to react with BTV-15 VP7. Recombinant BTV-15 VP7 protein produced from Escherichia coli was largely insoluble, but maintained its immunogenicity. Polyclonal mouse sera raised against the recombinant VP7 protein reacted strongly with VP7 of BTV-15, but weakly with that of BTV-1.

Phylogenetic characterisation of bluetongue viruses from naturally-infected insects, cattle and sheep in Australia.

The polymerase chain reaction was used to detect the presence of bluetongue virus (BTV) in a number of clinical and insect samples collected in the Northern Territory of Australia. Sequence analyses of the amplified BTV genes differentiated endemic Australian and exotic viruses. Two potential exotic BTV were detected as a result of PCR analyses of blood from sentinel animals and of the insect vector, Culicoides wadai. The detection of BTV in C wadai was the first direct demonstration of the presence of BTV in this potential vector. This new technology can significantly reduce the time taken for a diagnosis from a clinical sample and increase the amount of useful information obtained on a BTV isolate by using rapid sequencing techniques. Sequence data were used to differentiate between BTV20 isolated in 1975 and two isolates of the same serotype, isolated in 1992, and indicated that the latter were probably a recent incursion into Australia from Indonesia due to their greater VP3 sequence homology to the BTV9 (Java) than to Australian BTV isolates.

Phylogenetic analyses of the complete nucleotide sequence of the capsid protein (VP3) of Australian epizootic haemorrhagic disease of deer virus (serotype 2) and cognate genes from other orbiviruses.

The complete nucleotide sequence of the minor capsid protein (VP3) of epizootic haemorrhagic disease of deer virus (EHDV; Australian serotype 2) was determined using a combination of cloning and sequencing methods. Gene segment 3 that coded for the EHDV VP3 capsid protein was 2768 nucleotides in length with a coding region of 2697 nucleotides flanked by 5' and 3' non-coding regions of 17 and 53 nucleotides, respectively. A protein of 899 amino acids (Mr 103,160) having no overall charge at neutral pH was deduced from the nucleotide sequence. Comparisons with equivalent regions from the other Australian EHDV serotypes showed the VP3 genes and the segments that coded for them were similar, varying by a maximum of 5%. Comparisons with known cognate genes from bluetongue viruses showed that their VP3 genes and the proteins translated from them were remarkably similar to those of EHDV, having approximately 70% to 80% homology at either level, respectively. In an attempt to delineate the evolution of orbiviruses, we have obtained sequence data from the VP3 genes from representative members of all Australian orbiviruses now known. Computer analyses of this data enabled a phylogenetic tree for the orbiviruses to be proposed that incorporated the concept of topotypes.

Relationships amongst bluetongue viruses revealed by comparisons of capsid and outer coat protein nucleotide sequences.

Sequence data from the gene segments coding for the capsid protein. VP3, of all eight Australian bluetongue virus serotypes were compared. The high degree of nucleotide sequence homology for VP3 genes amongst BTV isolates from the same geographic region supported previous studies (Gould, 1987; 1988b, c; Gould et al., 1988b) and was proposed as a basis for "topotyping" a bluetongue virus isolate (Gould et al., 1989). The complete nucleotide sequences which coded for the VP2 outer coat proteins of South African BTV serotypes 1 and 3 (vaccine strains) were determined and compared to cognate gene sequences from North American and Australian BTVs. These VP2 comparisons demonstrated that BTVs of the same serotype, but from different geographical regions, were closely related at the nucleotide and amino acid levels. However, close inter-relationships were also demonstrated amongst other BTVs irrespective of serotype or geographic origin. These data enabled phylogenic relationships of the BTV serotypes to be analysed using VP2 nucleotide sequences as a determinant.