L.I. Pritchard

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

Summary.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.

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.

Peruvian horse sickness virus and Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia.

During 1997, two new viruses were isolated from outbreaks of disease that occurred in horses, donkeys, cattle and sheep in Peru. Genome characterization showed that the virus isolated from horses (with neurological disorders, 78% fatality) belongs to a new species the Peruvian horse sickness virus (PHSV), within the genus Orbivirus, family Reoviridae. This represents the first isolation of PHSV, which was subsequently also isolated during 1999, from diseased horses in the Northern Territory of Australia (Elsey virus, ELSV). Serological and molecular studies showed that PHSV and ELSV are very similar in the serotype-determining protein (99%, same serotype). The second virus (Rioja virus, RIOV) was associated with neurological signs in donkeys, cattle, sheep and dogs and was shown to be a member of the species Yunnan orbivirus (YUOV). RIOV and YUOV are also almost identical (97% amino acid identity) in the serotype-determining protein. YUOV was originally isolated from mosquitoes in China.

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.

The complete nucleotide sequence of the outer coat protein, VP5, of the Australian bluetongue virus (BTV) serotype 1 reveals conserved and non-conserved sequences.

The complete sequence of the outer coat protein, VP5, of the Australian BTV serotype 1 was determined and found to be 1634 nucleotides in length. One single open reading frame of 526 amino acids was observed defining a protein of Mr 59,252 and having a charge of +0.5 at neutral pH. When compared to VP5 of BTV serotype 10 from the United States of America (US) (Purdy et al., 1986, J. Gen. Virol. 67, 957) a homology of 68% at the nucleotide level and 76% at the amino acid level, was observed. However, this conservation at the protein level was more apparent in certain regions of the gene. In four main regions the conservation varied from 83-91% while in the remaining regions the homology dropped to between 56-62%. Many of the amino acid substitutions were conservative in nature, raising the apparent overall homology to 87%. Comparisons of the hydropathy profiles of the two proteins again revealed a remarkable degree of conservation. The importance of these observations is discussed.

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 Mareks disease, infectious laryngotracheitis virus and goose herpesvirus.

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.