Viruses of the family Rhabdoviridae infect a broad range of hosts from a variety of ecological and geographical niches, including vertebrates, arthropods, and plants. The arthropod-transmitted members of this family display considerable genetic diversity and remarkable genomic flexibility that enable coding for various accessory proteins in different locations of the genome. Here, we describe the genome of Holmes Jungle virus, isolated from Culex annulirostris mosquitoes collected in northern Australia, and make detailed comparisons with the closely related Ord River and Wongabel viruses, with a focus on identifying very small open reading frames (smORFs) in their genomes. This is the first systematic prediction of smORFs in rhabdoviruses, emphasising the intricacy of the rhabdovirus genome and the knowledge gaps. We speculate that these smORFs may be of importance to the life cycle of the virus in the arthropod vector.

Middle Point orbivirus (MPOV) is a recently described Australian arbovirus, related to Yunnan orbivirus from China. Analysis of genetic variation within the major serotype gene of MPOV isolates collected from sentinel cattle has identified eight co-circulating strains. The pattern of strain isolation from individual animals during the study period was consistent with an interpretation of persistent MPOV infection of up to five months, featuring episodes of quiescence (below levels required for virus isolation) followed by viral recrudescence. This is significant with regard to current interpretations of infection, persistence and recrudescence during natural infections of orbiviruses, including bluetongue virus.

Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle in Australia and TIBV has also been isolated from the biting midge Culicoides brevitarsis. Complete genomic sequencing revealed that the viruses share a novel genome structure within the family Rhabdoviridae, each virus containing two additional putative genes between the matrix protein (M) and glycoprotein (G) genes and one between the G and viral RNA polymerase (L) genes. The predicted novel protein products are highly diverged at the sequence level but demonstrate clear conservation of secondary structure elements, suggesting conservation of biological functions. Phylogenetic analyses showed that TIBV and CPV form an independent group within the 'dimarhabdovirus supergroup'. Although no disease has been observed in association with these viruses, antibodies were detected at high prevalence in cattle and buffalo in northern Australia, indicating the need for disease monitoring and further study of this distinctive group of viruses.

ABSTRACT Orbiviruses infect a wide range of hosts, including humans. The ability to detect them has been hampered by their diversity. Here we present a simple consensus reverse transcription (RT)-PCR method targeting the polymerase gene for orbivirus recognition and characterization. Phylogenetic assignment is achieved by automated Web-based sequence analysis of amplification products.

Ngaingan virus (NGAV) was isolated from a pool of biting midges that were collected in the tropics of northern Australia. Reported here is the full-length sequence of the NGAV genome, which, at over 15.7 kb, is the largest in any rhabdovirus described to date and contains 13 genes, the highest number of genes observed in any (-) ssRNA virus. Seven of these putative genes show no significant homology to known proteins. Like viruses in the genus Ephemerovirus, NGAV possesses a second glycoprotein gene (G(NS)). Phylogenetic analyses, however, place NGAV within the yet to be classified "Hart Park" group containing Wongabel and Flanders viruses, which do not contain a second glycoprotein gene. Screening of various animal sera from northern Australia has indicated that NGAV is currently circulating in macropods (wallabies, wallaroos and kangaroos), highlighting the need for further studies to determine its potential to cause disease in these species.

Stretch Lagoon orbivirus (SLOV) was isolated in 2002 from pooled Culex annulirostris mosquitoes collected at Stretch Lagoon, near the Wolfe Creek national park in the Kimberley region of Western Australia. Conventional serological tests were unable to identify the isolate, and electron microscopy indicated a virus of the genus Orbivirus, family Reoviridae. Here, a cDNA subtraction method was used to obtain approximately one-third of the viral genome, and further sequencing was performed to complete the sequences of segment 1 (viral polymerase) and segment 2 (conserved inner-core protein). Phylogenetic analysis showed that SLOV should be considered a new species within the genus Orbivirus. A real-time RT-PCR test was designed to study the epidemiology of SLOV in the field. Six additional isolates of SLOV were identified, including isolates from four additional locations and two additional mosquito species. Horses, donkeys and goats were implicated as potential vertebrate hosts in a serological survey.

In the mid 1990s an emerging disease characterised by the development of proliferative lesions around the face of Tasmanian devils (Sarcophilus harrisii) was observed. A multi-disciplinary approach was adopted to define the condition. Histopathological and transmission electron microscopic examination combined with immunohistochemistry help define Devil Facial Tumour Disease (DFTD) as a neoplastic condition of cells of neuroendocrine origin. Cytogenetic analysis of neoplastic tissue revealed it to be markedly different from normal devil tissue and having a consistent karyotype across all tumours examined. Combined with evidence for Major histocompatability (MHC) gene analysis there is significant evidence to confirm the tumour is a transmissible neoplasm.

Middle Point orbivirus (MPOV) was isolated in 1998 from a healthy cow pastured at Beatrice Hill farm, Middle Point (formerly Coastal Plains Research Station), 50 km east of Darwin in Australia's Northern Territory. The isolate could not be identified by using conventional serological tests, and electron microscopy indicated that it belongs to the family Reoviridae, genus Orbivirus. Genetic sequencing of segments 2 and 3 revealed that this virus is related to Yunnan orbivirus, an orbivirus known only from China and not previously associated with a vertebrate host. A real-time RT-PCR test was developed to study the epidemiology of this virus in the field. Over 150 previously unidentified viruses isolated from cattle between 1994 and 2006 were positively identified as isolates of MPOV. Serology was used to demonstrate the development of antibody responses to MPOV in cattle from multiple locations across the Northern Territory.

DNA microarrays have three main potential diagnostic uses in clinical microbiology: detection of known pathogens, pathogen typing and novel pathogen discovery. Although DNA microarray platforms offer the ability to screen for a large number of agents in parallel, sensitivity is dependent on the ability to obtain adequate amounts of pathogen nucleic acids from collected samples. In general, high levels of sensitivity require a PCR amplification step using specific primer sets, subsequently reducing the overall scope of the microarray assay. At present, relatively high costs, restricted sample throughput capabilities and validation difficulties are also major factors limiting the implementation of DNA microarray assays in diagnostic microbiology laboratories.

The DNA polymerase gene of a novel herpesvirus, vulture herpesvirus (VHV), isolated from an Indian Gyps vulture was completely sequenced using primer walking and transposon insertion strategies. DNA sequencing analysis revealed a single open reading frame (ORF) of 3660 nucleotides (53% G-C content) able to encode 1219 amino acids. Identification was based on a nucleotide sequence identity of approximately 50% to other herpesvirus sequences found in Genbank. Nine motifs were identified that are conserved amongst all known herpesviruses and are found within the 3′–5′ exonuclease and DNA binding functional domains of the DNA polymerase enzyme. Phylogenetic analysis using Clustal W with neighbour-joining revealed VHV to group within the subfamily Alphaherpesvirinae, more closely related to the avian herpesviruses than to those of other species. Partial sequence data also revealed VHV to contain other genes fundamental to the structure and replication of all herpesvirus genomes. A Real Time PCR Taqman assay specific for the VHV DNA polymerase gene was designed to detect the presence of VHV genomic material in post mortem tissue samples from diseased birds. Positive tissues included the spleen, rectum, thymus, kidney and brain. A herpesvirus specific to vultures may pose a threat to the management of captive breeding programs being established to assist the survival of wild populations of Gyps vultures.

RNA arbitrarily primed polymerase chain reaction (RAP-PCR) has been used extensively to identify differentially regulated genes (1–6). The RAP-PCR method begins with conversion of RNA into cDNA, followed by arbitrarily primed PCR. The technique uses arbitrarily primed PCR (7–11) to amplify cDNA stretches lying between sequences that, by chance, match arbitrarily chosen oligonucleotide primers well enough to initiate primer extension. In earlier applications of the method, the complex mixture of products was resolved by polyacrylamide gel electrophoresis (PAGE), yielding highly reproducible fingerprints characteristic of the RNA source. Differences between fingerprints resulting from differentially expressed genes were verified by Northern blot analysis or reverse transcription (RT)-PCR.KeywordscDNA ArraySuccinic AnhydrideArbitrary PrimerReverse Transcription MixtureQIAshredder ColumnThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.