Genomic analysis of bluetongue virus episystems in Australia and Indonesia.

The distribution of bluetongue viruses (BTV) in Australia is represented by two distinct and interconnected epidemiological systems (episystems)-one distributed primarily in the north and one in the east. The northern episystem is characterised by substantially greater antigenic diversity than the eastern episystem; yet the forces that act to limit the diversity present in the east remain unclear. Previous work has indicated that the northern episystem is linked to that of island South East Asia and Melanesia, and that BTV present in Indonesia, Papua New Guinea and East Timor, may act as source populations for new serotypes and genotypes of BTV to enter Australia's north. In this study, the genomes of 49 bluetongue viruses from the eastern episystem and 13 from Indonesia were sequenced and analysed along with 27 previously published genome sequences from the northern Australian episystem. The results of this analysis confirm that the Australian BTV population has its origins in the South East Asian/Melanesian episystem, and that incursions into northern Australia occur with some regularity. In addition, the presence of limited genetic diversity in the eastern episystem relative to that found in the north supports the presence of substantial, but not complete, barriers to gene flow between the northern and eastern Australian episystems. Genetic bottlenecks between each successive episystem are evident, and appear to be responsible for the reduction in BTV genetic diversity observed in the north to south-east direction.

Evolution of bluetongue virus serotype 1 in northern Australia over 30 years.

UNLABELLED: Bluetongue virus serotype 1 (BTV 1) was first isolated in Australia from cattle blood collected in 1979 at Beatrice Hill Farm (BHF), Northern Territory (NT). From long-term surveillance programs (1977 to 2011), 2,487 isolations of 10 BTV serotypes were made. The most frequently isolated serotype was BTV 1 (41%, 1,019) followed by BTV 16 (17.5%, 436) and BTV 20 (14%, 348). In 3 years, no BTVs were isolated, and in 12 years, no BTV 1 was isolated. Seventeen BTV 1 isolates were sequenced and analyzed in comparison with 10 Australian prototype serotypes. BTV 1 showed an episodic pattern of evolutionary change characterized by four distinct periods. Each period consisted primarily of slow genetic drift which was punctuated from time to time by genetic shifts generated by segment reassortment and the introduction of new genome segments. Evidence was found for coevolution of BTV genome segments. Evolutionary dynamics and selection pressure estimates showed strong temporal and clock-like molecular evolutionary dynamics of six Australian BTV genome segments. Bayesian coalescent estimates of mean substitution rates clustered in the range of 3.5 × 10(-4) to 5.3 × 10(-4) substitutions per site per year. All BTV genome segments evolved under strong purifying (negative) selection, with only three sites identified as under pervasive diversifying (positive) selection. The obligate replication in alternate hosts (insect vector and vertebrate hosts) imposed strong evolutionary constraints. The dominant mechanism generating genetic diversity of BTV 1 at BHF was through the introduction of new viruses and reassortment of genome segments with existing viruses. IMPORTANCE: Bluetongue virus (BTV) is the causative agent of bluetongue disease in ruminants. It is a disease of concern globally and is transmitted by biting midges (Culicoides species). Analysis of the evolutionary and selection pressures on BTV 1 at a single surveillance site in northern Australia showed strong temporal and clock-like dynamics. Obligate replication in alternate hosts of insect and vertebrate imposed strong evolutionary constraints, with all BTV genome segments evolving under strong purifying (negative) selection. Generation of genetic diversity of BTV 1 in northern Australia is through genome segment reassortment and the introduction of new serotypes.

Bovine ephemeral fever rhabdovirus α1 protein has viroporin-like properties and binds importin ß1 and importin 7.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.

Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia.

Bluetongue virus (BTV) is transmitted by biting midges (Culicoides spp.). It causes disease mainly in sheep and occasionally in cattle and other species. BTV has spread into northern Europe, causing disease in sheep and cattle. The introduction of new serotypes, changes in vector species, and climate change have contributed to these changes. Ten BTV serotypes have been isolated in Australia without apparent associated disease. Simplified methods for preferential isolation of double-stranded RNA (dsRNA) and template preparation enabled high-throughput sequencing of the 10 genome segments of all Australian BTV prototype serotypes. Phylogenetic analysis reinforced the Western and Eastern topotypes previously characterized but revealed unique features of several Australian BTVs. Many of the Australian BTV genome segments (Seg-) were closely related, clustering together within the Eastern topotypes. A novel Australian topotype for Seg-5 (NS1) was identified, with taxa spread across several serotypes and over time. Seg-1, -2, -3, -4, -6, -7, -9, and -10 of BTV_2_AUS_2008 were most closely related to the cognate segments of viruses from Taiwan and Asia and not other Australian viruses, supporting the conclusion that BTV_2 entered Australia recently. The Australian BTV_15_AUS_1982 prototype was revealed to be unusual among the Australian BTV isolates, with Seg-3 and -8 distantly related to other BTV sequences from all serotypes.

Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals.

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.

Evolutionary history of Simbu serogroup orthobunyaviruses in the Australian episystem.

Orthobunyaviruses of the Simbu serogroup are transmitted by insects (primarily biting midges) and infect mammals and/or birds. Many have been associated with disease in livestock or humans. The orthobunyavirus genome comprises three negative-sense RNA segments (L, M and S). We report the complete coding sequences of 57 isolates of Simbu serogroup viruses collected in Australia during 1968-1984. Phylogenetic analysis identified novel genogroups of Akabane virus (AKAV), Aino virus (AINOV) and Peaton virus, and provided evidence of constrained movement of AKAV between epidemiological systems in the northern and eastern regions of the continent. Differential clustering of AKAV isolates in trees inferred from L, M and S segments was indicative of intratypic segment reassortment. Similarly, intertypic segment reassortment was detected between AKAV and Tinaroo virus, and between AINOV and Douglas virus. L segments representing novel genogroups were detected in AINOV reassortants, suggesting the presence of unidentified Simbu group viruses in the episystem.

Detection of respiratory viruses and subtype identification of influenza A viruses by GreeneChipResp oligonucleotide microarray.

Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.