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Bluetongue virus (BTV) is an arbovirus (genus: Orbivirus ) that occurs worldwide. It infects domestic and wild ruminant species and can cause disease in livestock, producing high economic impact. Recently, it gained extra prominence throughout Europe, with disease occurring in regions traditionally free of BTV. BTV enters Australia from Southeast Asia via wind-borne infected Culicoides spp. The first Australian isolation was 1975 (BTV-20) and further serotypes were isolated between 1979-86 (BTV-1, -3, -9, -15, -16, -21, -23). Despite increased, more sensitive, monitoring, no more were detected in over two decades, implying a stable BTV episystem of eastern ancestry. Isolations of BTV-2, -7 and -5 then occurred between 2007-15, with the latter two possessing genome segments with high sequence identity to western isolates. We report on the first isolation and genomic characterization of BTV-12, which revealed that three more novel western topotype gene segments have entered northern Australia.

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 degrees S and 5 degrees N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

An improved bluetongue antigen capture ELISA (BTACE) technique was evaluated for its ability to detect the full range of 24 bluetongue (BLU) serotypes. The BTACE detected all 24 serotypes in cell culture fluids, including eight serotypes where the representative strains originated from both Australia and also from the South African reference collection. The amount of infectious virus required to obtain a positive BTACE result varied between 100-1000 TCID50. This was approximately 10-fold more sensitive than the antigen capture test described previously (Hosseini, M., Hawkes, R.A., Kirkland, P.D., Dixon, R., 1998. J. Virol. Methods 75, 39-46.). The BTACE method was compared with conventional passage in cell culture to detect the presence of virus in the tissues of embryonated chicken eggs (ECEs) which had been inoculated intravenously with the blood of sheep and cattle infected experimentally with the eight Australian serotypes of BLU (1, 3, 9, 15, 16, 20, 21, and 23). The BTACE method was at least as sensitive as the conventional cell culture detecting virus in ECEs, obviating the need for prolonged cell culture passage to detect the virus. A comparison of the amount of antigen detected in different embryo tissues indicated that liver homogenates gave the highest positive to negative ratios in the BTACE and were selected as the specimen of choice. In studies of sheep infected with all 24 South African reference BLU serotypes this new BTACE was able to detect viraemia with all serotypes. Finally, the BTACE was validated in surveillance programs for BLU in both New South Wales, Australia and in Yunnan Province, People's Republic of China. Blood samples from sentinel cattle were inoculated into ECEs. Homogenised ECE livers were tested by BTACE and those positive were passaged subsequently in cell culture for virus isolation and identification. This protocol led to the efficient isolation of field isolates of many serotypes. The high sensitivity and broad reactivity of the method indicates that it should be valuable for BLU diagnosis and surveillance programs.

Rapid serotyping of bluetongue virus (BTV) isolates is required to facilitate the choice of an appropriate serotype-specific vaccine in a disease situation or to improve surveillance of BTV serotype prevalence. This communication describes the development and validation of a bluetongue virus fluorescent inhibition test (BTV FIT) as a rapid method to serotype Australian BTV isolates. The BTV FIT uses virus neutralisation principles similar to those used in the rabies rapid fluorescent focus inhibition test. The BTV FIT has the ability to provide an accurate serotype identification within 24 h thereby abbreviating the serotyping process by 3-4 days relative to conventional virus neutralisation assays and making the BTV FIT comparable time-wise with the polymerase chain reaction technique. The development of the BTV FIT is described using BTV reference viruses which have been isolated in Australia, and validation of the assay by assessment of five Australian BTV isolates of unknown serotype by comparison with the plaque inhibition method. The use of the BTV FIT readily facilitated rapid and accurate serotype identification of Australian BTV reference viruses and five unknown BTV isolates with results indicating full agreement with the plaque inhibition method.

Two of the 5 serotypes of bluetongue virus (BTV) known from Australia have been isolated from field collected insects. Serotype 20 was isolated in 1975 from a mixed pool of 214 insects containing several Culicoides species. Serotype 1 has been isolated from C. (Avaritia) fulvus Sen & Das Gupta collected at Beatrice Hill in the Northern Territory and from C. (Avaritia) brevitarsis Kieffer collected at Peachester in southeast Queensland. All other isolates of bluetongue (BT) group viruses have been made from sentinel cattle. An additional 2 species of the subgenus Avaritia, C. wadai Kitaoka and C. actoni Smith, 1 species of the subgenus Culicoides, C. peregrinus Kieffer and 1 species of the Schultzei group, C. oxystoma Kieffer have been infected in the laboratory. Serotype 20 was transmitted from sheep to sheep by C. fulvus and serotype 1 by C. fulvus and C. actoni. The infection rates established for Culicoides fed on sheep infected with serotype 20 were C. fulvus 62%; C. wadai 11%; C. actoni 2% and C. brevitarsis 0.3%. All species of insects successfully infected are widely distributed in the Oriental region. Attempts to infect species that are restricted in range to the Australasian region have been unsuccessful. The 3 species with highest experimental infection rates: C. fulvus, C. wadai and C. actoni, are confined in Australia to areas with an annual summer rainfall in excess of 800 mm, and do not penetrate to the drier areas where sheep are commercially husbanded. C. brevitarsis is the vector responsible for transmission in the coastal dairying areas, and although it does occur where sheep are reared, it is an inefficient vector. It breeds in discrete cow dung pats on pasture and is more closely associated with cattle than with sheep when the 2 hosts occur together. More than 1 species of Culicoides is responsible for the transmission of BT group viruses in Australia and no BT disease of sheep has been recorded because the more efficient vector species are not present in sheep rearing areas.

Laboratory-reared C. brevitarsis (biting midges) were fed on sheep which had been experimentally infected with bluetongue serotype 1 (CSIRO 156), bluetongue serotype 20 (CSIRO 19) or bluetongue serotype 21 (CSIRO 154), or on cattle experimentally infected with bluetongue serotype 20 (CSIRO 19). Approximately 77 000 C. brevitarsis were exposed to sheep and 9000 to cattle. The average percentage feeding on sheep was 54% and on cattle 47%. In attempts to transmit virus by bite 3360 C. brevitarsis which had fed on viraemic sheep were held for 11-15 days before exposure to susceptible sheep. Although 11% of these insects fed, transmission of virus from sheep to sheep was not demonstrated. Estimated infection rates of C. brevitarsis for each serotype from sheep and serotype 20 from cattle were similar at 0.4% or lower. These low infection rates are one of the factors which make it unlikely that C. brevitarsis could be an efficient vector of bluetongue viruses in sheep in the field.

In the 200 years that cattle and sheep have been present in Australia no evidence of bluetongue (BT) disease has ever been reported. The discovery in 1977 that a virus isolated from Culicoides species collected in 1975 was bluetongue virus (BTV) triggered a search for BT viruses and their vectors throughout the country. Between 1975 and 1983, 46 strains of 5 serotypes of BTV were isolated by various workers from the blood of subclinically infected sentinel cattle or from Culicoides species. The serotypes are 1, 20, 21 and 2 others not yet fully characterized. By testing stored sera from cattle and buffaloes, antibodies to serotype 21 were found in sera collected in 1958, but antibodies to serotypes 20 and 1 were not found earlier than 1973 and 1969 respectively. Discrepancies between the results of BTV group antibody and neutralizing antibody surveys indicate that other serotypes may yet be found. Neutralizing antibodies to BTV have been found in cattle, buffaloes, deer, goats and sheep, but mammals indigenous to Australia are apparently not being infected. In the course of the search, Ibaraki virus and 4 other viruses of the epizootic haemorrhagic disease (EHD) group were found. Infection with EHD viruses has produced heterotypic antibodies detected in tests for BT group antibodies. The prevalence of antibodies to BT and EHD viruses in cattle is greater in the northern 1/3 of the continent than the middle 1/3 while the southernmost 1/3 is free. By the sequential sampling of sentinel cattle it has been determined that infection with BT and EHD viruses occurs mainly in the latter part of summer, and autumn. The seasons of the year, and the locations from which BT and EHD viruses originated were consistent with the serological findings. Isolations of BT and EHD viruses have also been made from insect vectors, BTV serotype 1 from C. fulvus and C. brevitarsis, plus serotype 20 from a mixed pool of Culicoides species and 2 different EHD viruses from C. brevitarsis. The susceptibility of other Culicoides species to experimental infection with BT and EHD viruses suggests that they may be involved in the natural spread of these viruses. BT and EHD viruses appear to be non-pathogenic for cattle under experimental conditions, but mildly or moderately pathogenic for sheep. The clinical signs produced are consistent with those produced experimentally in other countries.

Since 1975 3 serotypes of bluetongue (BT) virus (BTV) have been identified in Australia: BTV1 (CSIRO156), BTV20 (CSIRO19) and BTV21. At present 2 further BT viruses (DPP90 and DPP192) have been isolated from the blood of healthy cattle in the Northern Territory (NT) and are undergoing identification. There is serological evidence for BTV15 infection in Western Australia (WA) and the NT, and a background level of serological activity to BTV serotypes 1 to 17. In addition, over 50 isolations of epizootic hemorrhagic disease of deer (EHD) viruses (EHDV) have been made and can be divided into 5 main serotypes, CSIRO157, CSIRO439, CSIRO753, CSIRO775 and DPP59. All 5 serotypes occur within the ecological range of Culicoides brevitarsis, however, as for BTV, the high prevalence of antibodies in cattle in the Kimberley region of WA and the top end of the NT suggests other vectors may also be responsible. CSIRO439 has been shown to be closely related serologically to Ibaraki virus while the other EHD viruses were distinct from Ibaraki and from each other. The complex serological relationships between BTV, EHDV and Palyam virus groups are currently being examined using serological and biochemical techniques. It is often difficult to determine the differences between BTV and EHDV serogroup members. Antibodies to both BTV and EHDV have been found in cattle, buffaloes and deer but there has been no evidence of clinical disease in any of these species or in sheep under field conditions in Australia.