Prolonged Detection of Bovine Viral Diarrhoea Virus Infection in the Semen of Bulls.

Infection of bulls with bovine viral diarrhoea virus (BVDV) can result in the development of virus persistence, confined to the reproductive tract. These bulls develop a normal immune response with high neutralizing antibody titres. However, BVDV can be excreted in the semen for a prolonged period. Although relatively rare, in this study we describe six separate cases in bulls being prepared for admission to artificial breeding centres. Semen samples were tested in a pan-Pestivirus-reactive real-time PCR assay and viral RNA was detected in semen from five of the bulls for three to eight months after infection. In one bull, virus was detected at low levels for more than five years. This bull was found to have one small testis. When slaughtered, virus was only detected in the abnormal testis. The low levels of BVDV in the semen of these bulls were only intermittently detected by virus isolation in cell culture. This virus-contaminated semen presents a biosecurity risk and confirms the need to screen all batches of semen from bulls that have been previously infected with BVDV. The use of real-time PCR is recommended as the preferred laboratory assay for this purpose.

Identification of a novel nidovirus as a potential cause of large scale mortalities in the endangered Bellinger River snapping turtle (Myuchelys georgesi).

In mid-February 2015, a large number of deaths were observed in the sole extant population of an endangered species of freshwater snapping turtle, Myuchelys georgesi, in a coastal river in New South Wales, Australia. Mortalities continued for approximately 7 weeks and affected mostly adult animals. More than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a very small proportion of the population had survived, severely threatening the viability of the wild population. At necropsy, animals were in poor body condition, had bilateral swollen eyelids and some animals had tan foci on the skin of the ventral thighs. Histological examination revealed peri-orbital, splenic and nephric inflammation and necrosis. A virus was isolated in cell culture from a range of tissues. Nucleic acid sequencing of the virus isolate has identified the entire genome and indicates that this is a novel nidovirus that has a low level of nucleotide similarity to recognised nidoviruses. Its closest relatives are nidoviruses that have recently been described in pythons and lizards, usually in association with respiratory disease. In contrast, in the affected turtles, the most significant pathological changes were in the kidneys. Real time PCR assays developed to detect this virus demonstrated very high virus loads in affected tissues. In situ hybridisation studies confirmed the presence of viral nucleic acid in tissues in association with pathological changes. Collectively these data suggest that this virus is the likely cause of the mortalities that now threaten the survival of this species. Bellinger River Virus is the name proposed for this new virus.

Rabbit Hemorrhagic Disease Virus 2 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 Months of Its Arrival.

Rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit hemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations.IMPORTANCE This study describes the spread and distribution of Rabbit hemorrhagic disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

Detection and Circulation of a Novel Rabbit Hemorrhagic Disease Virus in Australia.

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses.

Hendra Virus Infection in Dog, Australia, 2013.

Hendra virus occasionally causes severe disease in horses and humans. In Australia in 2013, infection was detected in a dog that had been in contact with an infected horse. Abnormalities and viral RNA were found in the dog's kidney, brain, lymph nodes, spleen, and liver. Dogs should be kept away from infected horses.

Longitudinal study of the detection of Bluetongue virus in bull semen and comparison of real-time polymerase chain reaction assays.

Infection with Bluetongue virus (BTV) is a significant impediment to the global movement of bovine semen. Repeat testing of blood from donor animals is specified in the World Organization for Animal Health (OIE) Manual for the export of semen from regions where BTV may be present. Screening of blood or semen samples has usually been carried out by virus isolation (VI) either by inoculation of chicken embryos followed by passage onto insect and mammalian cell cultures or in vivo inoculation of sheep followed by serology to detect seroconversion. Direct testing of semen for BTV would enable earlier release of semen samples and avoid repeat testing of the donor, as well as provide an option for releasing batches of semen that were collected without certification of the donor. Quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) assays overcome most of the limitations of other methods and have the potential to provide higher sensitivity. The present study compared 5 qRT-PCR assays, including 2 commercially available kits, for the detection of BTV in semen serially collected from 8 bulls over a period of 90 days after experimental infection. The results of the study show that at least one of the qRT-PCR assays is extremely reproducible and has both very high sensitivity and specificity to reliably detect all available serotypes. The preferred qRT-PCR gave consistently superior results to VI, sheep inoculation, and conventional RT-PCR. Therefore, the assay can be recommended for the screening of bovine semen for freedom from BTV.

Identification and characterisation of an ostreid herpesvirus-1 microvariant (OsHV-1 µ-var) in Crassostrea gigas (Pacific oysters) in Australia.

Between November 2010 and January 2011, triploid Crassostrea gigas (Pacific oysters) cultivated in the Georges River, New South Wales, experienced >95% mortality. Mortalities also occurred in wild diploid C. gigas in the Georges River and shortly thereafter in the adjacent Parramatta River estuary upstream from Sydney Harbour. Neighbouring Saccostrea glomerata (Sydney rock oysters) did not experience mortalities in either estuary. Surviving oysters were collected to investigate the cause of mortalities. Histologically all oysters displayed significant pathology, and molecular testing revealed a high prevalence of ostreid herpesvirus-1 (OsHV-1). Quantitative PCR indicated that many C. gigas were carrying a high viral load at the time of sampling, while the load in S. glomerata was significantly lower (p < 0.001). Subsequent in situ hybridisation experiments confirmed the presence of a herpesvirus in C. gigas but not S. glomerata tissues, suggesting that S. glomerata is not susceptible to infection with OsHV-1. Naïve sentinel triploid C. gigas placed in the Georges River estuary in January 2011 quickly became infected and experienced nearly 100% mortality within 2 wk of exposure, indicating the persistence of the virus in the environment. Phylogenetic analysis of sequences derived from the C2/C6 region of the virus revealed that the Australian strain of OsHV-1 belongs to the microvariant (µ-var) cluster, which has been associated with severe mortalities in C. gigas in other countries since 2008. Environmental data revealed that the Woolooware Bay outbreaks occurred during a time of considerable environmental disturbance, with increased water temperatures, heavy rainfall, a toxic phytoplankton bloom and the presence of a pathogenic Vibrio sp. all potentially contributing to oyster stress. This is the first confirmed report of OsHV-1 µ-var related C. gigas mortalities in Australia.

Characterization of virulent West Nile virus Kunjin strain, Australia, 2011.

To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNV(NSW2011), that is most closely related to WNV Kunjin (WNV(KUN)), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNV(NSW2011) is substantially more neuroinvasive than the prototype WNV(KUN) strain. In WNV(NSW2011), this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNV(KUN). Additional studies are needed to determine the relationship of the WNV(NSW2011) strain to currently and previously circulating WNV(KUN) strains and to confirm the cause of the increased virulence of this emerging WNV strain.