Revisiting the Importance of Orthobunyaviruses for Animal Health: A Scoping Review of Livestock Disease, Diagnostic Tests, and Surveillance Strategies for the Simbu Serogroup.

Orthobunyaviruses (order Bunyavirales, family Peribunyaviridae) in the Simbu serogroup have been responsible for widespread epidemics of congenital disease in ruminants. Australia has a national program to monitor arboviruses of veterinary importance. While monitoring for Akabane virus, a novel orthobunyavirus was detected. To inform the priority that should be given to this detection, a scoping review was undertaken to (1) characterise the associated disease presentations and establish which of the Simbu group viruses are of veterinary importance; (2) examine the diagnostic assays that have undergone development and validation for this group of viruses; and (3) describe the methods used to monitor the distribution of these viruses. Two search strategies identified 224 peer-reviewed publications for 33 viruses in the serogroup. Viruses in this group may cause severe animal health impacts, but only those phylogenetically arranged in clade B are associated with animal disease. Six viruses (Akabane, Schmallenberg, Aino, Shuni, Peaton, and Shamonda) were associated with congenital malformations, neurological signs, and reproductive disease. Diagnostic test interpretation is complicated by cross-reactivity, the timing of foetal immunocompetence, and sample type. Serological testing in surveys remains a mainstay of the methods used to monitor the distribution of SGVs. Given significant differences in survey designs, only broad mean seroprevalence estimates could be provided. Further research is required to determine the disease risk posed by novel orthobunyaviruses and how they could challenge current diagnostic and surveillance capabilities.

Immune Priming of Pacific Oysters (Crassostrea gigas) to Induce Resistance to Ostreid herpesvirus 1: Comparison of Infectious and Inactivated OsHV-1 with Poly I:C.

Pacific oyster mortality syndrome (POMS), which is caused by Ostreid herpesvirus 1 (OsHV-1), causes economic losses in Pacific oyster (Crassostrea gigas) aquaculture in many countries. Reducing the mortality in disease outbreaks requires changing the host, pathogen and environment interactions to favor the host. Survivors of natural exposure to OsHV-1 are able to survive subsequent outbreaks. This has been replicated under laboratory conditions, suggesting the existence of an immune response. The aim of the present study is to compare the effects of prior exposure to infectious OsHV-1, heat-inactivated OsHV-1 and the chemical anti-viral immune stimulant poly I:C on mortality following exposure to virulent OsHV-1. All treatments were administered by intramuscular injection. Oysters were maintained at 18 °C for 14 days; then, the temperature was increased to 22 °C and the oysters were challenged with virulent OsHV-1. Heat-inactivated OsHV-1, infectious OsHV-1 and poly I:C all induced significant protection against mortality, with the hazard of death being 0.41, 0.18 and 0.02, respectively, compared to the controls, which had no immune priming. The replication of OsHV-1 on first exposure was not required to induce a protective response. While the underlying mechanisms for protection remain to be elucidated, conditioning for resistance to POMS by prior exposure to inactivated or infectious OsHV-1 may have practical applications in oyster farming but requires further development to optimize the dose and delivery mechanism and evaluate the duration of protection.

The Resistance to Lethal Challenge with Ostreid herpesvirus-1 of Pacific Oysters (Crassostrea gigas) Previously Exposed to This Virus.

Pacific oyster (Crassostrea gigas) aquaculture has been economically impacted in many countries by Pacific oyster mortality syndrome (POMS), a disease initiated by Ostreid herpesvirus 1. The objectives of this study were to determine whether naturally exposed, adult C. gigas could act as reservoirs for OsHV-1 and explain the recurrent seasonal outbreaks of POMS and to test whether or not they were resistant to OsHV-1. In a laboratory infection experiment using thermal shock, OsHV-1 replication was not reactivated within the tissues of such oysters and the virus was not transmitted to naïve cohabitating spat. The adult oysters were resistant to intramuscular injection with a lethal dose of OsHV-1 and had 118 times lower risk of mortality than naïve oysters. Considered together with the results of other studies in C. gigas, natural exposure or laboratory exposure to OsHV-1 may result in immunity during subsequent exposure events, either in the natural environment or the laboratory. While adult C. gigas can carry OsHV-1 infection for lengthy periods, reactivation of viral replication leading to mortality and transmission of the virus to naïve oysters may require specific conditions that were not present in the current experiment. Further investigation is required to evaluate the mechanisms responsible for resistance to disease in oysters previously exposed to OsHV-1, whether immunity can be exploited commercially to prevent POMS outbreaks and to determine the source of the virus for recurrent seasonal outbreaks.

Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus.

Megalocytiviruses (MCVs) are double-stranded DNA viruses known to infect important freshwater and marine fish species in the aquaculture, food, and ornamental fish industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV) is the type species within the genus Megalocytivirus that causes red seabream iridoviral disease (RSIVD) which is a reportable disease to the World Animal Health Organization (WOAH). To better control the transboundary spread of this virus and support WOAH reporting requirements, we developed and partially validated a TaqMan real-time qPCR assay (ISKNV104R) to detect all three genotypes of ISKNV, including the two genotypes that cause RSIVD. Parameters averaged across 48 experiments used a 10-fold dilution series of linearized plasmid DNA (107-101 copies), carrying a fragment of the three-spot gourami iridovirus (TSGIV) hypothetical protein revealed that the assay was linear over 7 orders of magnitude (107-101), a mean efficiency of 99.97 ± 2.92%, a mean correlation coefficient of 1.000 ± 0.001, and a limit of detection (analytical sensitivity) of ≤10 copies of TSGIV DNA. The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was evaluated and compared to other published assays using a panel of 397 samples from 21 source populations with different prevalence of ISKNV infection (0-100%). The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was 91.99% (87.28-95.6; 95% CI) and 89.8% (83.53-94.84). The latent class analysis showed that the ISKNV104R qPCR assay had similar diagnostic sensitivities and specificities with overlapping confidence limits compared to a second TaqMan qPCR assay and a SYBR green assay. This newly developed TaqMan assay represents a partially validated qPCR assay for the detection of the three genotypes of the species ISKNV. The ISKNV104R qPCR assay once fully validated, will serve as an improved diagnostic tool that can be used for ISKNV surveillance efforts and diagnosis in subclinical fish to prevent further spread of MCVs throughout the aquaculture and ornamental fish industries.

Reduction in Virulence over Time in Ostreid herpesvirus 1 (OsHV-1) Microvariants between 2011 and 2015 in Australia.

Microvariant genotypes of Ostreid herpesvirus 1 (OsHV-1) are associated with mass mortality events of Pacific oysters in many countries. The OsHV-1 microvariant (µVar) emerged in France 2008 and caused significant economic losses as it became endemic and displaced the previously dominant OsHV-1 reference genotype. Recently, considerable genotypic variation has been described for OsHV-1 microvariants, however, less is known about variation in viral phenotype. This study used an in vivo laboratory infection model to assess differences in total cumulative mortality, peak viral load, transmissibility, and dose-response for three OsHV-1 isolates obtained between 2011 and 2015 from endemic waterways in Australia. This followed field observations of apparent reductions in the severity of mass mortalities over this time. Significantly higher hazard of death and cumulative mortality were observed for an isolate obtained in 2011 compared to isolates from 2014-2015. In keeping with other studies, the hazard of death was higher in oysters challenged by injection compared to challenge by cohabitation and the mortality was higher when the initial dose was 1 × 104 OsHV-1 DNA copies per oyster injection compared to 1 × 102 DNA copies. There was no difference in the quantity of OsHV-1 DNA at time of death that could be related to isolate or dose, suggesting similar pathogenetic processes in the individual oysters that succumbed to end-stage disease. While the isolates examined in this study were biased towards pathogenic types of OsHV-1, as they were collected during disease outbreaks, the variation in virulence that was observed, when combined with prior data on subclinical infections, suggests that surveillance for low virulence genotypes of OsHV-1 would be rewarding. This may lead to new approaches to disease management which utilize controlled exposure to attenuated strains of OsHV-1.

Prevalence of Infectious Spleen and Kidney Necrosis Virus (ISKNV), Nervous Necrosis Virus (NNV) and Ectoparasites in Juvenile Epinephelus spp. Farmed in Aceh, Indonesia.

A cross-sectional survey was used to estimate the prevalence of infections with the Infectious spleen and kidney necrosis virus (ISKNV, Megalocytivirus), nervous necrosis virus (NNV, Betanodavirus), and infestations with ectoparasites during the rainy season in juvenile grouper (Epinephelus spp.) farmed in Aceh, Indonesia. The survey was intended to detect aquatic pathogens present at 10% prevalence with 95% confidence, assuming 100% sensitivity and specificity using a sample size of 30 for each diagnostic test. Eight populations of grouper from seven farms were sampled. Additional targeted sampling was conducted for populations experiencing high mortality. Infection with NNV was detected at all farms with seven of the eight populations being positive. The apparent prevalence for NNV ranged from 0% (95% CI: 0-12) to 73% (95% CI: 54-88). All of the fish tested from the targeted samples (Populations 9 and 10) were positive for NNV and all had vacuolation of the brain and retina consistent with viral nervous necrosis (VNN). Coinfections with ISKNV were detected in five populations, with the highest apparent prevalence being 13% (95% CI: 4-31%). Trichodina sp., Cryptocaryon irritans and Gyrodactylus sp. were detected at three farms, with 66% to 100% of fish being infested. Hybrid grouper sourced from a hatchery were 5.4 and 24.9 times more likely to have a NNV infection and a higher parasite load compared to orange-spotted grouper collected from the wild (p < 0.001). This study found that VNN remains a high-impact disease in grouper nurseries in Aceh, Indonesia.

Different in vivo growth of ostreid herpesvirus 1 at 18 °C and 22 °C alters mortality of Pacific oysters (Crassostrea gigas).

Seasonally recurrent outbreaks of mass mortality in Pacific oysters (Crassostrea gigas) caused by microvariant genotypes of ostreid herpesvirus 1 (OsHV-1) occur in Europe, New Zealand and Australia. The incubation period for OsHV-1 under experimental conditions is 48-72 hours and depends on water temperature, as does the mortality. An in vivo growth curve for OsHV-1 was determined by quantifying OsHV-1 DNA at 10 time points between 2 and 72 hours after exposure to OsHV-1. The peak replication rate was the same at 18 °C and 22 °C; however, there was a longer period of amplification leading to a higher peak concentration at 22 °C (2.34 × 107 copies/mg at 18 hours) compared to 18 °C (1.38 × 105 copies/mg at 12 hours). The peak viral concentration preceded mortality by 72 hours and 20 hours at 18 °C and 22 °C, respectively. Cumulative mortality to day 14 was 45.9% at 22 °C compared to 0.3% at 18 °C. The prevalence of OsHV-1 infection after 14 days at 18 °C was 33.3%. No mortality from OsHV-1 occurred when the water temperature in tanks of oysters challenged at 18 °C was increased to 22 °C for 14 days. The influence of water temperature prior to exposure to OsHV-1 and during the initial virus replication is an important determinant of the outcome of infection in C. gigas.

The impact of pooling samples on surveillance sensitivity for the megalocytivirus Infectious spleen and kidney necrosis virus.

Movements of large volumes and species varieties make the ornamental fish industry a high-risk pathway for the transfer of aquatic pathogens to new geographical regions and naïve hosts, potentially resulting in emergency disease events. Infectious spleen and kidney necrosis virus (genus Megalocytivirus) is considered exotic to Australia despite documented incursions since 2003. There are current import controls requiring freedom from infection for entry to Australia. The objective was to evaluate the effect of tissue pooling strategies for qPCR testing using a SYBR® assay for freedom from ISKNV at 2% expected prevalence with 95% confidence. Tissue homogenates from apparently healthy imported ornamental fish were tested as individuals and in pools of 5 and 10. Analytical sensitivity of the qPCR assay was reduced by two orders of magnitude when the nucleic acid extraction process was accounted for by spiking the plasmid in fish tissues and compared with molecular grade water. Diagnostic sensitivity of the assay was substantially reduced when testing tissues in pools compared with individual testing. For Population 1 (66% positive for ISKNV with moderate viral loads), surveillance sensitivity was only achieved using individual testing. For Population 2 (100% positive ISKNV with high viral loads), surveillance sensitivity was achieved using 260 fish in pools of 10 for a total of 26 tests or 200 fish in pools of 5 for 40 tests. Surveillance sensitivity could be maximized even when there was a reduction in pooled diagnostic sensitivity compared with diagnostic sensitivity for individual fish by increasing the sample size. Pooled sensitivity was influenced by the prevalence and variable virus load among fish with subclinical infections. Pooled testing is highly effective when the prevalence is >10% which should be informed by prior knowledge or pooling can be used for a screening test to rapidly identify populations with high prevalence.

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses.

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

Molecular epidemiology of Epizootic haematopoietic necrosis virus (EHNV).

Low genetic diversity of Epizootic haematopoietic necrosis virus (EHNV) was determined for the complete genome of 16 isolates spanning the natural range of hosts, geography and time since the first outbreaks of disease. Genomes ranged from 125,591-127,487 nucleotides with 97.47% pairwise identity and 106-109 genes. All isolates shared 101 core genes with 121 potential genes predicted within the pan-genome of this collection. There was high conservation within 90,181 nucleotides of the core genes with isolates separated by average genetic distance of 3.43 × 10-4 substitutions per site. Evolutionary analysis of the core genome strongly supported historical epidemiological evidence of iatrogenic spread of EHNV to naïve hosts and establishment of endemic status in discrete ecological niches. There was no evidence of structural genome reorganization, however, the complement of non-core genes and variation in repeat elements enabled fine scale molecular epidemiological investigation of this unpredictable pathogen of fish.

Complete Genome Sequence of a Bohle iridovirus Isolate from Ornate Burrowing Frogs (Limnodynastes ornatus) in Australia.

Bohle iridovirus (BIV) is a species within the genus Ranavirus, family Iridoviridae, first isolated from the ornate burrowing frog Limnodynastes ornatus in Australia. The BIV genome confirms it is closely related to isolates from boreal toad Anaxyrus boreas and leaf-tailed gecko Uroplatus fimbriatus within the United States and Germany, respectively.

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.