Kenneth A. McColl

Detection and characterisation of bluetongue virus using the polymerase chain reaction

Pairs of oligodeoxynucleotide primers whose sequences were based on those of RNA segment 3 that encodes the bluetongue virus serogroup-reactive protein VP3, were synthesized for three BTVs from different geographic regions of the world and for seven Australian orbiviruses. Each pair of primers was then tested for the synthesis of cDNA and in subsequent polymerase chain reactions (PCR) with all ten virus groups. All primers were serogroup-specific at low or high stringency. One pair of primers was specifically designed for its ability to serogroup a BTV isolate irrespective of its geographic origin. At either high or low stringency, this primer-pair resulted in a common and specific PCR product for each of the BTVs tested but not for the other orbiviruses. Eight pairs of primers based on RNA2 sequences (the gene segment encoding the serotype-specific protein VP2) were also synthesized for the eight Australian serotypes of BTV. Each primer-pair was serotype-specific at low or high stringency except for the BTV16A pair, which cross-reacted with BTV3A and also gave a non-specific product that differed in Mr from the authentic PCR product. Using the PCR and BTV1A RNA3-based primers, BTV1A was detected in blood samples from two sheep at 9 days post inoculation. Virus was found in the platelet, buffy-coat and packed red blood cell fractions, but not in whole blood.

Koi herpesvirus encodes and expresses a functional interleukin-10

ABSTRACT Koi herpesvirus (KHV) (species Cyprinid herpesvirus 3) ORF134 was shown to transcribe a spliced transcript encoding a 179-amino-acid (aa) interleukin-10 (IL-10) homolog (khvIL-10) in koi fin (KF-1) cells. Pairwise sequence alignment indicated that the expressed product shares 25% identity with carp IL-10, 22 to 24% identity with mammalian (including primate) IL-10s, and 19.1% identity with European eel herpesvirus IL-10 (ahvIL-10). In phylogenetic analyses, khvIL-10 fell in a divergent position from all host IL-10 sequences, indicating extensive structural divergence following capture from the host. In KHV-infected fish, khvIL-10 transcripts were observed to be highly expressed during the acute and reactivation phases but to be expressed at very low levels during low-temperature-induced persistence. Similarly, KHV early (helicase [Hel] and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes were also expressed at high levels during the acute and reactivation phases, but only low-level expression of the ITP gene was detected during the persistent phase. Injection of khvIL-10 mRNA into zebrafish (Danio rerio) embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10. Downregulation of the IL-10 receptor long chain (IL-10R1) using a specific morpholino abrogated the response to both khvIL-10 and zebrafish IL-10 transcripts, indicating that, despite the structural divergence, khvIL-10 functions via this receptor. This is the first report describing the characteristics of a functional viral IL-10 gene in the Alloherpesviridae.

Cloning and nucleotide sequence determination of the major envelope glycoprotein (gp.55) gene of hog cholera virus (Weybridge)

A 1.7 kb cDNA fragment corresponding to the coding region of the major envelope glycoprotein (gp55) of pestivirus hog cholera (Weybridge) was obtained using the polymerase chain reaction (PCR), and then cloned into pUC 8. The deduced amino acid sequence of gp55 showed a strong homology to that of HCV strains Brescia (94%) and Alfort (90%), and to a lesser extent to the closely related gp53 of bovine viral diarrhoea virus strain, NADL (65%). Eighteen cysteine residues were identified in the sequenced region, all of which were conserved between the gp55/gp53 sequences. This suggests that although the homology at the protein level may vary, there are strong conformational motifs which are conserved among the pestivirus envelope proteins.

Retrospective diagnosis of bluetongue virus in stored frozen and fixed tissue samples using PCR

Stored frozen (-70 degrees C) and formalin-fixed tissue samples constitute a valuable resource for retrospective studies of infectious diseases, or for diagnostic investigations. The polymerase chain reaction (PCR) affords an accurate and rapid method for detection of viral nucleic acids. It was applied to stored tissue samples collected from sheep inoculated with two Australian serotypes of bluetongue virus, BTV 1 and 23, and two North American serotypes, BTV 11 and 17. Specific nested PCR products were detected in both frozen and formalin-fixed samples from the Australian sheep after storage for 3.5 years. The tissues from sheep inoculated with the North American serotypes yielded specific nested PCR products after storage at -70 degrees C for 14 years. No specific primary PCR products were detected in any frozen or formalin-fixed samples. The PCR assay offers a potential benefit for epidemiological studies, and for screening of stored semen, embryos and tissue banks.

Characteristics of cyprinid herpesvirus 3 in different phases of infection: implications for disease transmission and control

Koi herpesvirus disease (KHVD) is an emerging and highly contagious viral disease of koi and common carp (Cyprinus carpio), causing mass mortalities and huge economic losses to the carp aquaculture industry. The disease has spread rapidly to 28 countries worldwide. However, mechanisms of koi herpesvirus (species Cyprinid herpesvirus 3; CyHV-3) transmission remain unclear. A potential experimental model of CyHV-3 infection in carp was used to characterise CyHV-3 in different phases of infection and to demonstrate that CyHV-3 persists in survivor fish and has the capacity to reactivate and transmit the disease to healthy fish. During acute infection, which occurred when fish were maintained at 22°C, viral genes were abundantly expressed and infectious virus was produced in association with tissue damage, clinical disease and mortality. In fish maintained at a lower temperature (11°C), viral DNA was present but viral gene expression was absent or greatly restricted, infectious virus was not recovered and there was no evidence of disease. Productive replication was re-initiated following an increase in water temperature to 22°C, resulting in 45% mortality. Shedding of reactivated virus killed 75% of cohabitating naïve fish, suggesting a potential risk for disease transmission.

EVALUATION OF ABALONE VIRAL GANGLIONEURITIS RESISTANCE AMONG WILD ABALONE POPULATIONS ALONG THE VICTORIAN COAST OF AUSTRALIA

ABSTRACT Between May 2006 and February 2010, abalone viral ganglioneuritis caused by abalone herpes virus (AbHV) spread along the coast of Victoria, Australia, and devastated wild abalone populations, causing high mortality (up to 90% in some areas). However, some abalone from the affected populations survived the epizootic and thus may be naturally resistant to the disease. To test this hypothesis, abalone (Haliotis rubra) from 5 reefs within the geographical range for AVG were collected and tested for resistance to infection and disease. Thus, mature survivors (abalone ∼160 mm in length) and juvenile “new recruits” (abalone ∼70 mm in length) were exposed to the virus using an experimental infectivity model to determine the presence of any potential resistance to the virus. Exposure to AbHV was performed by immersion using 3 serial viral dilutions to ensure that abalone were exposed to at least 1 viral concentration that would provide a morbidity dose of intermediate level. Results indicated that morbidity curves for the wild abalone groups (both mature and juvenile) were similar to those of the susceptible, naive, farmed hybrid (Haliotis laevigata × Haliotis rubra) abalone (positive control) groups. Histological lesions typical of abalone viral ganglioneuritis, and viral DNA, were detected in moribund, challenged abalone, confirming AbHV as the causative disease agent. Results suggest that the surviving wild abalone are not resistant to AbHV and were probably not exposed to pathogenic doses of the virus during the initial outbreak that commenced in 2006.

Abalone viral ganglioneuritis: Establishment and use of an experimental immersion challenge system for the study of abalone herpes virus infections in Australian abalone

In late 2005, acute mortalities occurred in abalone on farms located in Victoria, Australia. Disease was associated with infection by an abalone herpes virus (AbHV). Subsequently, starting in 2006, the disease (abalone viral ganglioneuritis; AVG) was discovered in wild abalone in Victorian open waters. Currently, it continues to spread, albeit at a slow rate, along the Victorian coast-line. Here, we report on experimental transmission trials that were carried out by immersion using water into which diseased abalone had shed infectious viral particles. At various time points following exposure, naïve abalone were assessed by an AbHV-specific real-time PCR and histological analyses including in situ hybridization (ISH). Results demonstrated that while exposed abalone began displaying clinical signs of the disease from 60 hours post exposure (hpe), they tested positive for the presence of viral DNA at 36 hpe. Of further interest, the AbHV DNA probe used in the ISH assay detected the virus as early as 48 hpe.

Australian abalone (Haliotis laevigata, H. rubra and H. conicopora) are susceptible to infection by multiple abalone herpesvirus genotypes

From 2006 to 2012, acute mortalities occurred in farmed and wild abalone (Haliotis spp.) along the coast of Victoria, Australia. The disease (abalone viral ganglioneuritis; AVG) is associated with infection by an abalone herpesvirus (AbHV). The relative pathogenicity of 5 known variants of AbHV was evaluated on abalone stocks from different states in Australia. Results indicated that all virus variants (Vic1, Tas1, Tas2, Tas3 and Tas4) cause disease and mortality in all abalone stocks tested (greenlip, blacklip and brownlip). In order to avoid further AVG outbreaks in Australian wild abalone, strict regulations on the transfer of abalone stocks must be implemented.

Molecular characterisation of Australasian isolates of aquatic birnaviruses.

An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 1998, has continued to be re-isolated on an infrequent but regular basis. Due to its low pathogenicity, there has been little urgency to undertake a comprehensive characterisation of this aquatic birnavirus. However, faced with possible incursions of any new aquatic birnaviruses, specific identification and differentiation of this virus from other, pathogenic, aquatic birnaviruses such as infectious pancreatic necrosis virus (IPNV) are becoming increasingly important. The present study determined the nucleic acid sequence of the aquatic birnavirus originally isolated in 1998, as well as a subsequent isolate from 2002. The sequences of the VP2 and VP5 genes were compared to that of other aquatic birnaviruses, including non-pathogenic aquatic birnavirus isolates from New Zealand and pathogenic infectious pancreatic necrosis virus isolates from North America and Europe. The deduced amino acid (aa) sequences indicate that the Australian and New Zealand isolates fall within Genogroup 5 together with IPNV strains Sp, DPL, Fr10 and N1. Thus, Genogroup 5 appears to contain aquatic birnavirus isolates from quite diverse host and geographical ranges. Using the sequence information derived from this study, a simple diagnostic test has been developed that differentiates the current Australian isolates from all other aquatic birnaviruses, including the closely related isolates from New Zealand.

Transcriptomic analysis of common carp anterior kidney during Cyprinid herpesvirus 3 infection: Immunoglobulin repertoire and homologue functional divergence

Cyprinid herpesvirus 3 (CyHV-3) infects koi and common carp and causes widespread mortalities. While the virus is a significant concern for aquaculture operations in many countries, in Australia the virus may be a useful biocontrol agent for pest carp. However, carp immune responses to CyHV-3, and the molecular mechanisms underpinning resistance, are not well understood. Here we used RNA-Seq on carp during different phases of CyHV-3 infection to detect the gene expression dynamics of both host and virus simultaneously. During acute CyHV-3 infection, the carp host modified the expression of genes involved in various immune systems and detoxification pathways. Moreover, the activated pathways were skewed toward humoral immune responses, which may have been influenced by the virus itself. Many immune-related genes were duplicated in the carp genome, and often these were expressed differently across the infection phases. Of particular interest were two interleukin-10 homologues that were not expressed synchronously, suggesting neo- or sub-functionalization. The carp immunoglobulin repertoire significantly diversified during active CyHV-3 infection, which was followed by the selection of high-affinity B-cells. This is indicative of a developing adaptive immune response, and is the first attempt to use RNA-Seq to understand this process in fish during a viral infection.