Melbourne Veterinary School - Research Publications
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ItemNo Preview AvailableReverse transcriptase-polymerase chain reaction for the detection equine rhinitis B viruses and cell culture isolation of the virus(SPRINGER WIEN, 2007-01)Equine rhinitis B virus (ERBV), genus Erbovirus, family Picornaviridae occurs as two serotypes, ERBV1 and ERBV2. An ERBV-specific nested reverse transcriptase-polymerase chain reaction (RT-PCR) that amplified a product within the 3D(pol) and 3' non-translated region of the viral genome was developed. The RT-PCR detected all 24 available ERBV1 isolates and one available ERBV2 isolate. The limit of detection for the prototype strain ERBV1.1436/71 was 0.1 50% tissue culture infectious doses. The RT-PCR was used to detect viral RNA in six of 17 nasopharyngeal swab samples from horses that had clinical signs of acute febrile respiratory disease but from which ERBV was not initially isolated in cell culture. The sequences of these six ERBV RT-PCR positive samples had 93-96% nucleotide identity with six other partially sequenced ERBV1 isolates and one ERBV2. ERBV was isolated from one of the six samples at fourth cell culture passage when it was shown that the addition of 20 mg/mL MgCl(2) to the cell culture medium enhanced the growth of the virus. This isolated virus was antigenically similar to ERBV2.313/75. Determination of the nucleotide sequence of the P1 region of the genome also indicated that the isolate was ERBV2, and it was therefore designated ERBV2.1576/99. This is the first reported isolation of ERBV in Australia. The study highlights the utility of PCR for the identification of viruses in clinical samples that may initially be considered negative by conventional cell culture isolation.
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ItemLow genetic diversity among historical and contemporary clinical isolates of felid herpesvirus 1(BIOMED CENTRAL LTD, 2016-09-02)BACKGROUND: Felid herpesvirus 1 (FHV-1) causes upper respiratory tract diseases in cats worldwide, including nasal and ocular discharge, conjunctivitis and oral ulceration. The nature and severity of disease can vary between clinical cases. Genetic determinants of virulence are likely to contribute to differences in the in vivo phenotype of FHV-1 isolates, but to date there have been limited studies investigating FHV-1 genetic diversity. This study used next generation sequencing to compare the genomes of contemporary Australian clinical isolates of FHV-1, vaccine isolates and historical clinical isolates, including isolates that predated the introduction of live attenuated vaccines into Australia. Analysis of the genome sequences aimed to assess the level of genetic diversity, identify potential genetic markers that could influence the in vivo phenotype of the isolates and examine the sequences for evidence of recombination. RESULTS: The full genome sequences of 26 isolates of FHV-1 were determined, including two vaccine isolates and 24 clinical isolates that were collected over a period of approximately 40 years. Analysis of the genome sequences revealed a remarkably low level of diversity (0.0-0.01 %) between the isolates. No potential genetic determinants of virulence were identified, but unique single nucleotide polymorphisms (SNPs) in the UL28 and UL44 genes were detected in the vaccine isolates that were not present in the clinical isolates. No evidence of FHV-1 recombination was detected using multiple methods of recombination detection, even though many of the isolates originated from cats housed in a shelter environment where high infective pressures were likely to exist. Evidence of displacement of dominant FHV-1 isolates with other (genetically distinct) FHV-1 isolates over time was observed amongst the isolates obtained from the shelter-housed animals. CONCLUSIONS: The results show that FHV-1 genomes are highly conserved. The lack of recombination detected in the FHV-1 genomes suggests that the risk of attenuated vaccines recombining to generate virulent field viruses is lower than has been suggested for some other herpesviruses. The SNPs detected only in the vaccine isolates offer the potential to develop PCR-based methods of differentiating vaccine and clinical isolates of FHV-1 in order to facilitate future epidemiological studies.
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ItemNo Preview AvailableEvidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1(SOC GENERAL MICROBIOLOGY, 2016-03)Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.