Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1.

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.

Accelerated vaccination schedule provides protective levels of antibody and complete herd immunity to equine influenza.

REASONS FOR PERFORMING STUDY: During the 2007 Australian equine influenza (EI) outbreak, an accelerated primary course 14 day intervaccination schedule was proposed, but not widely implemented. Expert opinion was divided as to the efficacy of such a schedule given the lack of published data. This study determined the level and duration of humoral immunity following administration of a recombinant canarypox-vectored vaccine (ALVAC-EIV) with a primary intervaccination interval of 14 days and booster at 105 days. OBJECTIVES: To examine whether protective levels of immunity of adequate duration were achieved following a primary course reduced from a minimum interval of 28 to 14 days. Antibody responses to 2 H3N8 American lineage virus strains (including A/equine/Sydney/6085/2007) were assessed and compared to previous challenge studies using ALVAC-EIV at conventional intervaccination intervals. METHODS: Fourteen Thoroughbred horses and 2 ponies from a rural racehorse training property in Victoria, Australia, were vaccinated with ALVAC-EIV on Days 0, 14 and 105. Serial blood samples were collected over the next 32 weeks and tested with haemagglutination inhibition and single radial haemolysis (SRH) in full assays to evaluate the serological response. RESULTS: All horses and ponies responded to the accelerated ALVAC-EIV vaccination schedule. Mean SRH antibodies remained above those consistent with clinical protection for the duration of the study period. All vaccinates demonstrated high SRH antibodies 14 days following V2, thereby achieving 100% herd immunity to homologous viral challenge. CONCLUSIONS: An accelerated vaccination schedule conferred long-lasting protective antibody levels despite a >50% reduction in the recommended V1-V2 interval. POTENTIAL RELEVANCE: High levels of rapidly acquired herd immunity are critical in containing an outbreak of such a highly contagious pathogen as EIV. In a strategic vaccination programme, it is important that horses remain protected for sufficient time to allow control programmes to succeed. An accelerated 14 day primary course intervaccination interval and booster at 105 days achieves both of these objectives.

Gammaherpesvirus infection in a free-ranging eastern grey kangaroo (Macropus giganteus).

A gammaherpesvirus was detected by polymerase chain reaction (PCR) in ocular, nasal and oropharyngeal swab samples collected from an adult free-ranging male eastern grey kangaroo (Macropus giganteus) with clinical signs of severe respiratory disease. This is the first time a gammaherpesvirus has been detected in a free-ranging macropod in Australia. The nucleotide sequence of a conserved region of the DNA polymerase gene of the detected virus showed a high degree of identity to a gammaherpesvirus recently detected in a zoological collection of eastern grey kangaroos in North America. The detection of this gammaherpesvirus in a free-ranging, native eastern grey kangaroo provides evidence that this species is a natural host.

Reverse transcriptase-polymerase chain reaction for the detection equine rhinitis B viruses and cell culture isolation of the virus.

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.

Prevalence of serum neutralising antibody to equine rhinitis A virus (ERAV), equine rhinitis B virus 1 (ERBV1) and ERBV2.

The objective of this study was to determine the incidence of serum neutralising (SN) antibody to ERAV, ERBV1 and ERBV2 in a population of horses from birth to 22 years of age. The prevalences of ERAV, ERBV1 and ERBV2 SN antibodies in 381 sera obtained from 291 horses were 37%, 83% and 66%, respectively. ERAV, ERBV1 and ERBV2 maternal antibody was present in foals 12 h postsuckling but by 10-12 months, ERAV SN antibody was not detected in any of the horses, while ERBV1 and ERBV2 SN antibodies were common (83% and 100%, respectively). Sera were obtained from 44 Thoroughbred horses when they were newly introduced into a training centre when their average age was 23 months and a second sample was obtained approximately 7 months later. ERAV SN antibody was present in 8 (18%) when first bled and in 27 (61%) when tested 7 months later. Accordingly 19 of the 44 horses (43%) seroconverted to ERAV within 7 months of entering the training stable. Among all the horses the average ERAV SN antibody titre was relatively high (3796) and in contrast, ERBV1 and ERBV2 titres were relatively low (average 84 and 45, respectively) and often fell to below detectable levels over time and at a rate comparable to new seroconversions in the same group of horses.

Glycoprotein G deletion mutants of equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus).

Glycoprotein G (gG) deletion mutants of EHV1 and EHV4, designated EHV1DeltagG and EHV4DeltagG, were constructed. The growth characteristics of the EHV1DeltagG mutants were similar to the parent virus. All of the EHV4DeltagG mutants grew more slowly in cell culture and produced plaques of different morphology including smaller size. The yields of both gG deletion mutant viruses in cell culture were similar to the parent viruses. Sequencing of the genes flanking gG, Southern blot, PCR and western blot analyses of the mutant viruses demonstrated that the deletions were as expected, except for EHV4DeltagG mutants, which in addition to deletion of gG contained unexpected deletions in the adjacent down stream gene ORF 71 (glycoprotein 2). Antisera to EHV1DeltagG and EHV4DeltagG neutralised the respective mutant and the parent viruses to the same titre and these antisera could be distinguished from antisera to the wild type viruses in a gG antibody detection ELISA. The mutant viruses may be useful as vaccine candidates and the deletion of gG may act as a marker to distinguish vaccinated from the naturally infected horses.