Jacquelyn Horsington

Evaluation of cross-protection between O1 Manisa and O1 Campos in cattle vaccinated with foot-and-mouth disease virus vaccine incorporating different payloads of inactivated O1 Manisa antigen.

Serology is used to predict vaccine induced protection against challenge with a heterologous strain of the same serotype of foot-and-mouth disease virus (FMDV). To evaluate the accuracy of such predictions, we compared the protection afforded to cattle vaccinated with the O(1) Manisa strain of FMDV against challenge with either a homologous (O(1) Manisa) or a heterologous strain (O(1) Campos). Serology by virus neutralization test (VNT) using O(1) Manisa antiserum predicted an acceptable protection against such a challenge. Two experiments were carried out to compare the results for consistency. A total of 78 naïve cattle were vaccinated with different antigen payloads (60-0.94 μg) of O(1) Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O(1) Manisa or O(1) Campos. Unvaccinated naïve control cattle (n=20) were also challenged with either the O(1) Manisa or O(1) Campos viruses and all developed generalized FMD. The protection results for the vaccinated cattle revealed that higher payloads of O(1) Manisa vaccine were needed to protect against heterologous challenge compared to that for homologous challenge. The 50% protective dose (PD(50)) values for the vaccine in experiments 1 and 2 were found to be 28.78 and 9.44 for the homologous challenge and 3.98 and 5.01 for heterologous challenge. Furthermore, protection against O(1) Campos required a higher level of vaccine-induced antibody against this virus compared to the level of O(1) Manisa neutralizing antibody associated with protection against homologous challenge. The 50% protective level of in vitro neutralizing antibody was found to be log(10)1.827 for O(1) Campos and log(10)0.954 for O(1) Manisa based on O(1) Manisa based virus neutralization test.

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.

Early protection in sheep against intratypic heterologous challenge with serotype O foot-and-mouth disease virus using high-potency, emergency vaccine.

In 2009-2011, spread of a serotype O foot-and-mouth disease virus (FMDV) belonging to the South East Asia topotype led to the culling of over 3.5 million cattle and pigs in Japan and Korea. The O1 Manisa vaccine (belonging to the Middle East-South Asian topotype) was used at high potency in Korea to limit the expansion of the outbreak. However, no data are available on the spread of this virus or the efficacy of the O1 Manisa vaccine against this virus in sheep. In this study, the early protection afforded with a high potency (>6 PD50) FMD O1 Manisa vaccine against challenge with the O/SKR/2010 virus was tested in sheep. Sheep (n=8) were vaccinated 4 days prior to continuous direct-contact challenge with donor sheep. Donor sheep were infected with FMDV O/SKR/2010 by coronary band inoculation 24h prior to contact with the vaccinated animals, or unvaccinated controls (n=4). Three of the four control sheep became infected, two clinically. All eight O1 Manisa vaccinated sheep were protected from clinical disease. None had detectable antibodies to FMDV non-structural proteins (3ABC), no virus was isolated from nasal swabs, saliva or oro-pharyngeal fluid and none became carriers. Using this model of challenge, sheep were protected against infection as early as 4 days post vaccination.

Identification of mixed equine rhinitis B virus infections leading to further insight on the relationship between genotype, serotype and acid stability phenotype

Equine rhinitis B virus (ERBV) is the single species in the genus Erbovirus, family Picornaviridae. Equine rhinitis B viruses exist in three serotypes and are associated with respiratory disease in horses. Members of the species vary in stability at acid pH. To date there has been discordance in genotype, serotype and acid stability phenotype groupings. To identify capsid regions associated with acid stability, two viruses were serially treated at pH 3.3 to isolate acid-stable mutants. An acid-stable mutant of the prototype acid-labile serotype 1 virus contained a single amino acid change in the C-terminus of VP1. Similar treatment with a separate isolate identified a multiple ERBV serotype infection with acid-labile serotype 1 and acid-stable serotype 3. Dual infections were subsequently identified in original swabs taken from the infected horse and from two further cell culture passaged viruses originally isolated in Switzerland. Serotype specific rat antisera were produced and used to examine a collection of isolates from a range of genotypes, acid stability phenotypes and serotypes. In contrast to previous reports, we showed viruses previously classified as acid-stable serotype 1 are in fact serotype 3 and that there is a clear association of serotype with genotype and acid stability phenotype in ERBVs. Additionally, we have shown that ERBV capsids dissociate into pentamers in acidic conditions below their threshold of stability, similarly to closely related viruses in the same family.

Equine picornaviruses: Well known but poorly understood

Of the many members that comprise the family Picornaviridae, only two species are known to infect horses: equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV). Each species now occupies a distinct phylogenetic branch within the family, with the single serotype of ERAV grouping with the aphthoviruses, such as foot-and-mouth disease virus (FMDV), and the three serotypes of ERBV as the sole members of the genus Erbovirus. The high seroprevalence of equine picornaviruses in horse populations worldwide contrasts with the relatively few reports of detection of these viruses and poor understanding of their contribution to disease. This review examines the current knowledge regarding the distribution and pathogenesis of these viruses and discusses recent advances in diagnostic methods that may lead to a better understanding of the role of these viruses as contributors to equine respiratory disease.

Low genetic diversity among historical and contemporary clinical isolates of felid herpesvirus 1

BackgroundFelid 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.ResultsThe 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 40xa0years. Analysis of the genome sequences revealed a remarkably low level of diversity (0.0–0.01xa0%) 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.ConclusionsThe 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.

Mapping B-cell epitopes in equine rhinitis B viruses and identification of a neutralising site in the VP1 C-terminus

Erbovirus is a genus of the family Picornaviridae and equine rhinitis B virus (ERBV) is the sole species. Erboviruses infect horses causing acute respiratory disease and sub-clinical and persistent infections. Despite the high seroprevalence and worldwide distribution of these viruses, the pathogenesis and antigenic structure of the three ERBV serotypes (ERBV1, 2 and 3) is poorly understood. To characterise linear epitopes on ERBV structural proteins, a set of fusion proteins were expressed in Escherichia coli. These proteins were tested in Western blot and ELISA and reactive proteins were also used to identify neutralisation epitopes. VP1 contained serotype specific epitopes whereas VP2 was highly cross-reactive across the serotypes. The C-terminus of VP1 accounted for most of the reactivity of full-length VP1 and was also the location of a neutralising site in each serotype.

Foot-and-mouth disease virus infection in fetal lambs: Tissue tropism and cytokine response

Foot-and-mouth disease virus (FMDV) can cause transplacental infection and death in fetal lambs. This study investigates the pathogenesis of FMDV infection in ovine fetuses using in-situ hybridization (ISH) to detect viral transcripts in tissue and real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays to quantify the fetal cytokine response to infection. FMDV ribonucleic acid (RNA) was localized mainly to the heart and skeletal muscles of fetuses and was only occasionally expressed in the lingual epithelium, demonstrating that FMDV has a different tissue tropism in the fetus compared with that in adult sheep. There was early expression of genes encoding anti-viral cytokines (IFN-alpha and IFN-beta) in fetuses at 2 and 4 days post-infection (dpi), followed by a marked rise in the transcription of pro-inflammatory cytokine genes (IFN-gamma, TNF-alpha and IL-1alpha) from 7 to 18 dpi, particularly in the heart. The degree of cytokine mRNA expression correlated with fetal infection and was likely to be a factor in fetal death. In contrast, cytokine gene expression in infected neonatal lambs was much less and mainly occurred between 2 and 4 dpi. This study identifies two key factors in the pathogenicity of FMDV in fetal lambs: viral tropism for cardiac and skeletal muscles, and a marked cytokine response following infection.

Seroprevalence study of Equine rhinitis B virus (ERBV) in Australian weanling horses using serotype-specific ERBV enzyme-linked immunosorbent assays

Respiratory infections are a major burden in the performance horse industry. Equine rhinitis B virus (ERBV) has been isolated from horses displaying clinical respiratory disease, and ERBV-neutralizing antibodies have been detected in 50–80% of horses in reported surveys. Current ERBV isolation and detection methods may underestimate the number of ERBV-positive animals and do not identify multiple serotype infections. The aim of the current study was to develop a serotyping ERBV antibody-detection enzyme-linked immunosorbent assay (ELISA) and examine the seroprevalence of ERBV in a group of Australian weanling horses. ELISAs with high sensitivity and specificity were developed. The seroprevalence of ERBV in the weanling horses was high (74–86%); ERBV-3 antibodies were most prevalent (58–62%) and ERBV-2 antibodies were least prevalent (10–16%). Many horses were seropositive to 2 or more serotypes. All 3 serotypes of ERBV were detected, and concurrent positivity to multiple serotypes was common.

Generation of mAbs to foot–and–mouth disease virus serotype A and application in a competitive ELISA for serodiagnosis

BackgroundFoot–and–mouth disease (FMD) is an economically devastating disease that severely limits international trade of animals. Of the seven FMD virus (FMDV) serotypes, serotype A is one of the most widespread cross the world. Currently antibodies to FMDV are detected in animals using the virus neutralization test (VNT) and the enzyme-linked immunosorbent assay (ELISA). The VNT is laborious, time–consuming and reliant on live virus and cell cultures, while ELISA has the advantage of using inactivated antigens and often provides more reproducible results. The aim of this study was to develop a reliable and rapid competitive ELISA (cELISA) for the detection of antibodies to FMDV serotype A (FMDV/A).ResultsA panel of FMDV/A specific monoclonal antibodies (mAbs) was generated and their ability to compete with a polyclonal serum from FMDV/A–infected cattle was examined. Two mAbs inhibited the binding of a polyclonal serum to FMDV/A viruses. The binding epitopes of each were determined as conformational and located on the VP2 viral capsid protein. The FMDV/A cELISA was developed using these two mAbs and FMDV/A inactivated virus as antigen. The diagnostic specificity and sensitivity were 99.7 and 99.3% (98.5–100%) respectively, based on a predetermined cut–off of 50% inhibition. When analysing sera from animals experimentally infected with FMDV/A, the cELISA detected antibodies from 5-days post infection (dpi) and remained positive for at least 21–28 days post infection. Comparison based on the Kappa coefficient showed strong agreement (90–94%) between cELISA and VNT.ConclusionThe cELISA results are comparable to the VNT for antibody detection making it a simple and reliable test to detect antibodies against FMDV/A.