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

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.

Genome sequences of equid herpesviruses 2 and 5.

We resequenced the genome of equid herpesvirus 2 (EHV2) strain 86/67 and sequenced the genomes of EHV2 strain G9/92 and equid herpesvirus 5 (EHV5) strain 2-141/67. The most prominent genetic differences are the dissimilar locations of the interleukin-10 (IL-10)-like genes and the presence of an OX-2-like gene in EHV5 only.

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.

Virion associated proteins of equine rhinitis B virus 1 (ERBV1): the non-structural protein 3C(pro) co-purifies with virions.

Equine rhinitis B virus (ERBV), genus Erbovirus, is most closely related to the Cardiovirus genus in the family Picornaviridae. The structural proteins (VP1-4) of erboviruses are not well described, but are predicted by sequence to be 35, 29, 26 and 7 kDa. Methods for the purification of cardioviruses (polyethylene glycol, trypsin treatment) were used to characterise the structural proteins of ERBV1. Only one of the virus proteins detected was an expected molecular mass, and this 26 kDa protein was identified as VP3 by N-terminal amino acid sequencing. N-terminal sequencing of the 56 and a 29 kDa protein identified sequences consistent with VP2 and VP1 respectively, despite these being 27 kDa larger and 6 kDa smaller than predicted. Virus purified without trypsin showed proteins more consistent with masses predicted for VP1, VP2 and VP3 at 35, 29 and 26 kDa respectively. These proteins were further identified with antibodies affinity purified to recombinant VP1, VP2, VP3 produced in E. coli. Interestingly, antibodies affinity purified to the non-structural protein 3C(pro), produced in insect cells, strongly detected a 27 kDa protein in western blots of virus purified with and without trypsin treatment, suggesting the non-structural 27 kDa 3C(pro) co-purifies with ERBV1 virions.

The order Herpesvirales.

The taxonomy of herpesviruses has been updated by the International Committee on Taxonomy of Viruses (ICTV). The former family Herpesviridae has been split into three families, which have been incorporated into the new order Herpesvirales. The revised family Herpesviridae retains the mammal, bird and reptile viruses, the new family Alloherpesviridae incorporates the fish and frog viruses, and the new family Malacoherpesviridae contains a bivalve virus. Three new genera have been created in the family Herpesviridae, namely Proboscivirus in the subfamily Betaherpesvirinae and Macavirus and Percavirus in the subfamily Gammaherpesvirinae. These genera have been formed by the transfer of species from established genera and the erection of new species, and other new species have been added to some of the established genera. In addition, the names of some nonhuman primate virus species have been changed. The family Alloherpesviridae has been populated by transfer of the genus Ictalurivirus and addition of the new species Cyprinid herpesvirus 3. The family Malacoherpesviridae incorporates the new genus Ostreavirus containing the new species Ostreid herpesvirus 1.

Sequence and function of canine herpesvirus alpha-transinducing factor and its interaction with an immediate early promoter.

The sequence of the alpha-transinducing factor (alpha-TIF) of canine herpesvirus (CHV-l) was determined. Alignment of the predicted CHV-1 alpha-TIF amino acid sequence with other alpha-TIF homologues reveals a core region of similarity with divergent amino and carboxyl termini. Analysis of the CHV-1 infected cell protein 4 promoter region identified a region containing nine copies of a 52 bp repeat that showed significant up-regulation of transcription by alpha-TIF. This region contained an imperfect 'TAATGARAT' motif, the binding site for herpes simplex virus 1 alpha-TIF, with an imperfect Oct-1 binding site immediately following. The infectious laryngotracheitis virus alpha-TIF was also shown to up-regulate transcription through this region of the promoter. Transfection of CHV-1 genomic DNA failed to yield infectious virus in canine kidney cell lines. Co-transfection of genomic DNA and an alpha-TIF expression plasmid resulted in virus plaques, indicating a potential essential role for alpha-TIF in CHV-1 infection.

Formerly unclassified, acid-stable equine picornaviruses are a third equine rhinitis B virus serotype in the genus Erbovirus.

Acid-stable equine picornaviruses (ASPs) were identified as a distinct serotype of equine picornaviruses that were isolated from nasal swabs taken from horses with acute febrile respiratory disease in the UK and Japan, and were placed in the group of unclassified picornaviruses. The nucleotide sequence of the P1 region, encoding the capsid proteins, was determined for three ASP isolates from the UK and the sequences were aligned with published sequences of Equine rhinitis B virus (ERBV), genus Erbovirus, including acid-labile ERBV1 and ERBV2 and the recently identified acid-stable ERBV1. The ASPs belong to the same phylogenetic group, composed of five acid-stable ERBV1 isolates. ERBV1 rabbit antiserum neutralized the ASP isolates at approximately 1/10 titre relative to acid-stable and acid-labile ERBV1 isolates, supporting prior findings that ASPs are a distinct serotype, albeit cross-neutralizing weakly with ERBV1. The genus Erbovirus therefore presently comprises three serotypes: ERBV1, ERBV2 and the proposed ERBV3.

Sequence variation divides Equine rhinitis B virus into three distinct phylogenetic groups that correlate with serotype and acid stability.

Equine rhinitis B virus (ERBV), genus Erbovirus, family Picornaviridae, occurs as two serotypes, ERBV1 and ERBV2, and the few isolates previously tested were acid labile. Of 24 ERBV1 isolates tested in the studies reported here, 19 were acid labile and five were acid stable. The two available ERBV2 isolates, as expected, were acid labile. Nucleotide sequences of the P1 region encoding the capsid proteins VP1, VP2, VP3 and VP4 were determined for five acid-labile and three acid-stable ERBV1 isolates and one acid-labile ERBV2 isolate. The sequences were aligned with the published sequences of the prototype acid-labile ERBV1.1436/71 and the prototype ERBV2.313/75. The three acid-stable ERBV1 were closely related in a phylogenetic group that was distinct from the group of six acid-labile ERBV1, which were also closely related to each other. The two acid-labile ERBV2 formed a third distinct group. One acid-labile ERBV1 had a chimeric acid-labile/acid-stable ERBV1 P1 sequence, presumably because of a recombination event within VP2 and this was supported by SimPlot analysis. ERBV1 rabbit antiserum neutralized acid-stable and acid-labile ERBV1 isolates similarly. Accordingly, three distinct phylogenetic groups of erboviruses exist that are consistent with serotype and acid stability phenotypes.

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses.

OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.

Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

Sialic acid acts as a receptor for equine rhinitis A virus binding and infection.

Equine rhinitis A virus (ERAV) is a member of the genus Aphthovirus, family Picornaviridae, and causes respiratory disease in horses worldwide. To characterize the putative receptor molecule(s) of the ERAV isolate 393/76 (ERAV.393/76) on the surface of Vero and other cells, an assay was developed to measure the binding of purified biotinylated ERAV.393/76 virions to cells by flow cytometry. Using this assay, the level of binding to different cell types correlated with the relative infectivity of ERAV in each cell type. In particular, equine fetal kidney cells, mouse fibroblast cells, rabbit kidney-13 and Crandell feline kidney cells bound virus at high levels and produced high virus yields (> or =10(7) TCID50 ml(-1)). Madin-Darby bovine kidney and baby hamster kidney cells showed little or no binding of virus, producing yields of < or =10(1.8) TCID50 ml(-1). Treatment of Vero and other cells with sodium periodate and the metabolic inhibitors tunicamycin, benzyl N-acetyl-alpha-D-galactosamide, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and proteases indicated that part of the receptor-binding and entry complex for ERAV.393/76 is on N-linked carbohydrates and that the carbohydrate is likely to be present on a protein rather than a lipid backbone. The effect of carbohydrate-specific lectins and neuraminidases on ERAV.393/76 binding and infection of Vero and other cell types implicated alpha2,3-linked sialic acid residues on the carbohydrate complex in the binding and infection of ERAV.

Identification of a neutralizing epitope in the betaE-betaF loop of VP1 of equine rhinitis A virus, defined by a neutralization-resistant variant.

Equine rhinitis A virus strain 393/76 (ERAV.393/76) was passaged in the presence of post-infection ERAV.393/76 equine polyclonal antiserum (EPA). Viruses with increased resistance to neutralization by EPA were obtained after 15 passages. Compared with the parent virus, five plaque-purified, neutralization-resistant mutant viruses, in addition to the non-plaque-purified viruses that were examined, had a Glu-->Lys change at position 658, which is located in the predicted betaE-betaF (EF) loop of VP1. Rabbit antiserum was prepared against the isolated EF loop of ERAV.393/76 VP1 expressed as a fusion protein with glutathione S-transferase. This antiserum bound to purified ERAV.393/76 in Western blots, but not to the neutralization-resistant mutant virus or to ERAV.PERV/62, a naturally occurring ERAV strain that has a Lys residue at position 658. These results suggest that the EF loop of VP1 is involved in a neutralization epitope of ERAV.

Mapping epitopes in equine rhinitis A virus VP1 recognized by antibodies elicited in response to infection of the natural host.

Equine rhinitis A virus (ERAV) is an important respiratory pathogen of horses and is of additional interest because of its close relationship and common classification with foot-and-mouth disease virus (FMDV). As is the case with FMDV, the VP1 capsid protein of ERAV has been shown to be a target of neutralizing antibodies. In FMDV VP1, such antibodies commonly recognize linear epitopes present in the betaG-betaH loop region. To map linear B cell epitopes in ERAV VP1, overlapping fragments spanning its length were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. These fusion proteins were tested for reactivity with sera from ERAV-infected horses and with polyclonal sera from ERAV-immunized rabbits and mice. Regions at the N- and C-termini as well as the betaE-betaF and the betaG-betaH loop regions contained B cell epitopes that elicited antibodies in the natural host. GST fusion proteins of these regions also elicited antibodies following immunization of rabbits and mice, which, in general, strongly recognized native ERAV VP1 but which were non-neutralizing. It is concluded that the N-terminal region of ERAV VP1, in particular, contains strong B cell epitopes.

Polymorphism of open reading frame 71 of equine herpesvirus-4 (EHV-4) and EHV-1.

Open reading frame (ORF) 71 genes of both equine herpesvirus-1 (EHV-1) and EHV-4 encode a unique glycoprotein, which has been described to vary in molecular mass from 200 to 450 kDa. Using PCR and nucleotide sequence analysis, it was shown that the ORF 71 genes of EHV-1 and EHV-4 are polymorphic due to a variable number of reiterated sequences in two regions, designated regions A and B. Region A was threonine-rich and was located near the N terminus. Region B comprised a 38 amino acid repeat near the C terminus that expanded following cell culture adaptation. Western blot analysis of viruses showed that EHV-4 gp2 was modified by glycosylation and that variation in region A resulted in the marked differences in the molecular mass of EHV-4 gp2.

Equine rhinitis B virus: a new serotype.

Equine rhinovirus serotype 3 isolate P313/75 was assigned, with an unclassified genus status, to the family PICORNAVIRIDAE: The sequence from the 5' poly(C) tract to the 3' poly(A) tract of P313/75 was determined. The sequence is 8821 bases in length and contains a potential open reading frame for a polyprotein of 2583 amino acids. Sequence comparison and phylogenic analysis suggest that P313/75 is most closely related to the prototype equine rhinitis B virus (ERBV) strain P1436/71, formerly named equine rhinovirus type 2. A high degree of sequence similarity was found in the P2 and P3 regions of the two genomes. However, the deduced amino acid sequences of the P1 region of P313/75 and ERBV strain P1436/71 contained significant differences, which presumably account for the serological segregation of the two viruses. It is suggested that P313/75 can be classified as a new serotype of the genus Erbovirus, tentatively named ERBV2. Seroepidemiological data indicate that ERBV2 infection of horses may be common (24%) in Australia.

Equine rhinitis A virus: structural proteins and immune response.

Equine rhinitis A virus (ERAV) is a picornavirus that has been reclassified as a member of the Aphthovirus genus because of its resemblance to foot-and-mouth disease virus at the level of nucleotide sequence and overall genomic structure. The N-terminal amino acid sequence of three of the four capsid proteins of ERAV was determined and showed that the proteolytic cleavage sites within the precursor P1 polypeptide occur exactly as those predicted for an aphthovirus-like 3C protease, which generates the capsid proteins VP1 and VP3. However, the autocatalytic cleavage site between VP4 and VP2, which is independent of 3C protease cleavage, was different from that predicted previously. ERAV.393/76 antisera from horses and rabbits showed different reactivity to the viral structural proteins in both serum neutralization assays and Western blots. High neutralizing antibody titres appeared to correlate with strong reactivity to VP1 in Western blots.