Elsevier

Virus Research

Volume 155, Issue 2, February 2011, Pages 506-513
Virus Research

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

https://doi.org/10.1016/j.virusres.2010.12.007Get rights and content

Abstract

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.

Research highlights

▶ Equine rhinitis B virus (ERBV) infects horses and has three known serotypes. ▶ Previously reported discordance in genotype/phenotype groupings was clarified. ▶ We show a clear correlation between serotype, genotype and acid stability phenotype. ▶ Dual serotype infections were identified in horses and cell culture grown viruses. ▶ ERBV capsids dissociate into pentamers in acidic conditions.

Introduction

The Picornaviridae is a large viral family classified into several genera with extensive diversity in pathogenesis, antigenicity and physical properties. Members of the Picornaviridae cause significant diseases in humans and various animal species. Important members include poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth disease virus (FMDV). Picornaviruses are small non-enveloped viruses with positive-sense RNA genomes. The viral capsid structure is generally conserved across the family and comprises 60 copies each of four proteins – VP1, VP2, VP3 and VP4, the latter of which is a smaller, internal protein.

Equine picornaviruses, formerly known as the equine rhinoviruses, comprise equine rhinitis A virus (ERAV), a member of the genus Aphthovirus, and equine rhinitis B virus (ERBV), the sole species in the genus Erbovirus. Equine rhinitis B virus has been isolated from horses with acute respiratory disease and a high seroprevalence has been reported in horses from Europe, North America, the Middle East and Australasia (Black et al., 2007a, Carman et al., 1997, de Boer et al., 1979, Ditchfield and McPherson, 1965, Dynon et al., 2007, Fukunaga et al., 1983, Holmes et al., 1978, McCollum and Timoney, 1992, Mumford and Thomson, 1978, Newman et al., 1977, Rose et al., 1974, Wernery et al., 1998). Despite this apparent abundance, there have been few reports on ERBV and little is known of the pathogenesis of these viruses.

There is significant variation in acid stability within the picornavirus family. Such physicochemical properties in conjunction with serological relationships have traditionally been the basis for picornavirus taxonomy. More recently, analysis of viral genomic sequence data, in preference to physical properties, has resulted in significant reclassification within the family. Viruses with differing stability in acidic environments have been classified within the same genus, most importantly within the genus Enterovirus (http://www.picornaviridae.com; http://www.ncbi.nlm.nih.gov/ICTVdb). In the genus Erbovirus, both acid-labile and acid-stable phenotypes of ERBV have been reported (Ditchfield and McPherson, 1965, Fukunaga et al., 1983, Newman et al., 1977, Plummer, 1962). The acid stability of a picornavirus is generally an important determinant of the route of virus infection. It also influences cell entry mechanisms. Acid-stable viruses commonly infect the gastrointestinal tract, whereas acid-labile viruses infect primarily through the respiratory tract (Racaniello, 2007). Despite the variation of acid stability phenotypes amongst picornaviruses, conformational changes in the capsid induced by acid are often necessary for the release of the viral genome, either by a stable conformational change [reviewed by Belnap et al. (2000), Neubauer et al. (1987), and Prchla et al. (1994)] or before capsid dissociation into pentameric subunits (Baxt and Bachrach, 1980, Tuthill et al., 2009). The effect of acid on the ERBV capsid and the relevance of the acid-stable phenotype in pathogenesis have not been described.

There are currently three known serotypes of ERBV, termed ERBV1, ERBV2 and ERBV3. While there is a strong correlation between genotype and acid stability phenotype, the correlation of these properties with the virus serotypes is less clear. The serological difference between ERBV1 and ERBV2 has been well defined (Black et al., 2005, Fukunaga et al., 1983, Steck et al., 1978) yet the relationship between ERBV1 and ERBV3 is less apparent. Low-level cross-reactivity between ERBV1 and ERBV3 has been reported in virus neutralisation (VN) assays using rabbit and equine ERBV antisera (Mumford and Thomson, 1978). More recently, five acid-stable viruses with ERBV3-like genomes were assigned to the ERBV1 serotype based on VN assays with ERBV1 rabbit hyper-immune antiserum (Black et al., 2005, Black and Studdert, 2006). In contrast, two previous studies found no cross-reactivity in VN assays with ERBV1 and ERBV3 sera produced in guinea pigs, rabbits and horses (Fukunaga et al., 1981, Fukunaga et al., 1983).

This study provides the first report of the detection of dual erbovirus infections with viruses of different serotype and acid stability phenotype in clinical samples, a finding which significantly contributes to explaining the previous confusion in this area of virus taxonomy. We clarify the relationship between erbovirus genotype, serotype and acid stability phenotype and describe an amino acid residue associated with increased acid stability. The five viruses previously defined as acid-stable ERBV1 are shown to clearly cluster into the ERBV3 serotype.

Section snippets

Viruses and cells

Viruses used in this study are detailed in Black et al. (2005). Clinical and cell culture history of many isolates was unknown. ERBV1.2225/03 was isolated in 2003 from a yearling thoroughbred displaying clinical signs consistent with viral respiratory disease. ERBV isolates were propagated in Rabbit Kidney (RK13) cells grown in maintenance medium (MM) containing Eagle's minimal essential medium (MEM; Sigma–Aldrich) with 10 mM NaHCO3, 50 μg/ml ampicillin (Sigma) and 2% (v/v) foetal bovine serum

Viruses previously classified as acid-stable ERBV1 are ERBV3

Previous reports have documented four types of erboviruses: acid-labile ERBV1, acid-stable ERBV1, acid-labile ERBV2 and acid-stable ERBV3. Despite being defined serotypically as ERBV1 (Black et al., 2005), the ERBV1 viruses with an acid-stable phenotype group with ERBV3 by genotype. High titre, monospecific rat antisera to purified viruses were used to clarify the relationship of the serotype to the genotype and acid stability phenotype of ERBVs (Table 1). Previously, only serum to acid-labile

Discussion

In an attempt to isolate an acid-stable quasispecies variant from an Australian ERBV1 clinical isolate (ERBV1.2225/03), a dual infection with acid-labile ERBV1 and acid-stable ERBV3 was identified. This represents the first reported case of a dual serotype ERBV infection in horses and is the first ERBV3 to be isolated in Australia. The presence of ERBV3.2225AS in two nasal swabs collected 6 months apart, and seroconversion to ERBV1 14 days after the first ERBV3 positive swab, suggested that the

Acknowledgements

We thank Nino Ficorilli and Cynthia Brown for technical assistance. J.J.H. was the recipient of a Melbourne Research Scholarship. Other funding support was from the Special Virology Fund, Centre for Equine Virology, The University of Melbourne.

References (56)

  • R. Acharya et al.

    The three-dimensional structure of foot-and-mouth disease virus at 2.9 A resolution

    Nature

    (1989)
  • D.M. Belnap et al.

    Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus

    J. Virol.

    (2000)
  • P. Bharaj et al.

    Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India

    Virol. J.

    (2008)
  • J.L. Bittle et al.

    Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence

    Nature

    (1982)
  • W.D. Black et al.

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

    J. Gen. Virol.

    (2005)
  • W.D. Black et al.

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

    Arch. Virol.

    (2007)
  • W.D. Black et al.

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

    J. Gen. Virol.

    (2006)
  • R. Burrows et al.

    Observations of picornavirus, adenovirus, and equine herpesvirus infections in the Pirbright pony herd

  • S. Carman et al.

    Infectious agents in acute respiratory disease in horses in Ontario

    J. Vet. Diagn. Invest.

    (1997)
  • C. Carrillo et al.

    Comparative genomics of foot-and-mouth disease virus

    J. Virol.

    (2005)
  • S. Curry et al.

    Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids

    J. Virol.

    (1995)
  • G.F. de Boer et al.

    Prevalence of antibodies to equine viruses in the Netherlands

    Tijdschr. Diergeneesk.

    (1979)
  • J. Ditchfield et al.

    The properties and classification of two new rhinoviruses from horses recovered in Toronto

    Cornell Vet.

    (1965)
  • Drummond, A.J., Ashton, B.B.S., Cheung, M., Heled, J., Kearse, M., Moir, R., Stones-Havas, S., Thierer, T., Wilson, A.,...
  • K. Dynon et al.

    Detection of viruses in nasal swab samples from horses with acute, febrile, respiratory disease using virus isolation, polymerase chain reaction and serology

    Aust. Vet. J.

    (2007)
  • F.M. Ellard et al.

    Evidence for the role of His-142 of protein 1C in the acid-induced disassembly of foot-and-mouth disease virus capsids

    J. Gen. Virol.

    (1999)
  • Y. Fukunaga et al.

    Isolation of picornavirus from horses associated with Getah virus infection

    Nippon Juigaku Zasshi

    (1981)
  • Y. Fukunaga et al.

    Equine Picornaviruses: isolation of virus from the oral cavity of healthy horses

    Bull. Equine Res. Inst.

    (1983)
  • Cited by (10)

    • Veterinary Medicine, Eleventh Edition

      2016, Veterinary Medicine, Eleventh Edition
    • Update on viral diseases of the equine respiratory tract

      2015, Veterinary Clinics of North America - Equine Practice
    • Equine picornaviruses: Well known but poorly understood

      2013, Veterinary Microbiology
      Citation Excerpt :

      There is direct correlation between genotype, serotype and acid-stability phenotype with no cross-neutralisation observed between the three serotypes (Fukunaga et al., 1981, 1983; Horsington et al., 2011). Previously, a small group of viruses that were genetically and phenotypically similar to ERBV3 were classified on the basis of serology as “acid-stable ERBV1” (Black et al., 2005) however, more recent studies have shown this to be a case of misidentification due to the occurrence of dual serotype infections (Horsington et al., 2011). Linear B cell epitopes of ERBV show VP1 contains serotype-specific epitopes and VP2 is highly cross-reactive across the serotypes.

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

      2012, Veterinary Microbiology
      Citation Excerpt :

      All three serotypes have been isolated from horses world-wide and neutralising antibodies have been detected in 50–80% of horses tested and in all age groups (Black et al., 2007; Carman et al., 1997; Dunowska et al., 2002; Dynon et al., 2007; Fukunaga et al., 1981; Holmes et al., 1978; McCollum and Timoney, 1992; Mumford and Thomson, 1978; Rose et al., 1974; Steck et al., 1978; Wernery et al., 1998). Simultaneous infection with multiple serotypes has been reported and neutralising antibodies to two or all three serotypes in the one horse is common (Black et al., 2007; Dynon et al., 2007; Horsington et al., 2011). Antibodies are the major effectors of protection against picornavirus infection, although cell-mediated immunity plays an essential role in the immune response to at least some of these viruses (Glezen et al., 1969; McCullough et al., 1992; Pay and Hingley, 1987; Rossi et al., 1991).

    View all citing articles on Scopus
    View full text