Comparing the genetic diversity of ORF30 of Australian isolates of 3 equid alphaherpesviruses

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Abstract

A single nucleotide polymorphism (SNP) has been previously associated with EHV-1 neurological disease in several countries around the world. This disease is very uncommon in Australia and little information is available about the presence of this SNP in Australian EHV-1 isolates. The ORF30 sequence of 66 Australian EHV-1 isolates was determined and the genotype was compared to the disease manifestation of the case from which the virus was isolated. Of the 66 isolates, 61 were from cases of abortion and 5 were cases associated with equine herpesvirus myeloencephalopathy (EHM). There was no association between pathotype and genotype in these isolates. In total, 64 of the 66 isolates encoded N752, including 4 isolates from EHM cases. The ORF30 sequence was also determined for 14 EHV-4 isolates, including 2 isolates from confirmed EHV-4 abortion cases. All 14 EHV-4 isolates had aspartic acid at the position equivalent to EHV-1 AA752. Aspartic acid was also confirmed in this position for the single isolate of AHV-3 sequenced in this study. The nucleotide sequence of ORF68 was also determined and showed considerable genetic heterogeneity in the EHV-1 isolates, however, this ORF was highly conserved among the 14 EHV-4 isolates sequenced, with only one SNP identified among 7 isolates.

These results confirm that the EHV1 ORF30 N752 is unique and that the D752 sequence is most likely to be the true parent strain of this virus. We suggest that the abortigenic form of EHV-1 should be considered to be the more recently emerged mutant.

Introduction

Herpesviruses infect a wide variety of domestic and wild animals, with nine distinct herpesviruses identified in equids (Davison et al., 2009). The alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and -4), are the most economically significant of these equid herpesviruses (Allen et al., 2004, Roizman et al., 1992). Equine herpesvirus-1 and EHV-4 have a high degree of nucleotide and antigenic homology (Telford et al., 1992, Telford et al., 1998) and were originally thought to be different forms of the same virus until the early 1980s (Sabine et al., 1981, Studdert et al., 1981). Both EHV-1 and EHV-4 are respiratory pathogens that initiate infection in the upper respiratory tract and cause local respiratory disease. Equine herpesvirus 4 typically remains restricted to the respiratory system, while EHV-1 infection progresses further. Equine herpesvirus 1 spreads rapidly from the respiratory tract to the local lymph nodes from where the infection becomes systemic with leucocyte infection in the local lymph nodes resulting in a cell associated viraemia (Allen and Bryans, 1986, Kydd et al., 1994a, Kydd et al., 1994b, Matsumura et al., 1992). This cell-associated viraemia disseminates the virus to capillary beds around the body and results in systemic diseases such as abortion and equine herpesvirus myeloencephalopathy (EHM) (Allen et al., 2004, Edington et al., 1991, Patel and Heldens, 2005, Smith et al., 2001).

A third equine alphaherpesvirus, asinine herpesvirus 3 (AHV-3) (also know as EHV-8) was first isolated from donkeys following high-dose corticosteroid administration (Browning et al., 1988). Phylogenetic analysis has shown that AHV-3 is more closely related to EHV-1 than EHV-4 (Browning et al., 1988, Ficorilli et al., 1995). This virus has been associated with rhinitis in experimentally infected donkeys (Browning et al., 1988).

Comparison of the nucleotide sequence of EHV-1 isolates from both abortions and neurological disease identified a single nucleotide polymorphism (SNP) at nucleotide position 2254 of the ORF30 gene encoding the DNA polymerase that was detected more frequently in isolates associated with cases of neurological disease than from isolates associated with abortion (Nugent et al., 2006). This nucleotide change resulted in an amino acid substitution from asparagine (N) to aspartic acid (D) at amino acid position 752 of the DNA polymerase. Equine herpesvirus 1 abortion isolates were more likely to encode N752 (95% of abortion isolates), while 86% of isolates from neurological cases encoded the D752 in the original study (Nugent et al., 2006). Subsequent comparative studies of the D/N752 substitution found significant changes in the level and duration of viraemia following experimental challenge (Allen and Breathnach, 2006) and that the D752 change was sufficient to induce neurovirulence (Goodman et al., 2007, Van de Walle et al., 2009).

Recently, studies investigating the frequency of this substitution in North America, South America and Europe reported a frequency of the D752 substitution in archived neurological and abortion isolates between 2.7% and 24% (Fritsche and Borchers, 2011, Perkins et al., 2009, Pronost et al., 2010, Smith et al., 2010, Tsujimura et al., 2011, Vissani et al., 2009). Outbreaks of EHM have been reported with increasing frequency in recent years, especially the US where large outbreaks of EHM have occurred (Anonymous, 2007, Anonymous, 2013).

Interestingly, the D752 residue associated with neuropathogenic potential in EHV-1 is a highly conserved amino acid in mammalian and non-mammalian herpesviruses in which the DNA polymerase sequence is known (Nugent et al., 2006). In contrast, it is the SNP associated with EHV-1 abortions (N752) that is unique and as such is likely to be a more recent mutation. It is not known whether polymerase variants also exist in equid alphaherpesviruses most closely related to EHV-1, such as AHV-3 and EHV-4. Specifically, EHV-4 strains occasionally isolated from abortions might give further insights into the relationship between this unique ORF30 SNP and the pathogenesis of abortigenic viruses.

Since the first isolation of EHV-1 in Australia in 1977 (Dixon et al., 1977), the prevalence of EHM in Australia has been extremely low, in stark contrast to many other countries. The aim of this study was to determine the ORF30 sequence of archived Australian EHV-1 isolates and that of two other closely related viruses to examine ORF30 polymorphisms and their relationship to disease. The sequence of a region of the ORF68 gene is also included in this study since this ORF has been described as a marker for strain variability that has shown some geographic restriction (Nugent et al., 2006).

Section snippets

Samples

Viruses used in this study were isolated from clinical samples submitted to the Centre for Equine Infectious Disease, (formerly the Centre for Equine Virology) since 1969. This archive included 61 isolates from EHV-1 abortion or perinatal foal death and 5 isolates from cases associated with EHM. It also included 12 EHV-4 isolates from cases of respiratory disease and 2 EHV-4 isolates associated with abortion (Studdert, 1983). The single AHV-3 in the archive was isolated from a nasal swab taken

Origin of isolates

In total 66 Australian EHV-1 isolates, 14 EHV-4 isolates and one AHV-3 isolate were included in this study. Fifty-three of the 66 Australian EHV-1 isolates (80.0%) originated from Victoria or New South Wales, which contain the largest horse breeding populations in Australia. All EHV1 isolates were submitted to the laboratory between 1977 and 2011. The majority of archived EHV-1 isolates (61/66, 92.4%) were from clinical cases of abortion or perinatal foal deaths, which were the dominant

Discussion

Although outbreaks of EHM are being described with increasing frequency in North America and Europe, neurological disease due to EHV-1 infection remains an unusual disease manifestation in Australian horses. The primary aim of this study was to investigate a large number of archived Australian EHV-1 isolates to determine if any of these isolates had an ORF30 genotype that has been associated with neuropathogenic potential (Nugent et al., 2006). The frequency of the D752 amino acid substitution

Acknowledgments

This work was funded in part by the Rural Industries Research and Development Corporation and the Special Virology Fund. The authors gratefully acknowledge Professor Michael J. Studdert for his contribution to the collection of isolates used in this study.

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