Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks

Abstract

Equid herpesvirus 1 (EHV-1) can cause a wide spectrum of diseases ranging from inapparent respiratory infection to the induction of abortion and, in extreme cases, neurological disease resulting in paralysis and ultimately death. It has been suggested that distinct strains of EHV-1 that differ in pathogenic capacity circulate in the field. In order to investigate this hypothesis, it was necessary to identify genetic markers that allow subgroups of related strains to be identified. We have determined all of the genetic differences between a neuropathogenic strain (Ab4) and a nonneuropathogenic strain (V592) of EHV-1 and developed PCR/sequencing procedures enabling differentiation of EHV-1 strains circulating in the field. The results indicate the occurrence of several major genetic subgroups of EHV-1 among isolates recovered from outbreaks over the course of 30 years, consistent with the proposal that distinct strains of EHV-1 circulate in the field. Moreover, there is evidence that certain strain groups are geographically restricted, being recovered predominantly from outbreaks occurring in either North America or Europe. Significantly, variation of a single amino acid of the DNA polymerase is strongly associated with neurological versus nonneurological disease outbreaks. Strikingly, this variant amino acid occurs at a highly conserved position for herpesvirus DNA polymerases, suggesting an important functional role.

FIG. 1.

Regions of sequence variation for ORF68 are shown, with a representative isolate for each different sequence type displayed. N indicates the number of independent isolates identified with each particular sequence. Sequences are aligned with reference to the prototype Ab4 (GB80_1_2) sequence (GenBank accession number AY665713), with dots indicating sequence identity and dashes indicating gaps in the alignment. Nucleotide positions are indicated above the alignment, in accordance with the Ab4 (GB80_1_2) sequence. Vertical dotted lines indicate breaks between contiguous blocks of sequence. Nucleotide positions characteristic of each ORF68 group are indicated by boxes (further details in text). Isolate GB86_3_2 has one of the two nucleotides (T₇₅₅) which distinguish between group 4 and group 6 and cannot be assigned to either of these groups. Isolate GB87_1_1 has a stretch of five nucleotide substitutions (boxed), none of which are seen with any other isolate. This may be a representative of a separate group, but in the absence of other isolates sharing the signature five substitutions, this isolate has not been assigned to a group.

FIG. 2.

Reduced median network for panel of EHV-1 isolates. Polymorphism data were derived from a set of ORFs containing at least one nonsynonymous substitution and from ORF68 sequences in a panel of 25 isolates representing neuropathogenic and nonneuropathogenic outbreaks and all six ORF68 groupings. Isolates are labeled as shown in Table 6, and the area of each circle is in proportion to the frequency of isolates sharing the same genotype. The positions of substitutions at ORF30 nt 2254 are shown (30.1’), and other substitutions are to scale but unlabeled. The parallelogram at top left indicates the alternative mutational paths involving two variable sites. Groupings based on ORF68 are indicated by dashed lines.

FIG. 3.

An alignment of amino acid sequences for various herpesvirus DNA polymerases is shown, generated using ClustalW (run using the BioEdit software [21]). For each sequence, the name of the virus, database accession number, and residue number of the first amino acid are shown (the residue number in italics refers to a partial polymerase sequence). The conserved aspartate residue, corresponding to D/N₇₅₂ of the EHV-1 polymerase, is highlighted by gray shading. BoHV-1, bovine herpesvirus 1; FeHV-1, felid herpesvirus 1; HHV-3, human herpesvirus 3; CeHV-9, cercopithecine herpesvirus 9; SimVZV, simian VZV; SuHV-1, suid herpesvirus 1; PRV, pseudorabies virus; GaHV-2, gallid herpesvirus 2; MDV-1, Marek's disease virus type 1; MeHV-1, meleagrid herpesvirus 1; HVT, herpesvirus of turkeys; MuHV-1, murid herpesvirus 1; MCMV, mouse cytomegalovirus; RCMV, rat cytomegalovirus; PorcCMV, porcine cytomegalovirus; RhCMV, rhesus monkey cytomegalovirus; GPCMV, guinea pig cytomegalovirus; ElephHV-1, elephantid herpesvirus 1; EBV, Epstein-Barr virus; CalHV-3, callitrichine herpesvirus 3; AlHV-1, alcelaphine herpesvirus 1; MHV-68, mouse herpesvirus strain 68; SaHV-2, saimiriine herpesvirus 2; HVS, herpesvirus saimiri; AtHV-2, ateline herpesvirus 2; PorcLTHV-1, porcine lymphotropic herpesvirus 1; EHV-2, equine herpesvirus 2; HawGrTurtHV, Hawaiian green turtle herpesvirus; and TortHV, tortoise herpesvirus.