Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks

Abstract

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Keywords: equine herpesvirus type 1; sequencing, diversity.

Figure 1

EHV‐1 genome structure showing functional ORFs, inverted repeats and tandem repeats (adapted from GenBank accession NC_001491.2). Inverted repeats are shown as black rectangles, and unique regions as black lines above the ORFs. Regions used for subsequent sequence analysis are indicated with an asterisk (*)

Figure 2

ML phylogenetic trees of (a) EHV‐1 U_L and (b) EHV‐1 U_S, midpoint rooted. Bootstrap values obtained after 100 replicates are shown at the major nodes. Clades are indicated by continuous bars on the right and numbered. Accession numbers and disease phenotype for new sequences are listed in Table 1. Sequences obtained from PubMed are indicated with an asterisk (*), country of isolation is noted in italics, and disease phenotypes are indicated in parentheses as respiratory (R), neurological (N) or abortigenic (A) if known

Figure 3

Variation in EHV‐1 protein sequences. The mean number of amino acid (aa) substitutions (relative to the consensus), normalized to protein length, is shown for the 78 strains. The ORF number is indicated on the x‐axis. The mean number of substitutions for the combined ORFs is marked by a dotted line. ORF64‐ORF68 were not included, due to low sequence coverage for several strains. Nonsense mutations were counted as single substitutions for this analysis, and frameshifts were not included. Repeat regions are not included because of variation within strains and low sequence coverage

Figure 4

Mutations in the DNA polymerase catalytic subunit (ORF30). (a) Variable amino acid residues for EHV‐1 strains are shown in a sequence alignment and are identified by residue numbering at the top. (b) ML tree of the amino acid sequences created using PhyML v 3.1. Strains isolated from neurological disease outbreaks are identified by red circles, and strains with the G2254/D752 genotype are identified by asterisks. (c) Ribbon diagram of the crystal structure of HSV‐1 DNA polymerase catalytic subunit (2gv9.pdb) with mutations mapped on the surface. P81T in EHV‐1 corresponds to position 102 in HSV‐1, R151S to position 175, C249Y to position 254 and H250R to position 255. Residues 643 and 694 are not resolved and are therefore omitted. N752D corresponds to position 751, Y753S to position 752, Y769 to position 768 and E990K to position 989 [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

Phylogenetic network based on EHV‐1 U_L, created using Splitstree v. 4. Clades are indicated as in Figure 2a. Clade 7 includes a magnified view. Sequences obtained from PubMed are labelled with an asterisk (*). Viruses isolated from respiratory disease or conjunctivitis outbreaks are coloured blue, neurological disease outbreak viruses are coloured red, and viruses from abortions are coloured black [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Recombination in EHV‐1. Similarity plots of selected regions of EHV‐1 strains (a) Devon/97/2012 (nucleotides 113835‐118298) compared to EHV‐4 strain NS80567, (b) Suffolk/123/2005 (nucleotides 83081‐86533) compared to EHV‐8 strain wh, analysed by Simplot. Arrows indicate the position and orientation of ORFs in relation to the regions of recombination, with the spliced ORF47_44 indicated with a dotted line