Equid herpesvirus 8: Complete genome sequence and association with abortion in mares

Abstract

Equid herpesvirus 8 (EHV-8), formerly known as asinine herpesvirus 3, is an alphaherpesvirus that is closely related to equid herpesviruses 1 and 9 (EHV-1 and EHV-9). The pathogenesis of EHV-8 is relatively little studied and to date has only been associated with respiratory disease in donkeys in Australia and horses in China. A single EHV-8 genome sequence has been generated for strain Wh in China, but is apparently incomplete and contains frameshifts in two genes. In this study, the complete genome sequences of four EHV-8 strains isolated in Ireland between 2003 and 2015 were determined by Illumina sequencing. Two of these strains were isolated from cases of abortion in horses, and were misdiagnosed initially as EHV-1, and two were isolated from donkeys, one with neurological disease. The four genome sequences are very similar to each other, exhibiting greater than 98.4% nucleotide identity, and their phylogenetic clustering together demonstrated that genomic diversity is not dependent on the host. Comparative genomic analysis revealed 24 of the 76 predicted protein sequences are completely conserved among the Irish EHV-8 strains. Evolutionary comparisons indicate that EHV-8 is phylogenetically closer to EHV-9 than it is to EHV-1. In summary, the first complete genome sequences of EHV-8 isolates from two host species over a twelve year period are reported. The current study suggests that EHV-8 can cause abortion in horses. The potential threat of EHV-8 to the horse industry and the possibility that donkeys may act as reservoirs of infection warrant further investigation.

Fig 1. Maximum likelihood phylogenetic tree of ORF30 amino acid sequences based on the JTT matrix-based model.

Details of the sequences are in S1 Table of the Supporting Information. The scale bar represents the number of substitutions per site. Bootstrap values after 1000 replications are indicated at major nodes.

Fig 2. Map of the equid herpesvirus 8 (EHV-8) genome.

The coordinates are based on the 149600 bp sequence of EHV-8/IR/2003/19. Predicted protein-coding regions are shown by coloured arrows with names below. Red open reading frames (ORFs) were inherited from the ancestor of family Herpesviridae and blue ORFs from the ancestor of subfamily Alphaherpesvirinae. Orange ORFs are specific to certain lineages within subfamily Alphaherpesvirinae. Inverted repeats TR_S/IR_S flanking U_S are coloured yellow, and inverted repeats TR_L/IR_L flanking U_L are shown by green vertical lines at 1–32 and 112999–113030 bp. A third green vertical line marks a third copy of the TR_L/IR_L sequence that is part of a more extensive inverted repeat at 1–87 and 547–633 bp. Tandem reiterations are marked by grey shading, and origins of DNA replication by vertical red lines.

Fig 3. Conserved sequences near the genome termini of equid varicelloviruses.

An alignment of sequences from the four Irish equid herpesvirus (EHV) 8 (EHV-8) strains, EHV-9 P19 (AP010838.1), EHV-1 AB4 (AY665713.1) and EHV-4 NS80567 (AF030027.1) is shown. In each sequence, TR_L is boxed, the terminal nucleotides are shaded black, and conserved regions AnTn and γ (which are present near the left and right termini, respectively) are underlined. Ellipses denote the remainder of the genome.

Fig 4. Phylogenetic tree of concatenated amino acid sequences of ORF3-ORF76 from the Irish equid herpesvirus 8 (EHV-8) strains, EHV-8 strain Wh and other equid alphaherpesvirus strains.

The tree was constructed by using the maximum likelihood method based on the JTT matrix-based model [30], and is drawn to scale. Bootstrap values from 1000 replicates are shown as percentages at major nodes.