Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1

Abstract

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.

Fig. 1.

Nucleotide sequence alignment for the complete genomes of Australasian EHV-1 isolates. Alignment of the complete genome sequences of EHV-1 isolates was performed using MAFFT. Our prototype strain 438-77 was used as the reference sequence. Vertical black lines indicate SNPs compared to the reference and dashes indicate sequence gaps.

Fig. 2.

Nucleotide sequence alignment for the complete genomes of Australasian EHV-4 isolates. Alignment of the complete genome sequences of EHV-4 isolates was performed using MAFFT. Our prototype strain EHV-4.405-76 was used as the reference sequence. Vertical black lines indicate SNPs compared to the reference and dashes indicate sequence gaps.

Fig. 3.

Recombination network trees generated from Australasian EHV-4 nucleotide alignments (excluding sequence repeats) using SplitsTree4. (a) Complete genome sequences, (b) UL region, (c) US region and (d) repeat region. The multiple reticulate networks indicate recombination events between the different isolates. The bar indicates the rate of evolution in sequence substitutions per site. The Phi test for detecting recombination, as implemented in SplitsTree4, was highly significant for the whole genome, and for the UL region, but not for the US region or the repeat region.

Fig. 4.

Recombination network trees generated from Australasian EHV-1 nucleotide alignments (excluding sequence repeats) using SplitsTree4. (a) Complete genome sequences, (b) UL region, (c) US region and (d) repeat region. The bar indicates the rate of evolution in sequence substitutions per site. The Phi test for detecting recombination, as implemented in SplitsTree4, was not significant for the whole genome, or for any of the individual genome regions.