Prevalence and sequence analysis of equid herpesviruses from the respiratory tract of Polish horses

Abstract

Background: Equid herpesviruses (EHVs) are widespread in equine populations worldwide. While the infection with equine α-herpesviruses (EHV-1 and EHV-4) has been linked to several clinical outcomes, the pathogenic potential for equine γ-herpesviruses (EHV-2 and EHV-5) is still unclear. The objective of the current study was to determine the prevalence of infection with EHVs among Polish horses, to investigate factors associated with EHV infections among horses sampled, and to determine genetic variability within Polish EHV-2 isolates.

Methods: Virus-specific real-time PCR assays were used for detection of EHV-1, EHV-2, EHV-4 and EHV-5 in nasal swabs collected from 540 horses from 13 national horse studs located throughout Poland. A proportion of EHV-2/5 positive samples were subjected to virus isolation followed by amplification and analysis of partial glycoprotein B sequence.

Results: Overall, 448/540 (83.0%) horses sampled were positive for at least one virus. The most prevalent was infection with EHV-2 (77.2%), followed by EHV-5 (47.0%), and EHV-4 (0.4%). None of the horses was positive for EHV-1. Approximately half of the virus-infected horses were positive for both EHV-2 and EHV-5. The proportion of EHV-2/5 positive horses varied by age, breed, and season. Only 8.0% of horses sampled, mostly Arabians, showed clinical signs of respiratory disease at the time of sampling. The viral load of both EHV-2 and EHV-5 DNA was highest in swabs from young horses, which was particularly evident for EHV-2 infected foals. Mean viral loads in nasal swabs collected from diseased horses were higher than in swabs from healthy horses. That was also true for EHV-2 when only diseased Arabian foals were considered, but the levels of EHV-5 DNA were lower in swabs from diseased than from healthy foals. In agreement with other studies, there was a considerable variability between Polish EHV-2 sequences, with no clustering of sequences from horses with different health status. The level of EHV-2 variability seemed to differ between different studs/breeds.

Conclusions: The presence of foals and yearlings on a property is likely to increase the risk of active EHV-2/5 infection among in-contact horses. The existence of breed-specific differences in susceptibility to EHV-2/5 infections should be further investigated, as it may provide one variable that needs to be considered in attempts to associate EHV-2/5 infections with disease. Overall, the data presented add to the existing knowledge of the epidemiology and biology of equine γ-herpesviruses, with the long-term goal of better understanding of the pathogenesis and the impact of infections with these viruses on the well-being of the horse.

Keywords: EHV-2; EHV-5; Equine herpesvirus; Phylogeny; Quantitative PCR; Virological survey.

Fig. 1

Frequency of detection of equid herpesvirus-2 (EHV-2) and EHV-5 in nasal swabs from horses included in the study (n = 540) based on virus-specific quantitative PCR

Fig. 2

Viral DNA load of equid herpesvirus-2 (EHV-2) and EHV-5 in nasal swab samples collected from horses included in the study (n = 540) stratified by age. The middle line in each box represents median, whiskers represent minimum and maximum values, with means indicated by ‘+’. Significance levels calculated using Kruskal-Wallis test with Dunn’s multiple comparison are indicated by the stars: < 0.05 (*),< 0.001 (***). The number of horses in each category is indicated above each box

Fig. 3

Viral DNA load of equid herpesvirus-2 (EHV-2) and EHV-5 in swabs from all Arabian horses tested (n = 101, a and d), Arabian foals (n = 39, b and e) and Arabian yearlings (n = 28, c and f), stratified by disease status. The middle line in each box represents median, whiskers represent minimum and maximum values, with means indicated by ‘+’. The number of virus-positive horses in each category is shown above each box

Fig. 4

Phylogenetic tree of equid γ-herpesviruses (EHV) based on 726 bp fragment from gB gene (nt 703–1429 in EHV-2 accession number NC_001650.2 and nt 700–1426 in EHV-5 accession number NC_026421.1). The sequences used included Polish EHV-2 sequences (n = 57), Polish EHV-5 sequences (n = 2), and international sequences of EHV-2 (n = 26) and EHV-5 (n = 7) sourced from GenBank. The Polish sequences obtained in the current study are labelled PL_EHV2_ID number_ stud number. Accession numbers for sequences from GenBank are included in the description of each sequence. The evolutionary history was inferred by using the maximum likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood (− 6036.44) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA5 [43]. The phylogenetic groups 1, 2, and 3 as defined by Sharp et al. [33] are shown on the right, with new groups labelled 4, 5, and 6. Clades within group 1 are labelled A, B, and C. Samples from the same stud are labelled with the rectangle of the same colour