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

Horses are natural hosts to at least five equid herpesviruses (EHVs) including equine α-herpesviruses EHV-1 and EHV-4, and equine γ-herpesviruses EHV-2 and EHV-5 [1]. Due to high economic impact, EHV-1 remains particularly important, as infection with this virus can result in various disease outcomes including upper respiratory tract disease, abortion, neonatal foal death, or neurological disease, termed equine herpesvirus myeloencephalopathy [2–4]. Infection with a closely related EHV-4 has been typically linked to upper respiratory disease only [5, 6].

The disease association for γ-herpesviruses EHV-2/5 has been difficult to establish [7, 8], as both viruses have been identified in samples from clinically normal horses, as well as from horses with respiratory disease [9–12]. It has been suggested that EHV-2/5 infections may be linked to poor performance in horses, with or without overt disease [13–15]. This was based on increased frequency of detection of EHV-2/5 from tracheal washes of horses affected by airway inflammation, although results of other studies failed to demonstrate such a relationship [16, 17]. In addition to respiratory disease, EHV-2/5 infections have been linked to keratoconjunctivitis [18, 19], although both viruses have also been detected from conjunctival swabs of clinically normal horses [20, 21]. Recently, EHV-2 and EHV-5 have also been detected in the gastric mucosa, which raised a question of their possible role in the development of gastric ulcers [22]. Equid herpesvirus 5 has been also implicated as an etiological agent for fibrotic lung disease in adult horses termed equine multinodular pulmonary fibrosis [23].

The controversy over EHV-2/5 involvement in equine respiratory disease likely reflects, at least in part, differences between various study designs including the type of samples collected, timing of sampling, definition of respiratory disease used, or the age-structure of the populations sampled. It is also possible that EHV-2/5 infections, even if subclinical on their own, predispose to infections with other pathogens. If so, clinical disease observed would depend on what other pathogens circulate among populations sampled. This seems to be supported by the fact that experimental vaccination with an iscom-based subunit vaccine against EHV-2 protected foals against a life-threatening pneumonia due to Rhodococcus equi infection [24]. Finally, discrepancies in the conclusions reached by various authors with regard to EHV-2/5 disease association may be due to the possible existence of viruses with different biological properties. This has been suggested based on marked genomic heterogeneity observed for both EHV-2 and EHV-5 [25–27].

Genomes of EHV-2 and EHV-5 consist of genes conserved between different herpesviruses that are interspersed with species-specific genes and non-coding regions [28]. The conserved herpesviral genes include that coding for glycoprotein B (gB). This protein is essential for virus replication and plays a role in virus entry into the cells [29]. The gB of both EHV-2 and EHV-5 is a dilsulphate-linked heterodimer that forms an integral part of the viral envelope [30, 31]. The variability in amino acid sequence between different EHV-2 gB sequences has been mapped to three main regions (sites I, II and III) using monoclonal antibodies, with the viruses examined forming two main antigenic groups: EHV-2.86/67-like and EHV-2.141-like based on variability at site I [32]. This site is located in the N-terminus of EHV-2/5 gB exposed on the surface of the virions and contains neutralizing epitopes that are important targets for the immune response [31, 32]. The highest degree of variability between various EHV-2/5 viruses was mapped to site III. This hypervariable site is located between amino acids 415 and 448 in EHV-2.86/67 gB sequence (NP_042604.1), immediately N-terminal to the conserved endoproteolytic cleavage site [31, 32]. In a subsequent study [33] three main phylogenetic lineages of EHV-2 (groups 1, 2, and 3) were described based on phylogenetic analysis of a region (aa 235–609 of EHV-2 gB and 234–605 of EHV-5 gB) that included hypervariable site III of 18 field isolates of EHV-2/5.

Equine herpesviruses have worldwide distribution including Poland [6, 34]. However, EHV-1 detection in Poland has been reported mainly as part of diagnostic investigations of cases of abortion or neonatal death [35–38] and no data are currently available on the frequency of detection of EHV-1/4 among other groups of horses. The only virological survey of EHV-2 among Polish horses was conducted approximately 15 years ago [39]. Information on EHV-5 infections among Polish horses is limited to one case report published in a national journal [40], and hence not easily accessible to non-Polish speaking readers.

Hence, the objective of the current study was to determine prevalence of infection with equine herpesviruses among Polish horses, to investigate factors associated with herpesvirus infections among horses sampled, and to determine genetic variability within Polish EHV-2 isolates.

The high prevalence of EHV-2/5 infection among Polish horses is consistent with data reported from other countries, although the relative frequency of EHV-2/5 detection varied between studies with EHV-2 [44, 45] or EHV-5 [12, 46–50] being most common. These differences may reflect true differences in the epidemiology of EHV-2/5 in different horse populations, but may also be a reflection of the age structure of the horses sampled or the type of samples collected.

The viral load for both EHV-2 and EHV-5 was highest in swabs from young horses (foals and yearlings) for both EHV-2 and EHV-5. This was similar to findings reported by Hue et al. [42], but in contrast to those reported by Laabassi et al. [44], who did not find any differences between viral loads for horses of different ages. This discrepancy may be explained by the fact that in the latter study all horses 3-year-old and younger were considered together in one category, or may reflect other host- or virus-related differences between the populations studied. In the current study, the viral DNA load of EHV-2 was approximately 2 logs higher in nasal secretions from foals than from horses in all other age groups. It was also considerably higher than the levels of EHV-5 DNA shed by foals (Fig. 2). The reasons for this apparent high level of EHV-2 replication in upper respiratory tract of EHV-2 infected foals remain to be elucidated, but may explain efficient transmission of EHV-2 between foals and other in-contact animals.

While both EHV-2 and EHV-5 infections were common in young horses, EHV-2 infection was most common among foals, while EHV-5 infection was most common among yearlings, with foals being less likely to be infected with EHV-5 than older animals. Similar dynamics of EHV-2 and EHV-5 infections have been described recently among French horses [51], and EHV-5 infection has been reported to occur later than EHV-2 infection in studies were foals were followed for a period of time [9, 52, 53]. The reasons for these apparent differences in the epidemiology of two closely related equine γ-herpesviruses remain unclear. Based on considerably higher loads of EHV-2 compared to EHV-5 in nasal secretions of foals, one could speculate that EHV-2 may outcompete EHV-5 in dual infections of foals. In support of this hypothesis, EHV-2 was reported to grow faster than EHV-5 in vitro [26]. Whatever factors may facilitate efficient replication of EHV-2 in respiratory tract of foals, this apparent advantage seems to disappear with age, with similar levels of EHV-2 and EHV-5 shedding detected in nasal secretions from yearlings sampled in the current study.

Although foals and yearlings were most commonly positive for at least one virus, approximately 80% of 71 horses older than 10 years were also shedding EHV-2 or EHV-5. This was similar to a recent report from Algeria [44] where nasal swabs from 83.7% of 43 horses older than 10 years were positive for EHV-2, and 60.5% for EHV-5. However, all horses sampled by Laabassi and others [44] showed clinical signs of respiratory disease at the time of sampling. In contrast, in a recent Ethiopian study only 1/14 healthy and 1/9 diseased horses in the same age group were positive for EHV-2, with 2/9 diseased horses and none of healthy horses positive for EHV-5 [50]. In the current study, all older horses sampled comprised mares with foals at foot. It is possible that local immunosuppression associated with pregnancy [54] or stress related to birth induced recrudescence of latent EHV-2/5 infections in some mares, with subsequent horizontal spread to their foals, as has been suggested by others. Considering high frequency of EHV-2/5 shedding among foals, combined with high viral DNA load detected in nasal secretion of foals compared with older horses, it is likely that the presence of foals facilitated further spread of these viruses among in-contact animals, including other foals and mares, which resulted in the relatively high proportion of older horses positive for EHV-2/5 in the current study.

Similarly, the apparent seasonality of EHV-2 shedding (Table 3) was likely to be related to the seasonality of the breeding season and associated fluctuation in the age structure of horses present at studs, with most foals being born in spring.

Out of the breeds included in the study, Silesian and Polish Konik horses were less likely to be infected with either EHV-2 or EHV-5 than horses of other breeds. For Silesian horses, which represent a heavy type of Polish warmbloods, that may reflect the relatively older population sampled at stud X (median age of 4 years as opposed to 0.5 to 2 years for most other studs sampled). However, the age structure of the population of Polish Koniks sampled was similar to the age structure of horses at studs with other breeds. In addition, the virus load in nasal secretion of Polish Koniks was similar to the virus load secreted by horses of other breeds. Thus, factors other than the age structure or virus load must have influenced the comparatively limited spread of EHV-2/5 among Polish Koniks sampled.

We were unable to make any causative associations between EHV-2/5 infection and disease due to the fact that control healthy horses were not available for sampling on studs where diseased horses resided. In addition, as the samples were tested only for equine herpesviruses, we could not exclude the possibility that pathogens not tested for circulated among diseased horses and contributed to clinical disease observed, as suggested by results of a number of other studies [11, 55, 56]. Interestingly, there appeared to be differences in virus load in nasal secretions between diseased and healthy Arabian horses, which was particularly evident for EHV-2. This corresponds well with a higher blood EHV-2 DNA load in 2-month-old foals suspected of Rhodococcus equi pneumonia compared with healthy foals reported by others [57]. The reasons behind these findings, as well as the role of EHV-2 infection in development of respiratory disease needs further investigation.

Based on the phylogenetic analysis of a partial gB nucleotide sequence, Polish EHV-2 sequences clustered into at least four main groups, two of which were not originally described by Sharp et al. [33] based on the analysis of a comparable region that included hypervariable site III. This is not surprising considering a larger sample size used in the current study (83 versus 15 EHV-2 sequences), and highlights the high diversity of EHV-2 viruses. It is possible that the variability between Polish EHV-2 was even greater than reported, as DNA from 17 EHV-2 qPCR positive cultures did not yield expected gB products when tested with conventional primers, suggesting that there were nucleotide changes at the primer-binding sites that prevented binding of the primers used. Alternatively, lack of amplification of the conventional PCR product may reflect the quality of template DNA, as qPCR was designed to amplify a much shorter product (~ 78 bp) than conventional PCR (> 1300 bp). Altogether, our results agree with the results of others [9, 25, 47, 50, 58], and confirm high genetic variability between field EHV-2 viruses. The importance of this variability on biological properties of the virus and interactions with its equine host remains to be elucidated.

Interestingly, a group of four EHV-2 sequences including two sequences from Iceland [58] appeared to be closer related to EHV-5 than to EHV-2 based on the phylogenetic analysis, although the node separating these four EHV-2 sequences from EHV-5 sequences was positioned deep in the tree, with long branches separating the two groups. Another group of two EHV-2 viruses including Swe-2 was clearly separated from the remaining EHV-2 sequences. Based on a similar position of the Swe-2 sequence (group 3) on the phylogenetic tree constructed by Sharp et al. [33], the authors suggested that this virus may have arisen by recombination between EHV-2 and EHV-5. While this is one possible explanation that requires further investigation, it is also possible that viruses within group 3 and a newly designated group 4 represent EHV-2 viruses that diverged from other EHV-2 viruses early in the process of evolution.

It has been shown previously by several authors that foals can be hosts to more than one genotype of EHV-2 [25] and EHV-5 [27, 59]. Hence, the variability of EHV-2/5 among horses from the same stud (e.g. EHV-2 from stud IV, VII or stud IX and EHV-5 from stud IV) may represent ongoing introduction of viruses from outside sources, but may also simply indicate that a heterogenous population of EHV-2 viruses circulated among horses on those studs.

Interestingly, all four EHV-2 sequences from Polish Koniks were identical to each other, as were all three sequences from Silesian horses. Horses from these two breeds were significantly less likely to be infected with EHV-2 (and also EHV-5) than horses from other breeds. This observation is based on a very small number of samples, but the possibility that there is some as yet undefined relationship between breed and heterogeneity of EHV-2 warrants further investigation considering that similarly small number of samples from other studs/breeds yielded a more heterogenous population of EHV-2 sequences.

In the current study, 11/13 EHV-2 sequences obtained from horses with respiratory disease appeared to be closely related to each other based on the phylogenetic analysis. However, there was not a clear separation between these viruses and EHV-2 sequences from healthy horses, as evidenced by close similarity of EHV-2 sequences from sick and healthy Arabian horses from stud I. The possibility that genomic variability between EHV-2, and to lesser extent EHV-5, may explain somewhat poorly defended relationships between infection with these viruses and disease has been suggested by others, but, similarly to our results, there was no conclusive evidence that this may be the case [25].

The results of the present study demonstrate that, similarly to other countries, equine γ-herpesvirus infections are common among Polish horses of various breeds and ages. The frequency of infection and viral load in nasal secretions were highest in young horses, which was particularly evident for EHV-2 infected foals and EHV-5 infected yearlings. Together with the relatively high frequency of EHV-2/5 infection among adult horses sampled, this suggests that 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. This is also a likely explanation for an increased frequency of EHV-2 infections in spring, when the highest numbers of foals are typically present at the breeding studs. While our data suggested for the first time the existence of breed-specific differences in susceptibility to EHV-2/5 infections, this would need to be further confirmed. If true, such differences may need to be considered in assessment of associations between EHV-2/5 infection and disease, which are currently poorly defined. The viral load of EHV-2 DNA was higher in diseased Arabian horses compared to healthy Arabian horses of the same age. This was unlikely to be attributed to the differences between EHV-2 viruses circulating among those horses. Despite a relatively high level of heterogeneity observed overall among the viruses examined, there was not a clear difference between a limited number of EHV-2 genotypes from diseased and healthy horses. Increased levels of EHV-2 shedding by horses affected by respiratory disease compared with healthy horses, particularly evident for EHV-2 infected Arabian foals, suggested increased levels of EHV-2 replication in diseased foals/horses. It remains to be seen whether this was directly linked to the development of disease or merely a result of a permissive environment in the respiratory tract created by some other, as yet undefined, factors (e.g. co-infection with other respiratory pathogens).