Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland

Equid alphaherpesvirus 1 (formerly called equine herpesvirus 1, EHV-1) is one of the main infectious causative agents of abortion in mares [1]. Infection with EHV-1 can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM) [2]. EHV-1 can cause primary infection, but reinfection with a new strain or reactivation from latency was also shown to induce disease [3].

Infections with EHV-1 remain a major problem in horse breading studs in Poland as well as in other European countries. Admittedly, vaccines are available, but do not provide complete protection against abortion or EHM in mares [4, 5]. Moreover, in recent years, an increase in the number of EHM cases has been observed in many countries, e.g., France, Germany, Ethiopia, Argentina and the USA [6–10].

EHV-1 belongs to the subfamily Alphaherpesvirinae, a family of Herpesviridae. Its linear, double-stranded DNA genome of about 150kbp contains 80 open reading frames (ORFs), four of which are duplicated [11]. Although the mechanism of EHM development is not well understood, the potential to cause neuropathogenicity is significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase [12]. The A to G mutation in nucleotide (nt) position 2254 of the virus genome causes substitution of asparagine (N) by aspartic acid (D) at amino acid position 752 in the catalytic subunit of the viral DNA polymerase. This single amino acid mutation in the viral polymerase of EHV-1 causes higher tropism to lymphocytes and longer viremia in experimentally infected horses when compared to animals infected with EHV-1 lacking this particular mutation [13].

Although infection with N₇₅₂ can also cause EHM, infections with D₇₅₂ increase the risk of developing EHM [3, 6]. For this reason, EHV-1 N₇₅₂ is referred to as a non-neuropathogenic genotype, and D₇₅₂ as a neuropathogenic genotype. Other risk factors for EHM, beside the genotype of the virus, include host and environmental factors such as breed, age and sex of the horse [14].

In some neuropathogenic EHV-1 strains, another mutation, a substitution C to A in position 2258 in addition to A to G in position 2254 in the ORF30 gene was observed [15]. Infection with the neuropathogenic genotype of EHV-1 can also lead to severe abortion outbreaks [5]. Analysis of the whole EHV-1 genome sequences of reference neuropathogenic strain Ab4 and the less virulent V592 strain revealed that the highest variation rate occurs in the ORF68 gene, a homologue to the human herpes simplex virus type US2 region [12]. Analysis of 131 field isolates revealed that, indeed, this region of the EHV-1 genome has the highest mutation rate (2%), which allows its nucleotide sequence to be used as a genetic marker to classify virus strains into different groups [12]. The strains were divided into six groups (two strains remained unassigned) on the basis of SNPs in the polymorphic region of ORF68 and the number of G residues within this region (nucleotides 732–739). Therefore, sequencing of ORF68 replaced the restriction fragment length polymorphism in molecular and epidemiological analyses of the EHV-1 strains obtained during outbreaks [16, 17].

The aim of this study was to analyse the neuropathogenic potential and genetic relationship of EHV-1 strains isolated from aborted equine fetuses in Poland.

PCR products with a size of approximately 450 bp from 27 Polish EHV-1 strains were obtained from the ORF30 region of the virus genome. The SalI digestion of PCR products was negative for EHV-1 samples (Fig. 2). The sequencing of the PCR products further confirmed that all analysed sequences had adenine in position 2254. Guanidine was not observed in ORF30, meaning that all 27 strains isolated from aborted equine fetuses in Poland between 1993 and 2017 belonged to the non-neuropathogenic variant (N₇₅₂) of EHV-1. No other mutations, including substitution C to A in position 2258, were observed in analyzed ORF30 sequences. Sequences of EHV-1 ORF30 are added in Additional file 1.

PCR products with a size of approximately 900 bp were generated from the ORF68 region of the genome including approximately 600-bp-long polymorphic segments. Products were sequenced and aligned to identify SNPs. These SNPs are presented in Fig. 3. The nucleotide sequence of the Ab4 strain (GB80_1_1) as a member of EHV-1 Group 1 served as a basis for the comparison of nucleotide changes. The sequences obtained from sample 01_02 had lower quality despite multiple sequencing efforts; therefore this strain was excluded from the analysis. The sequences obtained for 93_04, 97_05, 97_06, 99_01 and 99_03 are shorter than the others, due to poorer sequencing coverage for region nt 720–760, but were included in the analysis. Out of 26 Polish EHV-1 sequences analysed in this study, five (19.2%) belonged to group II and one (3.8%) belonged to group IV, while the remaining 20 (76.9%) were not classified within any of the groups originally described by Nugent et al. [12] (Table 1 and Fig. 3). More than half of the EHV-1 sequences (57.7%) contained A₆₂₉ SNP. Ten Polish EHV-1 sequences (38.5%) possessed the same substitutions (A₆₂₉ and T₇₅₀) as the EHV-1 sequence of GB86_3_2, which was described as an unassigned sequence in the original study by Nugent et al. [12] and Polish EHV-1 sequence PL_1999_II from a recent study by Stasiak et al. [18]. Three sequences had the same SNPs (A₆₂₉, C₆₂₆ and T₇₅₀) as sequence PL_2013_V, while two sequences were similar to PL_2001_I from the Stasiak et al. [18] (Figs. 2 and 3).

All of the analysed ORF68 sequences possessed seven G residues in a homopolymeric tract compared to eight G residues in the Ab4 strain (nucleotides 732–739). The number of G residues was not established for 93_04, 97_05, 97_06, 99_01 and 99_03 due to the shorter sequences available for those strains.

A clear geographical distribution is difficult to establish (Table 1) since group “PL_1999_II” sequences were found in 7 different voievodenships in contrast to the group II described by Nugent et al. [12] which was found only in western voievodenships. Also, some sequences isolated from the same stud within a similar time period differed, e.g., in the 1997 Lubelskie voievodenship (LB) outbreak isolates 97_01–97_03 contained a A₆₂₉ SNP, vs. a T₅₆₂ SNP of isolates 97_04 –97_06. Two EHV-1 sequences isolated in the same stud over several years (B/WP- Table 1) clustered within group II, which may indicate that a similar virus was present in that region for 7 years.

The strains included in this study originated from abortion cases that occurred between October and May, which is the breeding season in mares (Table 1) [4].

In this study, none of the EHV-1 strains isolated from aborted fetuses between 1993 and 2017 belonged to the neuropathogenic genotype D₇₅₂, and all belonged to the non-neuropathogenic variant N₇₅₂. In two recent studies conducted in Poland, the neuropathogenic variant D₇₅₂ was found in 2 out of 20 cases (10%) and in none out of 37 (0%) EHV-1 isolates obtained from abortion cases [18, 20]. In those studies, Polish D₇₅₂ EHV-1 strains were isolated in the years 2009 and 2010. Our study includes older isolates than previous Polish studies done by Stasiak et al. [18, 20]. Summarizing all the analyses so far, on Polish EHV-1 sequences from years 1993 to 2017, the prevalence of the neuropathogenic genotype D₇₅₂ is very low, only 3.07% (2/65 EHV-1). In all three studies (this one and [18, 20]), the abortions were not associated with neurological symptoms in the horse premises. It seems that non-neuropathogenic EHV-1 strains are prevalent in Poland. To the author’s knowledge, EMH was never described in Poland, although horse practitioners report isolated incidents of neurological symptoms in horses, that are, however, not supported by any laboratory diagnosis of EHV-1. This is in contrast to other countries, where devastating outbreaks of EHM were reported [9, 10, 21, 22].

While studies indicate that the non-neuropathogenic N₇₅₂ (nt A2254) variants are more common, the prevalence of D₇₅₂ strains have increased in recent decades in the USA and some European countries [6, 15]. However, a similar increase has not been observed in other countries such as Japan or New Zealand [23, 24]. Recent studies suggest that neuropathogenic strains could have a selective advantage over non-neuropathogenic strains which have increased their prevalence in horse populations [25]. A very low prevalence of D₇₅₂ strains and an absence of EHM outbreaks in Poland could be due to the late introduction of these strains in Poland, therefore it is recommended to monitor the genotype of EHV-1 in the future. If the D752 strains were more prevalent, this could influence quarantine rules and infectious disease management in studs. It could also influence the amount of samples sent for diagnostics.

The use of the ORF68 sequence as a molecular marker associated with the geographical origin of EHV-1 was first proposed by Nugent et al. [12]. However, more data now available for this sequence indicate that many sequences cannot be classified within already established groups. In a similar study performed in Hungary, 65.7% of EHV-1 isolates were grouped according to the classification of Nugent et al. [12] into groups II, III and IV, while the remaining isolates formed four separate groups [17]. In a recent Polish study, the main EHV-1 groups were also III and IV, while the majority of sequences were matched either by the Nugent “unassigned” group or formed a separate group [18]. In our study, analysis of the geographical distribution of strains isolated in different regions in Poland have not revealed any pattern or clustering. In fact, similar sequences have been isolated from multiple regions, and the others appear only in one stud in a particular year. These findings are in agreement with previous studies conducted in Hungary and Poland [17, 18]. In this study, we observed different EHV-1 strains that caused abortions in the same stud within a similar time period. This could suggest that at least some of these abortions might have been caused by reactivation of a persistent EHV-1 infection rather than a single introduction of a new virus into a stud’s premises (stable A/LB, Table 1). It is proposed that EMH and abortions can be caused by virus shed during the reactivation of latency and transmission to susceptible horses in the stud [3, 26]. All the recent isolates of EHV-1 sequenced in this study belong to group PL_1999_II. This particular ORF68 sequence appeared in 1997 and is the only sequence isolated since 2001, suggesting that these EHV-1 strains possess advantages in transmission or more often induce abortion in mares.

In summary, equine abortion cases that occurred in Poland between 1993 and 2017 were caused by EHV-1 with a non-neuropathogenic ORF30 N₇₅₂ genotype. On the basis of ORF68 sequences, the majority of EHV-1 strains cluster within groups II, IV or cannot be assigned to any of the known groups, but show similarity to those already described in Poland.