Molecular Detection and Phylogenetic Analysis of Equine Herpes Virus-1 in Horses with History or Clinical Signs in Four Provinces of Iran

Introduction

Herpes virus infections are ubiquitous in equine populations worldwide, among them equine herpes virus-1 (EHV-1) is one of the major causes of economic loss in equine in- dustry (Ataseven, et al. 2009; Pusterla, et al. 2014; Slonska, et al. 2014). EHV-1 has a linear double stranded DNA genome from Alpha- herpesvirinae subfamily, genus varicellovirus. Infection with EHV-1 results in contagious re- spiratory disease, epidemic abortion, neonatal death and  myeloencephalopathy  (Ataseven, et al. 2009; Pusterla, et al. 2009; Carlson, et  al. 2013; Ko, et al. 2013; Hussey, et al. 2014; Vaz,  et al. 2016).  Upper respiratory disease  of foals and adults is associated with EHV-4 or, less commonly, EHV-1(Walter, et al. 2013; Stasiak, et al. 2015; Constable, et al. 2017; Laval, et al. 2017).

The biologic source of such virus orig- inates from a primary lytic infection or the shedding of virus after reactivation of a la- tent state (Pusterla, et al. 2009; Ma, et al. 2013; Jhonstone, et al.  2016;  Laabassi,  et al. 2017). Viral latency and reactivation are important features of EHV-1 epidemiology; this virus can establish latent infection in the host, then intermittent recrudescent and viral shedding from asymptomatic carrier to sus- ceptible horses (Ataseven, et al. 2009; Dami- ani, et al. 2014; Sarkar, et al. 2015; Sarkar, et al. 2016; Oladunni, et al. 2018). Therefore, different techniques have been used to detect and differentiate EHV-1 and there is a clear need for a specific and sensitive technique that allows the rapid diagnosis of clinical disease, as well as surveillance of suscepti- ble populations (Hussey, et al. 2006).

Real-time PCR is a flexible, rapid, sen- sitive, specific and quantitative method for diagnosis and surveillance of herpes viral diseases  (Hussey,  et al. 2006;  Diallo,  et al.

2007; Yilmaz, et al. 2012). Although there are some reports of EHV-1 infection in Iran, this study indicated  the presence  of EHV-1 in horses with signs associated with this vi- rus. Based on the authors’ findings, this is  the first study using real-time PCR TaqMan assay on nasal swab and blood samples of horses with clinical signs or clinical history associated with this virus and phylogenetic study in Iran.

This study focuses on estimating the fre- quency of EHV-1 in nasal swab and blood samples of horses from four provinces  of Iran using real-time PCR TaqMan assay and phylogenetic study of positive  samples.

Materials and Methods

Samples and sample  preparation

Blood samples and nasal swabs were tak- en from 150 horses (from different sex, breed and age) with clinical signs or history asso- ciated with this virus, including respiratory signs (fever, coughing and nasal discharge), neurologic signs (depression, ataxia, recum- bency and urinary incontinence) and abortion from different studs of four provinces of Iran that contain large horse population. Provinc- es include Golestan (north of Iran), Tehran (north of Iran), Khuzestan (south of Iran) and West Azerbaijan (northern west of Iran). The experimental procedures have been carried out in accordance with The Code of EU Di- rective 2010/63/EU for animal experiments. Number of horses has been sampled from each province: Golestan: 47, Khuzestan: 45, Tehran: 37 and West Azerbaijan: 21. Sam- pling was done between December 2015 and December 2016. Five milliliters of blood were collected from jugular vein in EDTA tubes.             Nasal  swabs  were  taken from both nostrils  then  transferred  to 1ml virus  trans-

port media containing PBS, penicillin  800 IU ml-1, streptomycin 800 µg ml-1 and 0.1% w/v fetal calf serum (OIE, 2015). Samples were immediately transported on ice to Lab- oratory of Virology in Faculty of Veterinary Medicine at University of Tehran.

The blood samples were centrifuged at 1500 g for 10 min, the buffy coat fraction was removed and stored at -70 ˚C. Nasal swabs with their transport media was stored at -70 ˚C (3). DNA was extracted from 100µl of each whole blood and nasal swab samples using DNA extraction kit (MBST, Iran) by the protocol described by the manufacturer. The quality of extracted DNA was confirmed by the agarose gel electrophoresis.

Reference strain used in this study was pu- rified DNA of EHV-1 strain 89c25 (Kawaka- mi, et al. 1962).

Primers and probes

EHV-1 specific real-time PCR (TaqMan assay) was performed on extracted DNA, isolated from buffy coat fraction and nasal swab solution. The glycoprotein B of Alpha Herpesvirinae was a conserved region and was selected because it contained highly specific sequence that could allow discrim- ination between the closely related equid herpes viruses EHV-1  and EHV-4  (Wanger et al., 1992).

Primers and probe targeting the glycopro- tein B gene of EHV-1 were used from OIE terrestrial, 2015 (OIE, 2015). (Tables  1)

Primers   and  probe   sequence  specificity

was confirmed by nucleotide – nucleotide Blast search in national reference for bio- technology information (NCBI) database and were synthesized by Sinacolon Ltd. Iran. The monoplex EHV-1 real-time PCR was performed as a 20µl reaction containing 0.4mM of each dNTPs, 3mM MgCl2, 1 unit TaqDNA polymerase, 0.3μm of each prim- ers and 5 μl of DNA template. The real-time was   performed   on   Rotor-Gene (Qiagene,

Germany) machine.

Cycling parameter

Real-time cycling parameters were used for initial denaturation at 95 ˚C for 5 min, the cycling consisted of 40 cycles at 95 ˚C for 15 s and 60 ˚C for 60 s. Results were interpreted based on CT values as follows: CT values below or equal to 35 were considered pos- itive, CT values above 35 were considered negative (Diallo et al., 2006). Four positive samples were sequenced for confirmation of results and phylogenetic study.

Phylogenetic analysis

In order to establish phylogenetic rela- tionship between EHV-1 strains, sequences results of EHV-1 strains obtained from this study were used for phylogenetic analysis using MEGA version 7.0 software.

Statistical analysis

Statistical analysis was performed by  SPSS software version 20. Statistical signif- icance was determined using Chi square and Fisher’s Exact test. A p≤0.05 was considered statistically  significant.

Table 1. Real-time PCR primers and probe

C

Primers used for sequence analysis on positive samples described by kirasawa et al.,1993

Results

Out of 150 sampled horses, a total of 14 (9.33%) were found positive for EHV-1, 7 (4.6%) buffy coat samples, 5 (3.33%) nasal swab samples and 2 (1.33%) of both nasal swab and buffy coat samples were found positive for EHV-1. There was not any sta- tistically significant difference between the nasal swab and blood samples with EHV-1 infection (p>0.05). Out of 14 positive sam- ples  of EHV-1,  10 (71.42%),  1 (7.14%),   3

(21.42%) were found with history or clinical signs of respiratory, abortion  and neurolog- ic disease respectively. Out of 47 samples from Golestan, 45 samples from  Khuzestan,

37 samples from Tehran and 21 samples from West Azerbaijan, 7 (14.89%),  0 (0%),  4 (10.8%) and 3 (14.28%) were positive for EHV-1 respectively. There was statistically significant difference between EHV-1infect- ed samples from Golestan and Khuzestan. There was no significant association among the other provinces (Table 2).

Sampled horses were divided into 3 differ-

ent age groups (≤ 5 years, 6–15 years and ≥16 years). Although frequency of infection with EHV-1 was higher in 6–15 years group, there was no significant association among differ- ent age groups. Out of 14 positive samples  of EHV-1, 9 were taken from crossbred, 1 from KWPN, 1 from Holstein, 3 from Thor- oughbred and 1 from Turkmen. There was significant difference between Turkmen and crossbred but there was not any significant difference among other breeds. Out of 14 positive samples for EHV-1, 8 belonged to female horses and 6 belonged to male hors- es. There was not any significant correlation between the male and female horses infected with EHV-1 (Table 2).

Phylogenetic tree designed based on gly- coprotein B gene of EHV-1 by using the real-time PCR sequence, neighbor joining method with bootstrap value equal to 1000 were used. Phylogenetic tree showed that the Iranian EHV-1 strains were homogenous and had a close relationship with the previously reported strains in gene bank (Fig.  1).

Figure 1. Phylogenetic tree of EHV-1 in Iran. It was generated by neighbor-joining method and Mega 7 software with 1000 bootstrap value. Black circles showed viruses detected in Iran and black diamond showed the out-group strain obtain from gene bank

Table 2. Frequency of infection with EHV-1 based on type of disease, province, age and breeds*.

C.S= Clinical sign, C.H= Clinical history, A= Affected, N= Non affected

.The heterogonous letters showed significant statistic differences*

Discussion

Real-time PCR was employed for detec- tion of EHV-1 based on its speed, high sen- sitivity and specificity (Hussy, et al., 2006; Diallo, et al., 2007; Pusterla, et al., 2009). Samples were taken from different studs in four provinces of Iran. These horses were used for race and jumping competition, breeding and riding. Based on the authors’ knowledge, this is the first study to present molecular detection of EHV-1 in nasal swab and blood samples of stud horses using re- al-time PCR TaqMan assay and phylogenet- ic study in Iran.

EHV-1 was detected from a total of 7.3% and 4.6% blood sample and nasal swab, re- spectively. Infection rate of EHV-1 in horses has been investigated worldwide and   differ-

ent results were found based on different re- gions and different detection methods were used (Ataseven, et al.,  2009;  Pusterla,  et  al., 2010; Yilmaz, et al., 2012; Turan, et al., 2012). In this study, some horses were pos- itive only with nasal swab, the others with blood sample and some with both, therefore different kinds of discussion based on their clinical status (clinical signs or history) and type of sample (nasal swab or blood) could be considered.

In a serological survey in Chaharma- hal-Bakhtiari, Iran, Momtaz and Hemmatza- deh, (2003) detected 39.08% EHV-1 positive horses. In molecular surveys the prevalence of EHV-1 in Isfahan, Chaharmahal-Bakhtiari and northeast of Iran were 13.2%, 8.1% and 0%, respectively  (Sarani,  et al.,  2012;  Tak-

taz, et al., 2015). Variation in detection rate among these studies could be due to climatic condition (moisture, temperature, etc.), fre- quency of EHV-1 infection in these provinc- es, immune status of horses, management factors and techniques used to detect this virus (Matsumura, et al., 1992; Gohering, et al., 2006).

The results of this study, like Friday et al., (2000), confirm the absence of significant association between different age groups, however Heninger et al., (2007) and Goher- ing et al., (2006) reported that, there is sig- nificant difference between age groups in- fected with EHV-1, nevertheless the results show that infection with EHV-1 in the age group of 5–16 years is higher than the other groups. We believe that with increase in age, possibility of EHV-1 infection increases. Ba- sically, the possibility of encountering EHV- 1 increases for adult horses used for compe- tition, breeding and training (Friday, et al., 2000; Gohering, et al., 2006; Heninger, et al., 2007; Taktaz, et al., 2015).

The results of this study were similar to Gohering et al., (2006), Lunn et al., (2009) and Taktaz et al.’s, (2015) studies that showed different frequencies of EHV-1 in different breeds of horses, nevertheless there is only    a significant difference between two breeds (Turkmen and crossbred);further, there  is not any significant difference among other breeds sampled in this study. Higher use of crossbred horses in competitions with stress- ful condition (poor housing, transportation, gathering a large number of horses) that de- crease function of immune system may be associated with higher frequency of EHV-1 infection in these horses.

The results show there is not any signif- icant association between sex and EHV-1 infection.  In  this  category,  our  results   are

in agreement with reports of Momtaz and Hemmatzadeh, (2003), Friday et al., (2009) and Taktaz, et al., (2015), on the other hand Gohering et al., 2006 reported sex is a fac- tor in the epidemiology of EHV-1 infection  in the Netherlands. Different results be- tween sexes in these studies may be due to differences in rate of exposure to the infec- tion, health status, age, previous vaccination or immune status of the horses sampled in these studies (Momtaz and Hemmatzadeh, 2003; Gohering, et al., 2006; Friday, et al., 2009; Taktaz, et al., 2015). In our study there is no significant association between blood and nasal swab samples of EHV-1 infected, however frequency of EHV-1 in blood was higher than nasal swabs. Results obtained from other parts of the world in this category are different (Brown, et al., 2007; Ataseven, et al., 2009; Turan, et al., 2012; Yilmaz, et  al., 2012).

In surveys performed in Turkey, Ataseven et al., (2009), identified frequency of EHV-   1 in nasal swabs 30% and in blood 14.3%, Yilmaz et al., (2012), reported frequency of EHV-1 in nasal swabs 13.6% and in blood 20%. Turan et al., (2012) and Brown et al., (2007) reported that analysis of swabs is  more sensitive than blood to detect EHV-1 infection.

Higher frequency of EHV-1 in blood sam- ples could be due to the higher tendency of this virus to produce viremia and subsequent- ly formation of latency in PBMC. However, it seems that based on sampling time, type of infection (lytic or latent), sensitivity of labo- ratory methods and sample size,  frequency of this virus in nasal swab and blood samples is variable and could not definitely conclude that frequency of EHV-1 is always higher in blood or nasal swab samples (Brown, et al., 2007;  Ataseven,  et  al.,  2009;  Turan,  et al.,