Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建

为了解新疆马疱疹病毒1型（EHV-1）主要毒力基因遗传进化情况并构建TK基因缺失株，本研究以EHV-1 XJ2015株DNA为模板，对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析，并扩增TK基因左右重组臂TKL和TKR，构建质粒pUC-TKLR，将扩增后的增强绿色荧光蛋白（EGFP，含有CMV+polyA）插入pUC-TKLR质粒，构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示，XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高，分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%；与EHV-3分离株同源性均最低，分别为72.9%、59.4%和62.1%；遗传进化分析显示，3个基因均与国外EHV-1同属于一个遗传进化分支，与EHV-9和EHV-4进化关系较近，但与EHV-3进化关系较远，表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显，没有明显的地域性特征，功能基因保守且进化缓慢，同源基因功能相同或相近；经PCR扩增、酶切、测序及转染鉴定，本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建，为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。     

Molecular data of UL24 homolog gene (ORF37) from Brazilian isolates of equine herpesvirus type 1

Equine herpesvirus type 1 (EHV-1) is associated with abortions, respiratory distress, and neurological disturbances in horses. The ORF37 of EHV-1 encodes a protein homolog to UL24 gene product of human herpesvirus that has been associated with neurovirulence. In the present work, ORF37 PCR fragments derived from two Brazilian EHV-1 isolates, a German isolate and an American reference strain were sequenced and characterized by molecular phylogenetic analysis. This genomic region is highly conserved an allowed to infer genetic distances between EHV-1 strains and other animal herpesvirus.     

Isolation and comparative restriction endonuclease DNA fingerprinting of equine herpesvirus-1 from cattle

Deoxyribonucleic acid fingerprinting analyses with 4 restriction endonucleases (EcoRI, BamHI, BglII, and HindIII) and serotest results have definitively indicated that 5 herpesviruses isolated from 1974 to 1986 from aborted bovine fetuses and from bovine tissues and nasal secretions were abortigenic subtypes of equine herpesvirus type 1 (EHV-1). The herpesviruses, designated BH1247, 3M20-3, G118, H1753, and 9BSV4, were neutralized by EHV-1-specific antiserum and could be propagated in cultures of either bovine or equine cells. Only minor differences in restriction endonuclease patterns were detected from the pattern of an Army 183 isolate of EHV-1 subtype 1 that had been passaged only in equine cells and from that of an attenuated EHV-1 subtype 1 (RQ) strain that had been passaged several hundred times in non-equine cells. The individual differences in the restriction endonuclease fragments of the 5 bovine isolates and the Army 183 and RQ strains mainly were attributable to alterations in the terminally repeated and the unique short nucleotide sequences of the EHV-1 genomes, which are known to be hot spots for deletions and tandem repeats. The BamHI restriction endonuclease pattern of the 1977 bovine isolate H1753 was identical to that of EHV-1 subtype-1 strains responsible for most of the virus abortions in vaccinated horses since 1981. Abortigenic EHV-1 strains have the ability to infect cattle and cause disease under natural conditions.     

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.     

Isolation and characterization of monoclonal antibodies against an attenuated vaccine strain of equine herpesvirus type 1 (EHV-1)

The production and differentiation of monoclonal antibodies (mabs) against the Rac-H strain of EHV-1 used as an attenuated live vaccine to prevent rhinopneumonitis and abortion is described. Seven different antigenic sites were detected by the 15 mabs produced. EHV-1 specific mabs as well as EHV-1 and -4 common mabs could be established, allowing easy typing of EHV isolates. One mab recognized the vaccine strain only. This reaction was used to investigate a possible involvement of the vaccine strain in cases of abortion. Common antigenic determinants with EHV-1,-3,-4 and BHV-1 could also be detected, indicating the presence of highly-conserved epitopes of alpha-herpesviruses.     

Comparison of the growth kinetics of neuropathogenic and nonneuropathogenic equid herpesvirus type 1 (EHV-1) strains in cultured murine neuronal cells and the relevance of the D/N(752) coding change in DNA polymerase gene (ORF30)

We compared the growth kinetics of neuropathogenic and nonneuropathogenic equine herpesvirus type 1 (EHV-1) strains in mouse cerebral cortex cells and investigated the relevance of the D/N amino acid change at position 752 of ORF30 in Japanese isolates. Neuropathogenic electropherotype P strains 01c1 and 89c25 exhibited similar growth kinetics to nonneuropathogenic P strain 90c16 in cultured neurons; however, the growth ability of type B strain 97c7 was lower than those of the other strains tested. The amino acid encoded at 752 of ORF30 in 01c1 was asparagic acid; asparagine was encoded in the other EHV-1 strains isolated from Japanese horses. The D/N(752) difference in ORF30 may not be related to replication ability in neurons.     

Equid herpesvirus 9 (EHV-9) isolates from zebras in Ontario,Canada, 1989 to 2007

The objective of this study was to identify and partially characterize 3 equid herpesviruses that were isolated postmortem from zebras in Ontario, Canada in 1989, 2002, and 2007. These 3 virus isolates were characterized by plaque morphology, restriction fragment length polymorphism (RFLP) of their genomic deoxyribonucleic acid (DNA), real-time polymerase chain reaction (PCR) assay, and sequence analyses of the full length of the glycoprotein G (gG) gene (ORF70) and a portion of the DNA polymerase gene (ORF30). The isolates were also compared to 3 reference strains of equid herpesvirus 1 (EHV-1). Using rabbit kidney cells, the plaques for the isolates from the zebras were found to be much larger in size than the EHV-1 reference strains. The RFLP patterns of the zebra viruses differed among each other and from those of the EHV-1 reference strains. Real-time PCR and sequence analysis of a portion of the DNA polymerase gene determined that the herpesvirus isolates from the zebras contained a G at nucleotide 2254 and a corresponding N at amino acid position 752, which suggested that they could be neuropathogenic EHV-1 strains. However, subsequent phylogenetic analysis of the gG gene suggested that they were EHV-9 and not EHV-1.     

Quantification by real-time PCR of the magnitude and duration of leucocyte-associated viraemia in horses infected with neuropathogenic vs. non-neuropathogenic strains of EHV-1

REASONS FOR PERFORMING STUDY: Neurological disease in horses caused by infection with certain 'paralytic' strains of equine herpesvirus-1 (EHV-1) is a potentially devastating condition the pathogenesis of which is poorly understood. Preliminary observations in both experimentally induced and naturally occurring cases of the central nervous system disease have revealed a more robust cell-associated viraemia in horses infected with paralytic isolates of EHV-1, relative to horses infected with abortigenic isolates. To investigate further this pathogenesis-relevant question, the present study was performed using a greater number of horses and a more precise method for quantification of EHV-1 DNA present in viraemic leucocytes. OBJECTIVE: To compare the magnitude and duration of leucocyte-associated viraemia in seronegative, age-matched foals following infection with paralytic vs. abortigenic isolates of EHV-1. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from 20 weanling foals at 2, 4, 7, 9, 11, 14 and 21 days after intranasal inoculation with either paralytic or abortigenic isolates of EHV-1. The amount of EHV-1 DNA present in each PBMC sample was measured by real-time quantitative PCR. RESULTS: Foals inoculated with paralytic strains of EHV-1 developed both a greater magnitude and longer duration of PBMC-associated viraemia than foals inoculated with abortigenic strains of the virus. CONCLUSIONS: Both the higher magnitude and longer duration of cell-associated viraemia contribute to the risk for development of neurological signs in horses infected with paralytic strains of EHV-1. POTENTIAL RELEVANCE: Our results provide empirically derived, scientific data that contributes to a better understanding of the pathogenetic basis for the differing abilities of paralytic and abortigenic strains of EHV-1 to cause post infection central nervous system disease in the horse. The findings identify the importance of minimising the quantitative burden of viraemic leucocytes that follows exposure to the virus, by the use of effective therapeutic antiviral drugs and efficacious prophylactic vaccines that stimulate cytotoxic immune responses against EHV-1 infected cells.     

Molecular Variability in Different Indian Isolates of Equine Herpesvirus-1

Three abortigenic Indian isolates of equine herpesvirus-1 (EHV-1) (Tohana, Hisar and Bikaner), along with two exotic abortigenic isolates (AB4 and V592) and another EHV-1 isolate (Jind) obtained from a case of perinatal foal mortality, were studied for variability. For this purpose, PCR and restriction endonuclease (RE) digestion techniques were used simultaneously as a DNA fingerprinting system. Nine different regions of EHV-1 virus were amplified by PCR using primer pairs specific for the regions and the products obtained from these regions were subsequently subjected to various restriction endonucleases to further assess the variability in the number of RE sites as well as in their positions. No difference was observed in all the four abortigenic isolates in terms of the size of different PCR products amplified by all the nine primer pairs, except for primer pairs ‘E’ and ‘C’. PCR products obtained with primer pair E revealed that Tohana and Bikaner isolates were most similar while Hisar isolate was like V592 isolate. However, the PCR product obtained from Jind isolate had a size between the PCR products of Hisar and Tohan/Bikaner isolates. The primer pair ‘C’ used to amplify the region between 1151 to 3679 in ‘Gene 1,2,3’ clearly differentiated the EHV-1 isolate obtained from a case of perinatal foal mortality from isolates obtained from abortion cases. This primer pair needs to be exploited more extensively for use as a potential marker for differentiating the EHV-1 isolates, mainly the abortion cases from perinatal foal mortality ones. Restriction endonuclease studies done with PCR product of all the isolates with various primer pairs did not reveal any changes in the position or number of RE sites present in the products amplified, indicating no variation in different RE sites within the amplified PCR products. However, this study clarified that all the Indian isolates belonged to the IP group of EHV-1.     

Molecular characterisation of the ORF68 region of equine herpesvirus-1 strains isolated from aborted fetuses in Hungary between 1977 and 2008

Equine herpesvirus-1 (EHV-1) can be classified into distinct groups by single nucleotide polymorphisms (SNPs) in their genomes. Only a few of these can be associated with a special attribute of the virus. Differences in the ORF30 region can determine the neuropathogenic potential, while by substitutions in the ORF68 region several strain groups can be made. In previous studies no connection was found between the neuropathogenic potential and the SNPs in ORF68, but the occurrence of members of distinct groups in different outbreaks can facilitate epidemiological investigations because the geographical distribution of a particular group is very often specific. The present study aimed at the molecular examination and grouping of 35 EHV-1 strains isolated from aborted equine fetuses in Hungary between 1977 and 2008. Genotyping was based on the comparison of nucleotide sequences of a polymorphic segment located in the ORF68 region, which had previously been found to be a useful tool for classification. After sequencing this region, the Hungarian EHV-1 isolates could be classified into seven groups. Only 23 of the 35 isolates belonged to the formerly described groups, while the SNPs of 12 isolates diverged, and four new groups could be set up. In addition, phylogenetic analysis was performed to compare the ORF68 sequences of the Hungarian strains with the sequences of isolates from Europe, America and Australia. The number of newly formed groups suggests that the further analysis of unknown EHV-1 isolates would involve the emergence of extended numbers of new groups, which can impair the usability of this grouping method.     

Derivation and characterisation of a live equid herpes virus-1 (EHV-1) vaccine to protect against abortion and respiratory disease due to EHV-1

A German abortion isolate of EHV-1 (strain M8) was grown in equine dermal (ED) cells at a low multiplicity of infection in presence of 5-bromo-2-deoxy uridine. The resulting stock was dialysed, titrated and cloned by terminal dilution in ED cells grown in 96-well microtitration plates. Of 192 clones each originating from a single focus, clone 147 (C147) was found to be restricted for growth at and above temperatures of 38.5 degrees C. It was also restricted for growth at 37 degrees C in rabbit kidney (RK-13) cells which are widely used for the isolation and titration of EHV-1; hence clone 147 was EHV-4-like.Clone 147 showed a remarkable efficacy as a vaccine in protecting conventional pregnant Welsh Mountain pony mares against abortions due to EHV-1. A single intranasal (IN) vaccination protected five out of six (83.3%), and four out of five (80%) of mares upon challenge 4 and 5-6 months, respectively, after the immunisation, whereas all six unvaccinated mares aborted between 9 and 19 days after IN EHV-1 challenge. With the exception of the day 9 abortion, foetuses of the remaining five mares were EHV-1 infected. Placenta from the early aborting mare was, however, EHV-1 positive. Both groups of vaccinated mares were also significantly protected against clinical reaction (notably pyrexia), nasal shedding and viraemia following challenge infection.     

Induction of a Th-1-biased IgG subclass response against equine herpesvirus type 1 in horses previously infected with type 4 virus

An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were na?ve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses na?ve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.     

Development of a Neutralizing Monoclonal Antibody-based Blocking ELISA for Detection of Equine Herpesvirus 1 Antibodies

A single-dilution, sensitive and specific monoclonal antibody-based blocking enzyme-linked immunosorbent assay (B-ELISA) was developed as an alternative to the cumbersome virus neutralization test (VNT) for detection of equine herpesvirus-1 (EHV-1) antibodies. Neutralizing monoclonal antibodies (1H6 and 9C6) raised against EHV-1 (Hisar-90-7 strain) and sera from 70 horses (30 known negative and 40 known positive for EHV-1 antibodies by VNT) were used for standardization of the B-ELISA. Using a single serum dilution of 1:250 in B-ELISA, 100% specificity was obtained with both monoclonal antibodies (Mabs) in comparison to VNT. Similarly, the sensitivity of the B-ELISA was 92.5% and 100% with 1H6 and 9C6 Mabs, respectively. A very high correlation coefficient (r = 0.85) was observed between B-ELISA and VNT that was significant at the p < 0.01 level. B-ELISA detected a more than 3-fold rise in antibody titres in paired serum samples collected from mares aborting owing to EHV-1 infection. Mab 9C6 was chosen for testing 231 field sera from apparently healthy vaccinated and non-vaccinated horses from organized breeding farms belonging to 11 Indian states, and from Bhutan, by B-ELISA and VNT. There was very good agreement between the results obtained by both VNT and B-ELISA (K = 0.9438). Of 231 field sera, 144 samples were negative for EHV- 1 antibodies by both VNT and B-ELISA and 81 were positive by both tests. Two samples negative by VNT were found positive in B-ELISA. On the other hand, four weakly positive samples in VNT (VN antibody titre 0.9 1.2 log10) were negative in B-ELISA. The Mab (9C6)-based B-ELISA was found to be a suitable alternative to VNT for screening large numbers of field sera and enabled confirmatory EHV-1 serodiagnosis.     

驴源马疱疹病毒8型的分离鉴定与生物信息学分析

本研究旨在分离1株驴源马疱疹病毒8型（Equine herpesvirus type 8，EHV-8）毒株并分析其遗传特征。从山东省聊城地区某驴场采集病驴肺脏组织，经PCR方法鉴定EHV-8的感染情况；对EHV-8感染阳性的肺组织进行研磨，经反复冻融后接种于兔肾细胞（RK-13）细胞中，盲传3代，待出现细胞病变（CPE）时收集细胞，并通过PCR、间接免疫荧光试验、透射电镜等技术对EHV-8进行鉴定。利用PCR扩增获得分离株的ORF70全基因组序列，并进行生物信息学分析。研究结果显示，经PCR鉴定获得EHV-8感染阳性的肺脏组织，病料接种易感细胞RK-13后出现典型CPE，分别收集前3代的细胞培养物，经PCR扩增，均获得与预期大小一致的ORF70基因片段，将分离获得的EHV-8毒株命名为SDLC66，序列上传GenBank，获得登录号：MW816102。经测序和序列比对发现，SDLC66毒株与AHV-3毒株（GenBank登录号：U24184.1）ORF70基因的相似性为99%，与国内的EHV-8 wh毒株（GenBank登录号：JQ343919.1）、国外EHV-8/IR/2015/40（GenBank登录号：MF431614.1）、EHV-8/IR/2003/19（GenBank登录号：MF431611.1）参考毒株的相似性最高，均为99.8%。经遗传进化树分析发现，SDLC66与EHV-8（EHV-8 wh、EHV-8/IR/2015/40和EHV-8/IR/2003/19株）在一个小分支上，亲缘关系最近；与EHV-1型参考毒株（Hong Kong/57/1984、United Kingdom/32/1982、Oxfordshire/206/2013株）序列来源于同一大分支，亲缘关系较近，与EHV-4毒株（91c1和TH20p株）亲缘关系较远。间接免疫荧光试验可见与病毒蛋白特异结合的红色荧光信号；透射电镜观察可见直径约110 nm的圆形病毒粒子，并且核衣壳外有一层亮晕，亮晕外有一层囊膜。说明本试验成功分离得到1株驴源EHV-8毒株，为进一步研究其致病性和致病机制奠定基础。     

Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool

In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus is no longer available. The sensitivity was improved by combining cycling optimization and visualization of PCR products in ethidium bromide and silver-stained acrylamide gels.     

Serological responses and clinical outcome after vaccination of mares and foals with equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) vaccines

Equine herpesvirus type 1 and type 4 (EHV-1 and EHV-4) cause infections of horses worldwide. While both EHV-1 and EHV-4 cause respiratory disease, abortion and myeloencephalopathy are observed after infection with EHV-1 in the vast majority of cases. Disease control is achieved by hygiene measures that include immunization with either inactivated or modified live virus (MLV) vaccine preparations. We here compared the efficacy of commercially available vaccines, an EHV-1/EHV-4 inactivated combination and an MLV vaccine, with respect to induction of humoral responses and protection of clinical disease (abortion) in pregnant mares and foals on a large stud with a total of approximately 3500 horses. The MLV vaccine was administered twice during pregnancy (months 5 and 8 of gestation) to 383 mares (49.4%), while the inactivated vaccine was administered three times (months 5, 7, and 9) to 392 mares (50.6%). From the vaccinated mares, 192 (MLV) and 150 (inactivated) were randomly selected for serological analyses. There was no significant difference between the groups with respect to magnitude or duration of the humoral responses as assessed by serum neutralization assays (median range from 1:42 to 1:130) and probing for EHV-1-specific IgG isotypes, although neutralizing responses were higher in animals vaccinated with the MLV preparation at all time points sampled. The total number of abortions in the study population was 55/775 (7.1%), 9 of which were attributed to EHV-1. Seven of the abortions were in the inactivated and two in the MLV vaccine group (p=0.16). When foals of vaccinated mares were followed up, a dramatic drop of serum neutralizing titers (median below 1:8) was observed in all groups, indicating that the half-life of maternally derived antibody is less than 4 weeks.     

Neutralizing and Complement-fixing Monoclonal Antibodies as an Aid to the Diagnosis of Equine Herpesvirus-1 Infection

One complement-fixing (C-MAb) and three complement-dependent neutralizing monoclonal antibodies (N-MAbs) were raised against Hisar-90-7 equine herpesvirus-1 (EHV-1) strain. The target antigen of the C-MAb (2A5) and two of the N-MAbs (1H6, 9C4) was identified as a 140 kDa polypeptide in Western blotting. The target antigen of N-MAb (9C6) could not be identified. Purified polypeptides of five EHV-1 strains isolated from different regions and at different times gave intense bands at 140 kDa when reacted with N-MAb (1H6) in Western blots. In sandwich ELISA, all four MAbs captured the viral antigen from clinical materials, giving a reliable and rapid diagnosis of EHV-1 infection in equines.