Equine herpesvirus type 1: detection of viral DNA sequences in aborted fetuses with the polymerase chain reaction

Primers and probes were selected from the gene encoding glycoprotein 13 (gp 13) of equine herpesvirus 1 (EHV-1). The polymerase chain reaction (PCR) was run on infected and noninfected cultured cells and on 63 specimens from 29 aborted equine fetuses. The results were evaluated by electrophoresis and dot-blot hybridization using an oligonucleotide probe labeled with biotin. In the infected samples electrophoresis showed a PCR product of about 280 base pairs. The dot-blot hybridization confirmed that this product contained EHV-1 DNA sequences. PCR took 4 h and hybridization another 14 h; the results were thus achieved within 24 h and were highly specific for EHV-1. Close concordance was found between the results of PCR and virus isolation.     

A new field strain of equine abortion virus (equine herpesvirus-1) among Kentucky horses

From restriction endonuclease characterization of the DNA of 317 isolates of equine abortion virus (equine herpesvirus-1; EHV-1) from 176 epizootically unrelated outbreaks of equine virus abortion occurring over 24 years in Kentucky, an epizootic pattern and variation of the virus have emerged. Two electropherotypes of EHV-1 (1P and 1B) accounted for greater than 90% of the nonvaccine-related abortion isolates examined. From 1960 to 1981, EHV-1 1P was the predominant isolate circulating in the central Kentucky area and the cause of greater than 80% of EHV-1-related abortions. In 1981, the occurrence of isolate 1B-related abortions began to increase and since 1982, 1B has become the most frequently recovered isolate of EHV-1 from aborted fetuses.     

Application of cloned fragments of equine herpesvirus type-1 DNA for detection of virus-specific DNA in equine tissues

Tissue specimens obtained from equine herpesvirus-1 (EHV-1), subtype 1-infected aborted foetuses were analysed for the presence of virus DNA by means of Southern blot and dot blot hybridisations. The specificity of the methods was confirmed although the sensitivity was inferior to classical techniques such as virus isolation. However, the possibility of detecting the state of the virus DNA and the ability to distinguish between subtypes were important features, and the dot blot method was shown to have potential for a rapid diagnostic test. This report demonstrates some potential practical applications of hybridisation methods for studying the pathogenesis and epidemiology of EHV-1 but also reveals limitations of the techniques.     

Detection of EHV-1 and EHV-4 in placental sections of naturally occurring EHV-1- and EHV-4-related abortions in the UK: use of the placenta in diagnosis

REASONS FOR PERFORMING STUDY: EHV-1 and EHV-4 abortion diagnosis is based upon detailed examination of the aborted fetus. However, in some cases, only the placenta is available for examination. Furthermore, the contribution of lesions in the placenta to pathogenesis and diagnosis of EHV-1 and EHV-4 abortion has been neglected. OBJECTIVES: To assess the utility of placental examination in equine herpesvirus-1 (EHV-1) and EHV-4 abortion diagnosis. METHODS: Sections of allantochorion from 49 herpesvirus abortions were analysed by PCR, in situ hybridisation and immunostaining. RESULTS: Virus-specific nested PCR confirmed the presence of viral DNA in 46 cases; 41 cases were EHV-1-positive and 5 EHV-4-positive. Microscopic changes were nonspecific. Examination of the PCR-positive sections of allantochorion revealed EHV-1 DNA by in situ hybridisation (ISH) in 21 cases and EHV-4 in 4 cases. In 2 samples, DNA of both viruses was present on PCR and ISH. Viral antigen was found by immunohistology in 15 cases. Regarding the localisation of virus in the placentae, both viral DNA and antigen of EHV-1 and EHV-4 were found in endothelial cells of chorionic villi and, occasionally, in trophoblast epithelium. In the stromal endothelium, only EHV-1 was found. CONCLUSIONS: The data indicate that examination of placentae is a useful diagnostic aid in EHV-1 and EHV-4 abortion diagnosis. POTENTIAL RELEVANCE: Virological examination of the placenta should become standard practice in equine abortion investigations, particularly in those cases where the fetus is not available for examination.     

A polymerase chain reaction for detection of equine herpesvirus-1 in routine diagnostic submissions of tissues from aborted foetuses

Equine herpesvirus 1 (EHV-1) is the causative agent of abortion, perinatal foal mortality, neurological and acute respiratory diseases in horses. Conventional laboratory diagnosis involving viral isolation from aborted foetuses is laborious and lengthy and requires processing of samples within 24 h of collection, which is problematic for samples that come from long distances. The aim of this study was to develop a polymerase chain reaction (PCR) assay useful in Argentina to detect DNA sequences of EHV-1 in different tissues from aborted equine foetuses with variable quality of preservation and without the use of conventional DNA fenolic extraction. Several DNA extraction protocols and primers were evaluated. The amplification method was standardized and its specificity was analysed using 38 foetal samples of variable quality of preservation. Of the 38 different foetal tissues, nine livers, six spleens and two lungs in good preservation and eight livers, one spleen and four lungs in a poor state of preservation were positive for PCR. EHV-1 was recovered only from the nine livers, five spleens and two lungs in good preservation. No virus was isolated from the samples that were poorly preserved. Viral isolation was confirmed by cytopathic effect and indirect immunofluorescence. The specificity of the PCR results was confirmed by the restriction endonuclease digestion of PCR products and hybridization.     

Molecular confirmation of an abortigenic strain of equine herpesvirus 1 (subtype 1) in a pregnant mare study

Four pregnant mares were inoculated intranasally and/or intravenously with equine herpesvirus 1 (EHV-1), subtype 1 during the third trimester of gestation. One mare aborted on postinfection day 15, one mare delivered a sick, weak full term foal, and two mares delivered healthy, full term foals. EHV-1, subtype 1 was isolated from several tissues of the aborted fetus and from the thymus of the sick foal. DNA restriction endonuclease patterns of the recovered EHV-1 viruses were identical to those of the EHV-1 challenge strain, documenting the origin of the abortigenic viruses.     

Comparison of methods for the diagnosis of equine herpesvirus type 1 infection

The objective of the investigations was to study the occurrence of the equine herpesvirus type 1 (EHV-1) infection in aborted equine fetuses and in newborn foals and to compare the sensitivity of virus isolation, immunohistochemistry and histology in 101 cases and of fetal serology in 68 cases in the diagnosis of the infection. Out of the 93 aborted equine fetuses and 8 weak foals, 15 (14.9%) (14 fetuses and 1 foal) proved to be EHV-1 infected by immunohistochemical and 13 (12.9%) by virological investigation. Characteristic microscopic changes were seen in several organs in all cases, while intranuclear inclusion bodies could be found only in 25 (35.2%) of the 71 virus-positive tissue samples. Four (5.9%) cases proved to be positive by fetal serological investigation, but none of these cases showed any EHV-1 specific lesions and in none of these cases could the virus be detected by virus isolation or by immunohistochemistry. According to the results, fetal serology does not seem to be a useful test in virus-positive cases, while the immunohistochemical method seems to be a reliable and a slightly more sensitive method than virus isolation in the diagnosis of EHV-1 infection.     

Equine parvovirus: initial isolation and partial characterization.

A viral agent was isolated from the fetal liver of an aborted equine fetus. The isolate hemagglutinated red blood cells from guinea pig, rhesus monkey and rooster. By hemagglutination inhibition tests, the isolate was shown to be antigenically distinct from parvoviruses of bovine and canine origin. Specific hemagglutination inhibiting antibody against the viral isolate was exhibited by 26 of 136 horse sera tested. The isolated virus showed properties compatible with those of an autonomous parvovirus including size, morphology, stability to ether treatment and heating to 56 degrees C, the presence of a 5300 base DNA genome, characteristic protein composition and density (1.405 g/mL). The virus was classified as an equine parvovirus.     

应用多重PCR检测和区分3个型的马疱疹病毒

针对马疱疹病毒（EHV）的EHV-1、EHV-2和EHV-4糖蛋白B基因序列，设计、合成了3对特异性引物进行多重PCR，不仅可以在数小时内分别检测这3个型的EHV，而且在同一反应系统内可以清晰地区分EHV-1、EHV-2和EHV-4，其PCR产物大小分别为226、333、570bp，符合预期的片段大小，序列分析证实与已发表的序列一致；该检测方法的灵敏度达到10^3 TCID50；分别从血清学阳性但病毒分离为阴性的1匹进口马组织样品和一些出口前检疫马的鼻咽样品检测到EHV-1和EHV-4特异性核酸。     

Isolation of equine herpesvirus type 5 in New Zealand

AIM: To report the first isolation of equine herpesvirus 5 (EHV-5) in New Zealand as part of a study of equine respiratory viruses in New Zealand. METHODS: Nasal swabs and peripheral blood leukocytes were collected from 114 foals and adult horses, inoculated on to equine fetal kidney, rabbit kidney and Vero cell lines and observed for cytopathic effect. EHV-5 isolates were identified using an EHV-5 specific polymerase chain reaction. All samples positive for EHV-5 were also checked for the presence of EHV-2, EHV-1 or EHV-4 DNA using published type-specific primers. The polymerase chain reaction results were further confirmed by dot blot and Southern hybridisation with specific DIG-labelled probes. RESULTS: EHV-5 was isolated from nasal swabs or peripheral blood leukocytes of 38 out of 114 horses sampled. From horses sampled more than once, EHV-5 was often isolated on more than one occasion. Most of the horses were infected with both EHV-2 and EHV-5 viruses. It was not possible to make an association between EHV-5 isolation and the presence of respiratory disease. CONCLUSION: EHV-5 is present in the New Zealand horse population. The exact role it plays in causing, or predisposing to, respiratory disease remains to be elucidated.     

Cloning of equine herpesvirus type 1 438/77 strain genome as a bacterial artificial chromosome

Equine herpesvirus type 1 (EHV-1) is a major cause of respiratory and reproductive diseases in horses worldwide. The genome of EHV-1 strain 438/77 (isolated from an aborted equine fetus) was cloned as a bacterial artificial chromosome (BAC) in E. coli without any gene deletions. The mini-F plasmid sequence was inserted in the middle of ORF19 and 20 via homologous recombination following co-transfection of viral DNA and plasmid pE19_20/HA into RK13 cells. Circular viral DNA was extracted from RK13 cells infected with purified recombinant virus expressing green fluorescent protein (GFP) and electrophorated into E. coli DH10B cells. The clone harboring the BAC was screened and analyzed by PCR and RFLP. Reconstitution of the recombinant virus was achieved successfully by transfection of the BAC DNA into RK13 cells. The mini-F sequence in the reconstituted virus was subsequently removed by homologous recombination between virus DNA and plasmid pE1920XM, inducing a point mutation in the Xbal site in ORF19. Comparison of RFLP profiles of the rescued, recovered and the wild-type viral genome demonstrated that no unexpected changes occurred during mutagenesis. In vitro replication assays showed that BAC-reconstituted virus mutant growth kinetics and plaque formation morphology/size were indistinguishable to those measured for wild-type virus.     

Isolation of equine herpesvirus type 5 in New Zealand

Aim. To report the first isolation of equine herpesvirus 5 (EHV-5) in New Zealand as part of a study of equine respiratory viruses in New Zealand.

Methods. Nasal swabs and peripheral blood leukocytes were collected from 114 foals and adult horses, inoculated on to equine fetal kidney, rabbit kidney and Vero cell lines and observed for cytopathic effect. EHV-5 isolates were identified using an EHV-5 specific polymerase chain reaction. All samples positive for EHV-5 were also checked for the presence of EHV-2, EHV-1 or EHV-4 DNA using published type-specific primers. The polymerase chain reaction results were further confirmed by dot blot and Southern hybridisation with specific DIG-labelled probes.

Results. EHV-5 was isolated from nasal swabs or peripheral blood leukocytes of 38 out of 114 horses sampled. From horses sampled more than once, EHV-5 was often isolated on more than one occasion. Most of the horses were infected with both EHV-2 and EHV-5 viruses. It was not possible to make an association between EHV-5 isolation and the presence of respiratory disease.

Conclusion. EHV-5 is present in the New Zealand horse population. The exact role it plays in causing, or predisposing to, respiratory disease remains to be elucidated.     

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.     

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC)

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.     

Demonstration of Chlamydia from an equine abortion

The isolation and identification of a chlamydial agent from an equine fetus is reported. The fetus was aborted by a mare with respiratory disease and fever in the 9th month of pregnancy. The serum of the mare was investigated by the compliment fixation test. Specific antibodies were detected for chlamydial antigen in a titer of > 1:40 and for equine herpes virus 1 antigen in a titer of 1:32. Pathological lesions were not found in the organs of the fetus. Chlamydiae were detected in the placenta by ELISA and subsequently isolated by cell culture. Using PCR technique the agent was identified as Chlamydophila psittaci.     

Comparative pathogenicity of selected bovine viral diarrhea virus isolates in gnotobiotic lambs

The infectivity and pathogenicity of selected bovine viral diarrhea virus (BVDV) isolates were determined in gnotobiotic, colostrum-deprived neonatal lambs. Five-day-old cesarean-derived gnotobiotic lambs were exposed to 1 of 10 BVDV isolates via aerosol suspension. These isolates were from tissues or secretions of calves or lambs affected with respiratory tract disease, weak neonatal calves, aborted bovine fetuses, or reference Singer or Draper BVDV. The pathogenicity of each isolate, relative to the others, was evaluated in lambs by measurement of the neutralizing antibody response, virus isolation from nasal secretions or tissues, and postmortem lesions. The BVDV isolates varied in their infectivity and pathogenicity. Singer, the cytopathic reference strain, was the most lymphotrophic isolate and stimulated the greatest neutralizing antibody response. Encephalitis was the most consistent lesion observed and was used as the final determinant of relative pathogenicity of the viruses. The most neuropathogenic isolates were the 2 viruses originating from lambs affected with respiratory tract disease, the 2 weak neonatal calf isolates, and 1 isolate from an aborted bovine fetus. The least pathogenic isolates were the 2 reference isolates, Draper and Singer; the 2 mucosal disease isolates; and 1 isolate originating from an aborted bovine fetus.     

Equine rhinopneumonitis virus (herpesvirus type 1): attenuation in stable monkey cell line.

An isolate of virulent equine herpesvirus (EHV) type 1 was adapted to Vero stable cell line by 13 serial passages at 37 C and 50 serial passages at 26 C. Characteristics of the attenuated EHV-1 were found to be avirulent, but immunogenic in horses if injected intramuscularly. The attenuated virus was regularly isolated from peripheral leukocytes in inoculated horses, but was not recovered from nasal turbinate tissues. A mild leukopenia was noticed. The attenuated virus produced characteristic large syncytia on primary isolation in rabbit kidney (RK13) or Vero cells at 37 C in contrast to cell rounding observed with virulent EHV-1. The syncytial marker was stable through 20 serial passages in Vero cells at 37 C. New application of double immunodiffusion test for distinguishing between EHV-1 and EHV-2 also is described.     

Clinical observations and management of a severe equine herpesvirus type 1 outbreak with abortion and encephalomyelitis

Latent equine herpesvirus type 1 (EHV-1) infection is common in horse populations worldwide and estimated to reach a prevalence nearing 90% in some areas. The virus causes acute outbreaks of disease that are characterized by abortion and sporadic cases of myeloencephalopathy (EHM), both severe threats to equine facilities. Different strains vary in their abortigenic and neuropathogenic potential and the simultaneous occurrence of EHM and abortion is rare. In this report, we present clinical observations collected during an EHV-1 outbreak caused by a so-called “neuropathogenic” EHV-1 G₂₂₅₄/D₇₅₂ polymerase (Pol) variant, which has become more prevalent in recent years and is less frequently associated with abortions. In this outbreak with 61 clinically affected horses, 6/7 pregnant mares aborted and 8 horses developed EHM. Three abortions occurred after development of EHM symptoms. Virus detection was performed by nested PCR targeting gB from nasal swabs (11 positive), blood serum (6 positive) and peripheral blood mononuclear cells (9 positive) of a total of 42 horses sampled. All 6 fetuses tested positive for EHV-1 by PCR and 4 by virus isolation. Paired serum neutralization test (SNT) on day 12 and 28 after the index case showed a significant (≥ 4-fold) increase in twelve horses (n = 42; 28.6%). This outbreak with abortions and EHM cases on a single equine facility provided a unique opportunity for the documentation of clinical disease progression as well as diagnostic procedures.     

Outbreak of equine herpesvirus type 1 myeloencephalitis: new insights from virus identification by PCR and the application of an EHV-1-specific antibody detection ELISA

Five of 10 pregnant, lactating mares, each with a foal at foot, developed neurological disease. Three of them became recumbent, developed complications and were euthanased; of the two that survived, one aborted an equine herpesvirus type 1 (EHV-1)-positive fetus 68 days after the first signs were observed in the index case and the other gave birth to a healthy foal on day 283 but remained ataxic and incontinent. The diagnosis of EHV-1 myeloencephalitis was supported by postmortem findings, PCR identification of the virus and by serological tests with an EHV-1-specific ELISA. At the time of the index case, the 10 foals all had a heavy mucopurulent nasal discharge, and PCR and the ELISA were used to detect and monitor EHV-1 infection in them. The status of EHV-1 infection in the five in-contact mares was similarly monitored. Sera from three of the affected mares, taken seven days after the index case were negative or had borderline EHV-1-specific antibody titres. In later serum samples there was an increase in the titres of EHV-1-specific antibody in two of the affected mares. In contrast, sera from the five unaffected in-contact mares were all EHV-1-antibody positive when they were first tested seven or 13 days after the index case.     

Potential of DNA-mediated vaccination for equine herpesvirus 1.

The potential of DNA-mediated immunisation to protect against equine herpesvirus 1 (EHV-1) disease was assessed in a murine model of EHV-1 respiratory infection. Intramuscular injection with DNA encoding the EHV-1 envelope glycoprotein D (gD) in a mammalian expression vector induced a specific antibody response detectable by two weeks and maintained through 23 weeks post injection. Immune responses were proportional to the dose of DNA and a second injection markedly enhanced the antibody response. EHV-1 gD DNA-injected mice developed neutralising antibodies, and a predominance of IgG2a antibodies after the DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunised with 50 microg of EHV-1 gD DNA were able to clear virus more rapidly from lung tissue and showed reduced lung pathology in comparison with control mice. The data indicate that DNA-mediated immunisation may be a useful strategy for vaccination against EHV-1.