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.     

The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding

ABSTRACT: Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) glycoprotein H (gH) has been hypothesized to play a role in direct fusion of the virus envelope with cellular membranes. To investigate gH's role in infection, an EHV-1 mutant lacking gH was created and the gH genes were exchanged between EHV-1 and EHV-4 to determine if gH affects cellular entry and/or host range. In addition, a serine-aspartic acid-isoleucine (SDI) integrin-binding motif present in EHV-1 gH was mutated as it was presumed important in cell entry mediated by binding to alpha4beta1 or alpha4beta7 integrins. We here document that gH is essential for EHV-1 replication, plays a role in cell-to-cell spread and significantly affects plaque size and growth kinetics. Moreover, we could show that alpha4beta1 and alpha4beta7 integrins are not essential for viral entry of EHV-1 and EHV-4, and that viral entry is not affected in equine cells when the integrins are inaccessible.     

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.     

First Report on Molecular Detection of Equine Upper Respiratory Infectious Viruses in Republic of Korea

The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.     

Genomic diversity among equine herpesvirus-4 field isolates

Infection with equine herpesvirus-4 (EHV-4) is a major cause of respiratory tract disease, equine rhinopneumonitis, in horses. Although the full sequence of EHV-4 has been reported, genomic differences among EHV-4 field isolates have not yet been characterized. In this study, the genomic diversity between 23 Japanese EHV-4 isolates was analyzed by digestion with restriction endonucleases (BamHI, BgIII, EcoRI, SacI, and SalI) and polymerase chain reaction (PCR). The restriction endonuclease digestion patterns of the EHV-4 field isolates showed distinct differences which included mobility shifts of some fragments as well as loss and/or gain of fragments. Two EHV-4 genes containing repeat sequences, ORFs 24 and 71, were amplified by PCR and the amplified fragments were compared among the field isolates. The sizes of the amplified fragments varied among epizootiologically unrelated isolates, while the fragments of related isolates had the same size. The observed genomic diversity among EHV-4 field isolates may be a useful tool for epidemiological study of equine rhinopneumonitis by EHV-4 infection.     

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.     

Comparison of Serum Amyloid A in Horses With Infectious and Noninfectious Respiratory Diseases

The acute phase protein serum amyloid A (SAA) has been shown to be a useful inflammatory parameter in the horse, but studies showing SAA responses to specific respiratory disease etiologies are limited. The goal of this study was to evaluate SAA responses in horses with infectious and noninfectious respiratory diseases as well as healthy, control horses. Two hundred seven horses were grouped into the following categories: equine influenza virus (EIV), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi ss equi), inflammatory airway disease (IAD), and healthy controls. Serum amyloid A concentrations were determined for all horses on serum using a stall-side lateral flow immunoassay test. Serum amyloid A levels were found to be significantly greater for infectious respiratory diseases (EIV, EHV-4, S. equi ss equi) and horses with IAD when compared to control horses. There was a significant difference between viral and bacterial infections and IAD. Although SAA values from horses with S. equi ss equi were significantly greater when compared to horses with viral infections (EIV/EHV-4), the wide range of SAA values precluded accurate classification of the infectious cases. In conclusion, SAA is more reliably elevated with infections of the respiratory tract rather than noninfectious airway conditions. This can facilitate early detection of respiratory infections, help track disease progression, and aid practitioners in making recommendations about proper biosecurity and isolation of potentially contagious horses.     

Surveillance programme for important equine infectious respiratory pathogens in the USA

The prevalence and epidemiology of important viral (equine influenza virus [EIV], equine herpesvirus type 1 [EHV-1] and EHV-4) and bacterial (Streptococcus equi subspecies equi) respiratory pathogens shed by horses presented to equine veterinarians with upper respiratory tract signs and/or acute febrile neurological disease were studied. Veterinarians from throughout the USA were enrolled in a surveillance programme and were asked to collect blood and nasal secretions from equine cases with acute infectious upper respiratory tract disease and/or acute onset of neurological disease. A questionnaire was used to collect information pertaining to each case and its clinical signs. Samples were tested by real-time PCR for the presence of EHV-1, EHV-4, EIV and S equi subspecies equi. A total of 761 horses, mules and donkeys were enrolled in the surveillance programme over a 24-month study period. In total, 201 (26.4 per cent) index cases tested PCR-positive for one or more of the four pathogens. The highest detection rate was for EHV-4 (82 cases), followed by EIV (60 cases), S equi subspecies equi (49 cases) and EHV-1 (23 cases). There were 15 horses with double infections and one horse with a triple infection. The detection rate by PCR for the different pathogens varied with season and with the age, breed, sex and use of the animal.     

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.     

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.     

Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a gamma-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an "alveolar-like" architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera-specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine gamma-herpesvirus, EHV-5.     

Lack of Detectable Equine Herpesviruses 1 and 2 in Paraffin-Embedded Specimens of Equine Sarcoidosis

Background: Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2).
Objective: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis.
Animals: Eight horses with sarcoidosis, reported previously.
Methods: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with EHV-1 and EHV-2. For both herpesviruses the target was their respective glycoprotein B gene. Positive controls consisted of DNA from viral cultures of culturettes from naturally occurring respiratory infections of EHV-1 and EHV-2.
Results: The PCR analyses for both equine herpesviruses' DNA were negative in all 8 horses.
Conclusion: The failure to detect DNA from EHV-1 and EHV-2 in paraffin-embedded skin of these 8 horses does not discount EHV-1 or EHV-2 as causing some cases of ES, but lends support to the presumably multifactorial etiologic nature of the disease.     

Mutations in the US2 and glycoprotein B genes of the equine herpesvirus 1 vaccine strain RacH have no effects on its attenuation]

The equine herpesvirus 1 (EHV-1) modified live vaccine strain RacH is apathogenic for both laboratory animals and the natural host. The apathogenicity of RacH was caused by serial passages of the virus in heterologous cells. When compared to the virulent parental strain RacL11 several changes in the RacH genome occurred. Previous results have shown that the loss of the IR6 gene correlated with the loss of virulence. Additional important mutations were observed within the US2 gene which is directly adjacent to the IR6 gene and within the glycoprotein B (gB) gene. To answer the question whether these mutations contribute to the attenuation of RacH several recombinant EHV-1 were constructed: The mutated genes in RacH were replaced by the wild-type US2 gene or the wild-type gB gene, respectively. In addition, a RacL11 recombinant expressing the mutated (RacH) gB instead of the wild-type gene was generated. All recombinant viruses were tested for virulence using the EHV-1 mouse model. The results were as follows: i) The insertion of the RacL11 US2 gene into the RacH virus did not restore virulence and none of the infected mice showed typical signs of EHV-1-caused disease (symptoms and body weight loss). ii) Exchanging gB genes between RacL11 and RacH did not alter their virulence phenotypes remarkably either. Therefore, it is concluded that attenuation of the EHV-1 vaccine strain RacH is caused solely by the absence of the IR6 gene and protein.     

马鼻肺炎病毒主要糖蛋白及其功能

马鼻肺炎(ER)是由亲缘关系密切的马疱疹病毒1型和马疱疹病毒4型(EHV-1/4)引起的马属动物几种高度接触传染性疾病的总称.其基因组包含约80个开放性阅读框,至少编码78种蛋白.目前发现EHV-1至少有12种糖蛋白,其中5个糖蛋白(gB、D、H、L、K)是病毒复制所必需的,另6个糖蛋白(gC、E、G、I、M、gp300)不是病毒在细胞培养物上生长所必需的,但存在于高毒力的野毒分离株.gB、C、D是重要的免疫原性抗原具有病毒中和表位,gM的功能是使病毒穿入和细胞间传播.论文就其中9种糖蛋白及其功能进行介绍,并对其应用前景进行了展望.     

Equine Viral Respiratory Pathogen Surveillance at Horse Shows and Sales

Equine respiratory viral infections cause significant worldwide disease and economic loss. Common causes include equine influenza virus (EIV) and equine herpesviruses-1 and -4 (EHV-1 and -4), and risk of exposure to these agents may be highest in young horses commingling at sales and competitive events. A surveillance study was conducted at two horse shows and two Thoroughbred sales to determine whether horses shed EHV-1, EHV-4, or EIV on arrival, or 2-4 days later, and whether shedding was associated with identifiable risk factors. Real-time polymerase chain reaction assays were used to detect EHV-1, EHV-4, and EIV nucleic acid in nasal swabs obtained from 369 horses at the four events. In response to evidence of clinical disease, 82 additional horses were sampled at two farms providing horses for one of the sales. On arrival at the events, shedding of EHV-1 was detected in 3.3%, EHV-4 in 1.1%, and EIV in 0.8% of horses. EHV-1 was detected at low levels, and EHV-1 and EHV-4 detection was not associated with clinical disease. EIV was detected only in horses at a Thoroughbred sale, in association with an outbreak of respiratory disease traced back to regional farms. On arrival at events, horses younger than 2 years had a significantly greater risk of shedding EHV-1 compared with older horses; no other significant risk factors associated with viral shedding were identified. Thus, there is a risk of exposure to EIV, EHV-1, and EHV-4 at equine events, and horses and events should be managed to mitigate this risk.     

Role of equine herpesviruses as co-infecting agents in cases of abortion, placental disease and neonatal foal mortality

Herpesviral infections frequently occur in horses. The objective of this study was to investigate the possible association of equine herpesviruses (EHV-1, EHV-2, EHV-3, EHV-4, EHV-5) with other causes of abortion, neonatal mortality or placental disorder. Sixty-seven abortions, 22 stillbirths, 14 cases of neonatal foal mortality and 3 cases of placental disease were investigated for infectious and non-infectious causes. Type-specific nested PCR assays and virus isolation were performed to detect EHV infections. A cause of fetal loss or placental disease was reached in 68 out 116 (58.7 %) cases. Twenty-seven cases were positive for EHV, and 22/27 (81.5 %) were positive for EHV-1 (16 neuropathogenic and 6 non-neuropathogenic strains), 4 (14.8 %) for EHV-2 and 3 (11.1 %) for EHV-5. The association between EHV infections and other etiological agents was statistically significant (two sided P?=?0.002). The odds ratio of EHV DNA associated with other diagnoses, especially with bacterial infection and premature placental separation, was 10.88 (95 % confidence interval: 2.15–55.16). EHV-1 was the main viral cause of pregnancy loss in this study, also associated with other etiological agents, including EHV-2 and EHV-5. The latter viruses in particular need to be more fully investigated to elucidate what role either or both may play as co-infecting agents with other established infectious causes of reproductive disease.     

Isolation of equine herpesvirus-2 from the lung of an aborted fetus.

This study describes the isolation of equine herpesvirus-2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti-EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause-effect relationship was established between the EHV-2 isolate and the abortion.     

Improving a Complement-fixation Test for Equine Herpesvirus Type-1 by Pretreating Sera with Potassium Periodate to Reduce Non-specific Hemolysis

Non-specific hemolysis has often been observed during complement-fixation (CF) tests for equine herpesvirus type-1 (EHV-1), even when the sera have virus-specific CF antibodies. This phenomenon has also been reported in CF tests for various infectious diseases of swine. We found that the sera from 22 of 85 field horses (25.9%) showed non-specific hemolysis during conventional CF testing for EHV-1. Because pretreatment of swine sera with potassium periodate (KIO₄) improves the CF test for swine influenza, we applied this method to horse sera. As we expected, horse sera treated with KIO₄ did not show non-specific hemolysis in the EHV-1 CF test, and precise determination of titers was achieved.