Antibody response to glycoprotein E after bovine herpesvirus type 1 infection in passively immunised, glycoprotein E-negative calves

This study was conducted to determine whether young calves with maternal antibodies against bovine herpesvirus type 1 (BHV-1) but without antibodies against glycoprotein E (gE) can produce an active antibody response to gE after a BHV-1 infection. Five calves received at birth colostrum from gE-seronegative cows which had been vaccinated two or three times with an inactivated BHV-1, gE-deleted marker vaccine. After inoculation with a wild-type virulent strain of BHV-1, all the passively immunised gE-negative calves shed virus in large amounts in their nasal secretions. All the calves seroconverted to gE within two to four weeks after inoculation and then had high levels of gE antibodies for at least four months. The development of an active cell-mediated immune response was also detected by in vitro BHV-1-specific interferon-gamma assays. All the calves were latently infected, because one of them re-excreted the virus spontaneously and the other four did so after being treated with dexamethasone. The results showed that under the conditions of this work the gE-negative marker could also distinguish between passively immunised and latently infected calves.     

Conventional and marked BHV-1 vaccines in Germany: a brief review

Since 1997 infections of cattle with bovine herpesvirus type 1 (BHV-1) are regulated by the German government with obligatory measures. Eradication of the virus is based on two different concepts: German federal states with a low BHV-1-seroprevalence comply with a selection concept, that provides deletion of BHV-1-positive cattle and only exceptional vaccination. States with a high BHV-1-prevalence attempt to eradicate the virus using the marker concept. The marker concept is based on the vaccination with glycoprotein E (gE)-deleted marker vaccines and the accompanying serological differentiation between vaccinated and infected animals. This review deals with both, conventional and marked BHV-1 vaccines, that are commercially available in Germany. Efficacy and safety of different inactivated vaccines and modified live vaccines have been evaluated in detail in several international experimental studies and field trials. Results of these studies are briefly reviewed and discussed in this article.     

广西陆川某猪场猪伪狂犬病病毒的分离鉴定及gE基因的分析

为了确诊猪伪狂犬病病毒(PRV)的感染,探讨目前广西猪群中流行的PRVgE基因的变异特征,为更好地防控猪伪狂犬病(PR)提供参考依据,本研究采集了广西陆川某猪场保育猪群发生呼吸道症状的肺脏组织,并用Vero细胞进行病毒分离,应用PCR方法对分离株的gE基因进行克隆和测序,根据测序结果证实分离到1个PRV毒株,命名为GXLC1。分离株在Vero细胞上增殖,细胞出现典型的病变;GXLC1株gE基因与GenBank上20株国内外具有代表性参考毒株的核苷酸序列同源性为97.1%~99.4%,氨基酸同源性为94.3%~99.6%;gE基因的遗传进化分析显示,GXLC1株与2012年的国内流行毒株亲缘关系较近,与欧美分离株亲缘关系较远;氨基酸序列分析显示,GXLC1株gE蛋白主要抗原表位区较之国内经典强毒株有3个氨基酸位点的变异,可能导致gE抗原性发生改变。本研究将分离到的1个PRV毒株进行了gE基因的分析,发现GXLC1株为近年来流行的变异株,gE蛋白在抗原表位区上有3个氨基酸位点的变异,这是否会影响GXLC1株毒力和抗原性的变化,还有待进一步研究;对gE基因变异特征的分析和病毒的分离为进一步丰富广西PRV的分子流行病学和疫苗的研制提供参考依据和重要材料。     

Isolation of a glycoprotein E-deleted bovine herpesvirus type 1 strain in the field

During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and PCR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.     

A study on latency in calves by five vaccines against bovine herpesvirus-1 infection

Four bovine herpesvirus-1 (BHV-1) commercial vaccines, three of which (vaccines B, D, E) were modified live vaccines (MLV) and one (vaccine A) identified as a live strain of BHV-1 gE negative, were used for vaccination of calves, using three calves for each vaccine. Three months after vaccination calves were subjected to dexamethasone (DMS) treatment following which virus was recovered from calves inoculated with vaccine B and from those given vaccine D. No virus reactivation was obtained in calves, which received vaccines A or E. The DNA extracted from the two reactivated viruses was subjected to restriction endonuclease analysis. The restriction pattern of the isolate obtained from calves vaccinated with vaccine D differs significantly from that of the original vaccine, whereas the reactivated virus from calves given vaccine B conserved the general pattern of the original vaccine strain. For each reactivated virus in this experiment (B and D) as well as for the isolate obtained from calves vaccinated with a further MLV (vaccine C) in a previous trial, three calves were inoculated. No clinical signs of disease were detected in any of the inoculated calves during the observation period. When the nine calves were exposed 40 days later to challenge infection with virulent BHV-1, they remained healthy and no virus was isolated from their nasal swabbings. These results indicate that some BHV-1 vaccines considered in the project can establish latency in the vaccinated calves, however, the latency does not appear to interfere with the original properties of the vaccines in terms of safety and efficacy.     

Establishment of latency associated with glycoprotein E (gE) seroconversion after bovine herpesvirus 1 infection in calves with high levels of passive antibodies lacking gE antibodies

This study was conducted to investigate the glycoprotein E (gE) antibody response raised after inoculation with a low infectious dose of bovine herpesvirus 1 (BHV-1) in six calves possessing high levels of passive immunity from cows repeatedly vaccinated with gE deleted marker vaccine. Four out of the six calves developed gE antibodies 3-5 weeks after infection, whereas the two other ones remained seronegative to gE. After 5 months of infection, the six calves were treated with dexamethasone. Virus was only re-excreted by the four calves which previously seroconverted against gE. The two other calves became seronegative against BHV-1, 30-32 weeks after infection. A second dexamethasone treatment performed 11 months after infection failed to demonstrate a latent infection in these two calves. Moreover, the lack of identification of a cell-mediated immune response, after the two dexamethasone treatments, and the failure to detect BHV-1 DNA sequences in trigeminal ganglia strongly suggest that these two calves were not latently infected. In conclusion, the presence of high levels of maternal immunity lacking gE antibodies does not prevent latency after infection with a low titre of BHV-1. Moreover, latency is associated with a serological response to gE. These results confirm that the gE deletion is a good marker to identify young calves latently infected with a field virus.     

Biological characterization of bovine herpesvirus 1 recombinants possessing the vaccine glycoprotein E negative phenotype

Intramolecular recombination is a frequent event during the replication cycle of bovine herpesvirus 1 (BoHV-1). Recombinant viruses frequently arise and survive in cattle after concomitant nasal infections with two BoHV-1 mutants. The consequences of this process, related to herpesvirus evolution, have to be assessed in the context of large use of live marker vaccines based on glycoprotein E (gE) gene deletion. In natural conditions, double nasal infections by vaccine and wild-type strains are likely to occur. This situation might generate virulent recombinant viruses inducing a serological response indistinguishable from the vaccine one. This question was addressed by generating in vitro BoHV-1 recombinants deleted in the gE gene from seven wild-type BoHV-1 strains and one mutant strain deleted in the genes encoding gC and gE. In vitro growth properties were assessed by virus production, one step growth kinetics and plaque size assay. Heterogeneity in the biological properties was shown among the investigated recombinant viruses. The results demonstrated that some recombinants, in spite of their gE minus phenotype, have biological characteristics close to wild-type BoHV-1.     

Differentiation of vaccine and field strains of bovine herpesvirus type 1 by restriction endonuclease analysis

Bovine herpesvirus type 1 (BHV-1) DNA molecules obtained from a limited number of infected cells were cleaved with a variety of restriction endonucleases. By use of selected DNA fragments in Bgl 1, Pst 1, Pvu 1 and Pvu 11 restriction patterns, one reference, two vaccine and three wild-type strains of BHV-1 were distinguished from one another. The simplified DNA fingerprinting method described here should be most useful, not only to control the genetic stability of BHV-1 vaccines during production, but also to differentiate the vaccine strains from other isolates in clinical cases.     

Diagnostic markers in the prevention of bovine herpesvirus type 1: possibilities and limitations

The usage of glycoprotein E (gE)-deleted BHV-1 marker or DIVA (differentiating infected from vaccinated animals) vaccines in combination with the accompanying determination of gE-specific antibodies is one of the basic parts of BHV-1 eradication programs in Germany. Following the adoption of the "Legislation for the protection of cattle holdings from an infection with Bovine Herpesvirus Type 1" in 1997, several million gE-antibody assays were conducted. Up to now, commercially available gE-blocking enzyme linked immunosorbent assays (ELISAs) are the only test systems available for the detection of gE-specific antibodies. However, there are some difficulties to meet the high requirements on their performance. Therefore, role and function of gE in the virus and the host as well as the principle of gE marker assays, the test design and the capability of the mainly used ELISAs are illuminated in this paper. Additionally, suggestions concerning the optimal usage and the correct application of gE-blocking-ELISAs are discussed.     

Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant

OBJECTIVE: To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1). PROCEDURE: The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests. RESULTS AND CONCLUSIONS: The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.     

A specific PCR to differentiate between gE negative vaccine and wildtype bovine herpesvirus type 1 strains

In the context of infectious bovine rhinotracheitis (IBR) control programmes using glycoprotein E (gE) deleted marker vaccines, a PCR assay was developed to allow the genotypic differentiation between wildtype bovine herpesvirus type 1 (BoHV-1) and gE negative strains. This assay is based on the PCR amplification of a 281 bp DNA fragment within the gE gene. The specificity of the amplification was confirmed by restriction endonuclease analysis and nucleotide sequencing of the PCR product. Its ability to determine the gE genotype of BoHV-1 strains was demonstrated on isolates coming from 20 experimental calves infected with four different BoHV-1 strains. This PCR assay may be a useful tool for monitoring the spread of live marker vaccine and the gE genotype of viral field isolates.     

豫南地区猪伪狂犬病病毒流行株的主要毒力基因遗传变异分析

为掌握豫南地区猪群中流行的伪狂犬病病毒（Pseudorabies virus,PRV）分子遗传特征,本研究利用PCR方法从PRV感染猪的组织中扩增其主要毒力基因gB、gE、gC、gD和TK,并进行核苷酸序列测定。利用MegAlign软件进行流行毒株的主要毒力基因与已发表的参考序列的相似性、进化树和关键位点氨基酸变异分析。测序结果表明,本研究成功从5份PRV感染猪组织中扩增出PRV的gB、gE、gC、gD和TK基因。氨基酸相似性和进化树分析结果表明,豫南地区的5株PRV流行株与国内流行毒株在G1群,与G1群国内流行毒株的gB、gE、gC和gD氨基酸相似性分别为98.5%~100%、97.2%~100%、97.5%~100%和98.3%~100%;与G2群亲缘关系较远（以Bartha、Becker、NiA3株为代表的欧美地区流行毒株）,与G2群内毒株的gB、gE、gC和gD氨基酸相似性分别为96.4%~97.4%、94.6%~95.7%、92.7%~94.0%和96.3%~99.0%。关键氨基酸变异分析结果表明,与Bartha株（或Becker和NiA3株等）相比,5株流行毒株的gB氨基酸存在75-77位"PGL"的缺失,94位"G"的插入;gE氨基酸存在48和496位有"D"的插入,gC氨基酸存在57-63位"VSGTTGA"的插入和65-69位"SPEAG"突变为"ASTPA",gD氨基酸存在278-281位"RP"或"RPRP"的插入。此外,流行株的gB、gE、gC、gD和TK氨基酸序列存在多个单位点的氨基酸突变。因此,豫南地区PRV流行株具有PRV变异毒株的分子遗传特征。上述结果证实,5株PRV流行毒株均为变异毒株,与疫苗毒株Bartha-K61株亲缘关系较远。     

猪伪狂犬病病毒囊膜糖蛋白E基因的序列结构分析

对猪伪狂犬病毒鲁A株(PRVLA株)gE基因进行了克隆和序列测定。应用DNAStar程序分析了PRV国内外各分离株以及PRV与HSV-1、BHV-1、EHV-1、CHV-1、MDV-1、SHV-SA8、SVV、ILTV的gE基因。PRV各分离株的gE基因有很高的同源性。不同疱疹病毒的gE核苷酸和氨基酸的同源性很低,但具有相似的结构。对上述α疱疹病毒的gE氨基酸序列进化树分析可以将其分为四个特定的组:单纯疱疹病毒组,水痘疱疹病毒组,类马立克氏病病毒组,传染性喉气管炎病毒组,推测α疱疹病毒的gE基因在进化上可能起源于ILTV的gE基因的或其前体基因。而且gE基因的进化趋势与其宿主的进化情况一致。     

Experimental infection of calves with a gI, gE, US9 negative bovine herpesvirus type 5

In this work, a role for the genes encoding glycoproteins I (gI) and E (gE) and the US9 protein of bovine herpesvirus type 5 (BHV-5) in neuropathogenicity and reactivation of latent infections was examined. Calves infected intranasally with a gI/gE/US9 deleted recombinant shed up to 10(2.85) TCID50/ml infectious virus in nasal secretions. Calves infected with the wild type BHV-5 parental virus shed up to 10(5) TCID50/ml virus. No signs of disease were observed in calves infected with the recombinant virus, whereas those infected with wild type virus displayed respiratory and neurological signs. The recombinant was only able to reach the basal portions of the central nervous system. In contrast, wild type virus was found widespread within the brain. Reactivation with dexamethasone 60 days post-infection resulted in reactivation of wild type virus, whereas the recombinant virus could not be reactivated. These studies demonstrate that genes gI, gE and US9 of BHV-5 are important for its neuropathogenicity and its ability to reactive from latency.     

Characterization of monoclonal antibodies directed against the bovine herpesvirus-1 glycoprotein E and use for the differentiation between vaccinated and infected animals

A panel of seven monoclonal antibodies (MAbs) directed against the bovine herpesvirus-1 (BHV-1) glycoprotein E (gE) was obtained. For that purpose, mice were either tolerized to BHV-1 gE-negative virus and then immunized with wild type BHV-1 or immunized with plasmid DNA expressing the gE and gI glycoproteins. The MAbs were characterized by their reactivity with the gE protein or the gE/gI complex and by competition experiments. Results showed that the MAbs were directed against three antigenic domains, two located on the gE glycoprotein and one on the gE/gI complex. Blocking experiments were performed with sera from experimentally vaccinated and infected cattle. A competition was observed between gE-positive bovine sera and six of the seven MAbs. The bovine sera thus recognized two of the three antigenic sites. Field sera were then tested in blocking enzyme-linked immunosorbent assay using one horseradish peroxidase-conjugated MAb. A specificity of 98.2% and a sensitivity of 98.2% compared to the commercially available test were observed.     

Tackling the issue of environmental survival of live Salmonella Typhimurium vaccines: Deletion of the lon gene

Vaccination is an important measure to control Salmonella contamination in the meat production chain. A previous study showed that both the ΔrfaJ and ΔrfaL strains are suitable markers and allow serological differentiation of infected and vaccinated animals. The aim of this study was to verify whether deletion of the lon gene in a Salmonella Typhimurium ΔrfaJ marker strain resulted in decreased environmental survival. Our results indicate that deletion of the lon gene in the ΔrfaJ strain did not affect invasiveness in IPEC-J2 cells and resulted in an increased susceptibility to UV, disinfectants (such as hydrogen peroxide and tosylchloramide sodium) and citric acid. Immunization of pigs with inactivated ΔrfaJ or ΔlonΔrfaJ vaccines allowed differentiation of infected and vaccinated pigs. Furthermore, deletion of the lon gene did not reduce the protection conferred by live wild type or ΔrfaJ vaccines against subsequent challenge with a virulent Salmonella Typhimurium strain in BALB/c mice. Based on our results in mice, we conclude that deletion of lon in ΔrfaJ contributes to environmental safety of the ΔrfaJ DIVA strain.     

伪狂犬病病毒FS-2015株gE和gB基因序列分析

为了解猪伪狂犬病病毒（porcine pesudorabies virus,PRV）gB和gE基因变异及遗传演化情况,本研究针对PRV FS-2015野毒株,应用"蚀斑法"对组织病料中病毒进行三轮纯化,应用全长扩增引物对FS-2015株gB和gE基因进行全基因扩增,并对PCR产物进行测序和序列分析。结果显示,PRV FS-2015株的gB、gE基因与国内外PRV参考毒株的核苷酸同源性分别为97.0%~100.0%和97.5%~99.7%,氨基酸同源性分别为96.4%~100.0%和95.3%~99.7%。氨基酸变异位点分析表明,FS-2015株的gB和gE基因均有位点突变和缺失。遗传进化分析表明,FS-2015株与国内近几年分离的PRV变异株GY、ZJ01、HB1201、HN1201、JS2012、BJ-YT和BP属于同一分支,同源性较高,亲缘关系较近;与PRV经典株Kaplan、Becker、NIA3、Kolchis、Bartha和Yangsan株属于不同分支,同源性较低,亲缘关系较远。从PRV FS-2015毒株与国内外经典毒株和当前国内流行的变异毒株的分析结果可知,PRV FS-2015毒株发生了一定的变异,属于当前国内流行变异毒株。本研究结果为广东省伪狂犬病分子流行病学调查、伪狂犬病的防控和疫苗株挑选工作提供参考数据。     

BHV-1 vaccine induces cross-protection against BHV-5 disease in cattle

Protection against BHV-5 disease induced by inactivated BHV-1 or BHV-5 based vaccines was analysed. Two groups of calves were subcutaneously immunized with an inactivated BHV-1 or BHV-5 based vaccine. A third group was not vaccinated and used as control. In the post-vaccination period, we studied the humoral and cellular immune response resulting similar to both groups. The efficacy of the vaccines was tested after intranasal challenge of the calves with a virulent Argentinean BHV-5 isolate (A-663). All control animals developed neurological signs associated with BHV-5 infection and high levels of virus shedding. Calves immunized with the BHV-1 and BHV-5 inactivated vaccines were protected against BHV-5 disease. Our study provides evidence that strongly support the existence of cross-protection between BHV-1 and BHV-5 in calves. Even though this has already been suggested by previous works, this is the first time an exhaustive study of the immune response is performed and typical clinical BHV-5 meningoencephalitis signs are reproduced in an experimental BHV-5 challenge trial.     

猪伪狂犬病病毒JSSQ2013株的分离鉴定及重要功能基因序列分析

为了解江苏省猪伪狂犬病病毒(Pseudorabies virus,PRV)野毒株的特点,本研究从2013年采自江苏省宿迁市的疑似PRV感染病料中分离纯化了一株PRV病毒,对其进行了PCR和间接免疫荧光法(IFA)鉴定,并进一步在Vero细胞上测定该分离株的病毒滴度TCID50和一步生长曲线,扩增其gB、gC、gD和gE基因进行序列比对及分子遗传进化分析,并将该分离株分别接种新西兰白兔和15日龄仔猪研究其致病性。结果显示,该病毒为一株PRV,命名为PRV JSSQ2013株,纯化后的病毒滴度为10^7.8 TCID50/ml;生长曲线测定显示在感染20h后病毒滴度即达到最高,为10^8.6 TCID50/ml。与我国近几年分离的PRV变异株序列相比,PRV JSSQ2013株的gB、gC、gD和gE基因核苷酸序列同源性分别为99.5~99.6%、99.5~99.6%、99.5~99.6%和98.7~99.7%,氨基酸序列同源性分别为98.9~99.0%、99.5~99.7%、99.0~99.2%和98.1~99.3%,均高于其与经典毒株(Ea、Fa和SC株)和欧美毒株(Becker、Kaplan、Bartha、Kolchis和NIA3)的同源性;基于gB、gC、gD和gE基因的遗传进化树分析均显示PRV JSSQ2013株与国内近几年分离的PRV变异株属同一分支。该病毒接种新西兰白兔后均出现典型的PR症状,如厌食、兴奋、啃咬或用爪挠接种部位等典型症状,且在48h内全部死亡;接种仔猪后第1天开始出现典型的PR症状,第5天全部死亡。以上结果证实,从江苏省宿迁市采集的疑似PRV感染病料中分离到一株强毒力的PRV变异株。本研究为了解江苏PRV分子流行特征、丰富我国PRV分子流行病学资料及新型疫苗的研制奠定了基础。     

Effects of hypervaccination with bovine herpesvirus type 1 gE-deleted marker vaccines on the serological response and virological status of calves challenged with wild-type virus

Twenty-four calves were immunised four times with gE-deleted infectious bovine rhinotracheitis marker vaccines before being challenged with small doses of wild-type bovine herpesvirus type 1 (BHV-1). The repeated vaccinations induced strong immunity that prevented detectable virus replication and gE-seroconversion after the challenge infection in most of the calves. The hypervaccinated calves that shed virus after the challenge infection showed no delay in gE-seroconversion compared with unvaccinated control calves. Using a sensitive nested PCR, BHV-1 gE sequences could be detected in the trigeminal ganglia of several of the gE-seronegative, challenge-infected calves, possibly indicating the presence of wild-type BHV-1 DNA.