Molecular characterization of equine herpesvirus 1 (EHV-1) isolated from cattle indicating no specific mutations associated with the interspecies transmission

Interspecies trasmission of equine herpesvirus 1 (EHV-1) from horse to cattle was shown by Crandell et al. (1988). Specific mutations related to the transmission were studied by comparison of five EHV-1 isolates in cattle (BH1247, 3M20-3, G118, G1753, and 9BSV4) using polymerase chain reaction and restriction fragment length polymorphism analysis with added sequencing. G118 and 3M20-3 were the genome type EHV-1 P, while G1753 was the genome type EHV-1 B. BH1247 and 9BSV4 might be other genome types. We could not identify specific mutations related to the interspecies transmission.     

应用一步PCR法检测并鉴定马疱疹病毒1型和4型

聚合酶链反应（PCR）可作为一种快速检测并鉴别马疱疹病毒1型（EHV－1）和马疱疹病毒4型（EHV－4）的诊断方法。PCR引物是根据编码EHV－1和EHV－4的糖蛋白B（ gB)基因上的共有的核苷酸序列或型特有的核苷酸序列设计的。利用这两种病毒的型特异性混合引物,在一步PCR反应中检测并鉴别EHV－1和EHV－4,而同一疱疹病毒科的单纯疱疹病毒1型（HSV－1）、伪狂犬病毒（PRV）、马立克氏病病毒（MDV）均无特异性扩增。应用建立的PCR方法检测了普氏野马流产胎儿病科,实验结果表明,这种PCR方法是一种直接检测并鉴定EHV－1和EHV－4的快速、敏感的诊断方法,同时,它可在一步PCR反应中直接鉴别这两种病毒,可用于病料中EHV－1和EHV－4检测的初步筛选。     

Growth and virulence alterations of equine herpesvirus 1 by insertion of a green fluorescent protein gene in the intergenic region between ORFs 62 and 63

Nucleotide sequences of the intergenic region between ORF 62 and ORF 63 of equine herpesvirus 1 (EHV-1) isolates were analyzed. The sequences of this region consisted of variable and conserved domains among EHV-1 isolates. An EHV-1 mutant, Ab4-GFP, was constructed by inserting a green fluorescent protein (GFP) expression cassette flanked by lox P at both ends into the intergenic region between ORF 62 and ORF 63. Another mutant, Ab4-loxP, which contains one lox P site, was constructed by excision of the GFP cassette from the Ab4-GFP virus genome by cre enzyme. The recombinant Ab4-GFP formed smaller plaques than the wild type in MDBK cells. Virus production also decreased for Ab4-GFP in multistep growth analyses. Virulence of Ab4-GFP in both mice and hamsters was weaker than that of the wild type. Ab4-loxP exhibited properties similar to those of the wild type. These results suggest that the intergenic region between ORF 62 and ORF 63 plays various roles in the virus growth.     

The equine herpesvirus 1 (EHV-1) UL3 gene, an ICP27 homolog, is necessary for full activation of gene expression directed by an EHV-1 late promoter.

We have previously reported that the equine herpesvirus 1 (EHV-1) XbaI G restriction fragment (nucleotides 1436 to 7943 relative to the left terminus of the EHV-1 genome [Kentucky A strain]) is required in combination with the EHV-1 immediate-early (IE) gene to achieve significant activation of two representative EHV-1 late promoter-chloramphenicol acetyltransferase (CAT) recombinants in transient expression assays. In this report, we demonstrate that the XbaI G-encoded UL3 gene (an ICP27 homolog) provides a trans-acting factor which acts (in combination with the EHV-1 IE gene product) to increase reporter gene expression directed by an EHV-1 late promoter-CAT recombinant plasmid. We show that cloned copies of UL3 can successfully substitute for the XbaI G fragment in CAT assays and that stop codon insertion within the UL3 open reading frame inhibits the ability of UL3 to activate reporter gene expression in trans.     

Regulatory function of the equine herpesvirus 1 ICP27 gene product.

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.     

Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction.

Although both equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) can be associated with respiratory disease, epizootics caused by EHV-1 are much more serious because the virus can cause abortions and paralysis. It is, therefore, important to identify the type of EHV involved in an outbreak by a test that is quick, sensitive, and reliable. We have adapted the polymerase chain reaction (PCR) to detect and distinguish between EHV-1 and EHV-4 in the same reaction. Primers for PCR were designed from the sequences of the glycoprotein B genes of EHV-1 and EHV-4. The PCR products derived from EHV-1 and EHV-4 were 135 and 326 base pairs, respectively, and could be readily separated by electrophoresis. The identity of the PCR products was confirmed by determining their nucleotide sequence, which agreed with the published sequence of the gB genes. The test could be performed directly on virus pelleted from small volumes (300 microL) of medium in which nasal swabs were transported and did not rely on the presence of infectious virus. The PCR was unaffected by conditions that reduced the infectivity of a virus preparation by 99%. The PCR detected EHV-4 in 5 of 10 nasal mucous samples taken from an outbreak of respiratory disease in race horses. Virus isolation in indicator cells was successful in detecting virus in four of the five samples positive by PCR.     

Coexpression by vaccinia virus recombinants of equine herpesvirus 1 glycoproteins gp13 and gp14 results in potentiated immunity.

The equine herpesvirus 1 glycoprotein 14 (EHV-1 gp14) gene was cloned, sequenced, and expressed by vaccinia virus recombinants. Recombinant virus vP613 elicited the production of EHV-1-neutralizing antibodies in guinea pigs and was effective in protecting hamsters from subsequent lethal EHV-1 challenge. Coexpression of EHV-1 gp14 in vaccinia virus recombinant vP634 along with EHV-1 gp13 (P. Guo, S. Goebel, S. Davis, M. E. Perkus, B. Languet, P. Desmettre, G. Allen, and E. Paoletti, J. Virol. 63:4189-4198, 1989) greatly enhanced the protective efficacy in the hamster challenge model over that obtained with single recombinants. The inoculum doses (log10) required for protection of 50% of hamsters were 6.1 (EHV-1 gp13), 5.2 (EHV-1 gp14), and less than 3.6 (vaccinia virus recombinant expressing both EHV-1 glycoproteins [gp13 and gp14]).     

Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4

The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T _m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15 pg/μl for EHV-1 and 5 copies/μl or 2.5 fg/μl for EHV-4. This assay was 50–1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.     

Mutational analysis of the ICP4 binding sites in the 5'' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene.

A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.     

TLR4全长及其截断体重组腺病毒的制备和功能鉴定

制备脂多糖 (LPS)Toll样受体 4 (TLR4 )全长及其胞内段缺失的TLR4截断体 (ΔTLR4 )的绿色荧光蛋白重组腺病毒并鉴定其功能 .用PCR方法扩增TLR4及ΔTLR4基因片段 ,酶切后亚克隆至腺病毒穿梭质粒中 ,形成带有目的基因的穿梭载体pAdTrack TLR4和pAdTrack ΔTLR4 .用BJ5 1 83细菌同源重组法将目的基因重组于腺病毒骨架载体中 ;将重组腺病毒质粒用PacⅠ酶切线性化后 ,用脂质体法转染HEK 2 93细胞进行腺病毒的包装扩增 .将重组腺病毒感染CHO K1细胞 ,采用荧光毒酶报告基因方法检测其对LPS诱导NF κB激活的影响 .酶切及测序表明 ,TLR4全长及其截断体ΔTLR4的重组腺病毒载体构建正确 .荧光素酶报告基因检测结果表明 ,TLR4全长及其截断体的重组腺病毒感染细胞对LPS诱导的反应具有不同的影响 ,Ad ΔTLR4明显抑制了LPS引起的NF κB激活 (P <0 0 5 ) ,Ad TLR4则使LPS引起的NF κB活性增强 (P <0 0 5 ) .LPS对细胞的激活作用依赖于TLR4的结构完整性     

Glycoproteins D of equine herpesvirus type 1 (EHV-1) and EHV-4 determine cellular tropism independently of integrins

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVβ3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.     

An improved cosmid vector for the cloning of equine herpesvirus DNA

We have modified the commercial cosmid vector, triple helix vector (THV), such that I-Sce-I restriction endonuclease sites flank the cloning site. I-Sce-I is a rare-cutting endonuclease which recognizes an 18-bp sequence. It does not restrict the genome of either of the equine herpesvirus 1 or 4 (EHV-1 and EHV-4) strains we have cosmid cloned. Thus, cosmid- cloned EHV fragments can be excised intact from the vector by I-Sce-I digestion, facilitating production of large overlapping EHV fragments for use in transfections to produce recombinant virus.     

Equid Herpesvirus Type 1 Activates Platelets

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.     

小鼠B7-H4基因的真核表达及其对淋巴细胞增殖的影响

目的构建小鼠B7-H4胞外段的真核表达载体,观察其在体外对淋巴细胞增殖的影响,为深入研究B7-H4在T细胞活化及移植排斥反应中的作用提供实验材料。方法提取小鼠肺、脾脏总RNA,RT-PCR反转录cDNA,以此为模板,扩增B7-H4胞外段基因,将其导入pGEM-T Easy载体,构建TA-mB7-H4质粒。用XBaI和HindIII双酶切后琼脂糖凝胶电泳分析和测序鉴定。将测序证实的mB7-H4酶切后装入MYC-HIS-EGFP-N荧光表达载体中,构建B7-H4-EGFP真核表达载体,转化JM109感受态细菌,提取重组质粒,酶切后琼脂糖凝胶电泳分析和测序鉴定。同时构建control-EGFP载体。应用脂质体法将重组质粒转染CHO细胞,经G418筛选,获得稳定表达B7-H4-EGFP的CHO细胞株,用MTT分析其分别对BALB/c小鼠、C57小鼠淋巴细胞和二者混合淋巴细胞增殖的影响。结果经测序证实,所克隆的小鼠B7-H4 cDNA和构建的重组质粒基因序列正确;转染的CHO细胞能稳定地表达跨膜型重组蛋白B7-H4;表达的B7-H4对淋巴细胞增殖具有明显抑制作用。结论成功构建了B7-H4真核表达系统,能表达有生物学活性的B7-H4分子,为进一步探讨B7-H4在T细胞活化和移植排斥反应中的作用奠定了基础。     

应用痘苗病毒为载体表达人白细胞介素4的研究

在真核细胞中表达近似于人类天然糖基化的白细胞介素４（ｈＩＬ－４）,为重要的研究课题。利用基因工程技术,以痘苗病毒作为载体,使之在真核细胞中表达ｈＩＬ－４,为生产糖基化的ｈＩＬ－４开辟新的可能途径。在痘苗病毒表达载体ｐＪ１２０质粒的基础上,组建了含有ｈＩＬ－４基因的ｐＪｌ２０／ｈＩＬ－４重组质粒,该质粒以痘苗病毒的胸腺嘧啶核苷激酶（ＴＫ）基因为侧翼序列,中间插入痘苗病毒的Ｐ１１和Ｐ２５两个转录方向相反的启动子,Ｐ２５的下游连接β－半乳糖苷酶（ＬａｃＺ）基因,ｈＩＬ－４基因在痘苗病毒启动子Ｐ１１控制下进行表达,将ＰＪ１２０／ｈＩＬ－４质粒ＤＮＡ用脂质体转染法导入ＴＫ阴性的骨髓瘤细胞（ＴＫ１４３细胞）,与预先感染的痘苗病毒在细胞内进行同源序列重组,并且用病毒的核酸杂交鉴定证实,ｈＩＬ－４基因已重组到痘苗病毒基因组中,用ｈＩＬ－４应答细胞株检测病毒感染细胞上清,发现重组病毒感染的细胞上清中含有高滴度的ｈＩＬ－４活性,并对此种情况下ｈＩＬ－４产生的条件进行了动态观察。     

Expression of equine herpesvirus 1 glycoprotein D by using a recombinant baculovirus.

Glycoprotein D (gD) of equine herpesvirus 1 (EHV-1) was expressed at the surface of insect cells infected by a recombinant baculovirus. EHV-1 gD was detected as multiple forms (56, 52, and 48 kDa) from 18 to 96 h postinfection. Laboratory animals inoculated with the recombinant EHV-1 gD developed neutralizing antibody responses against both EHV-1 and EHV-4.     

Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR)

In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility.Samples of stallion semen (n = 390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test.EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p < 0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs.There was a significant difference (p < 0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing.In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.