猪2型圆环病毒ORF1与ORF2基因和伪狂犬病毒基因的重组与表达的研究

根据GenBank发布的猪2型圆环病毒(PCV2 )序列(AY0 35 82 0 ) ,设计两对特异性引物,采用PCR方法,分别扩增了猪2型圆环病毒ORF1和ORF2基因。将ORF1和ORF2基因的PCR产物回收并酶切后,依次插入到伪狂犬病毒gE gI双缺失通用转移载体pIECMV中,构建了猪2型圆环病毒_伪狂犬病毒重组中间转移质粒pIEORF1-ORF2。采用脂质体介导法,将重组中间转移质粒pIEORF1_ORF2与伪狂犬病毒TK^- gE^- LacZ⁺ 基因组共转染IBRS_2细胞,待发生细胞病变后收集病毒液进行空斑纯化,利用检测PCV2ORF1基因和ORF2基因的PCR方法筛选重组病毒TK^- gE^- gI^- ORF1-ORF2⁺ ,用Southernblotting鉴定重组病毒,并用Westernblotting检测ORF1_ORF2融合蛋白的表达情况,在此基础上也测定了重组病毒在不同细胞上的增殖滴度。结果表明,外源基因ORF1和ORF2已成功插入到TK^- gE^- LacZ⁺ 亲本株的基因组中,并获得了表达,表达的蛋白可与PCV2阳性血清发生反应。同时发现ORF1和ORF2基因的插入不影响重组病毒的增殖特性,其毒力与亲本株相当。     

猪伪狂犬病毒鲁A株TK基因的序列测定及其结构功能与进化分析

对猪伪狂犬病毒鲁A株(PRV LA株)TK基因进行了克隆和序列测定,并分析比较了该序列与PRV NIA-3株、Ea株、SH以及HSV-1和VZV的同源性,结果表明:在全长1048bp的DNA序列中,包括着一个963bp的开放阅读框(ORF),可编码320个氯基酸组成的多肽;在整个TK基因的ORF内,PRV LA株与PRV NIA-3株、PRV Ea株、PRV SH株、HSV-1、VZV的TK基因比较,核苷酸的同源性分别为98.9%、99.5%、99.3%、36.4%、39.1%,氨基酸的同源性分别为98.4%、99.7%、98.7%、36.6%、37.2%.PRV LA株TK具有疱疹病毒胸苷激酶催化结构域的保守氨基酸共有序列和亚结构域特征序列.将PRV-LA TK、人类和小鼠的胸苷酸激酶、人类脱氧胞苷激酶、人类腺苷酸激酶的对应于这两个亚结构域的氨基酸用DNA Star分析的进化树表明,疱疹病毒的TK与人类和小鼠的胸苷酸激酶的亲缘关系比与人类脱氧胞嘧啶激酶的亲缘关系更近,因此疱疹病毒的TK基因在进化上可能起源于宿主细胞的胸苷酸激酶基因.     

Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1.

In the Alphaherpesvirinae subfamily, the gE and gI genes are conserved and encode membrane glycoproteins required for efficient pathogenesis (virulence). The molecular mechanism(s) responsible is not well understood, but the existence of similar phenotypes of gE and gI mutations in diverse Alphaherpesvirinae implies conservation of function(s). In this report, we describe construction of pseudorabies virus (PRV) recombinants that efficiently express the bovine herpesvirus 1 (BHV-1) membrane proteins gI and gE at the PRV gG locus. Each BHV-1 gene was cloned in a PRV mutant lacking both the PRV gI and gE coding sequences. All recombinant viruses expressed the BHV-1 proteins at levels similar to or greater than that observed after infection with parental BHV-1, and there were no observable differences in processing or ability to form gE-gI oligomers. The important observation resulting from this report is that the BHV-1 gE and gI proteins functioned together to complement the virulence defect of PRV lacking its own gE and gI genes in a rodent model, despite being derived from a highly restricted host range virus with a different pathogenic profile.     

Potential sites of virus latency associated with indigenous pseudorabies viruses in feral swine

Free-ranging feral swine (Sus scrofa) are known to be present in at least 32 states of the USA and are continuously expanding their range. Infection with pseudorabies virus (PRV) occurs in feral swine and the primary route of transmission in free-living conditions seems to be venereal. Between 1995 and 1999, naturally infected feral swine and experimentally infected hybrid progeny of feral and domestic swine, were kept in isolation and evaluated for occurrence of latent PRV indigenous to feral swine in sacral and trigeminal ganglia and tonsil. Sacral ganglia were shown, by polymerase chain reaction (PCR) amplification of the thymidine kinase (TK) gene of PRV, to be the most frequent sites of latency of PRV. Nine (56%) of 16 sacral ganglia, seven (44%) of 16 trigeminal ganglia, and five (39%) of 13 tonsils from naturally infected feral swine were positive for PCR amplification of TK sequences of PRV. These tissues were negative for PRV when viral isolation was attempted in Vero cells. DNA sequencing of cloned TK fragments from the sacral ganglia of two feral swine, showed only one nucleotide difference between the two fragments and extensive sequence homology to fragment sequences from various domestic swine PRV strains from China, Northern Ireland, and the USA. The hybrid feral domestic swine, experimentally inoculated with an indigenous feral swine PRV isolate by either the genital or respiratory route, acquired the infection but showed no clinical signs of pseudorabies. Virus inoculated into either the genital or respiratory tract could, at times, be isolated from both these sites. The most common latency sites were the sacral ganglia, regardless of the route and dose of infection in these experimentally infected hybrids. Nine of 10 sacral ganglia, six of 10 trigeminal ganglia, and three of 10 tonsils were positive for PCR amplification of TK sequences. No virus was isolated from these tissues in Vero cells. The demonstration of the sacral ganglia as the most common sites of latency of pseudorabies viruses indigenous to feral swine, supports the hypothesis that these viruses are primarily transmitted venereally, and not by the respiratory route as is common in domestic swine, in which the trigeminal ganglia are the predominant sites of virus latency.     

伪狂犬病病毒基因缺失疫苗制苗用毒种特性研究

本试验通过对PRV基因缺失株SA215(gE-/gI-/TK- )细胞培养特性、理化特性及形态发生过程进行研究,来确定gE、gI和TK 基因缺失对病毒特性的影响。结果表明,基因缺失对该毒株在培养细胞的吸附和穿入过程没有影响,但与亲本株相比,表现为生长掩蔽期延长,增殖速度减缓,但能达到相似的增殖滴度,并且基因缺失对病毒的理化特性影响很小。形态发生过程观察结果表明,PRV SA215 株在细胞培养上形态发生正常,能形成感染性病毒粒子,但在由核膜出芽和囊膜形成上受到一定程度的阻碍。本研究为疫苗研制和生产提供指导和依据。     

伪狂犬病毒gI基因的克隆表达及其对病毒增殖的影响

从伪狂犬病毒(PRV)国内地方分离Ea株基因组DNA片段中克隆了完整的gI基因,序列分析结果表明,gI基因编码区全长1101bp,可编码366个氨基酸残基,二级结构预测具有典型I型膜蛋白特征。与GenBank中收录的国外Rice株的同源比较发现,Ea株gI在核苷酸和氨基酸水平上均存在多处突变,尤其是潜在胞浆区中连续两个碱基的缺失导致移码突变,致使gI基因的读码框架后移,从而导致Ea株gI较rice株长16个氨基酸残基。将gI基因克隆到真核表达载体pcDNA31+中的人巨细胞病毒早期启动子下游,构建的真核表达质粒转染PK15细胞,间接免疫荧光检测证实gI获得正确表达。进一步测定天然缺失gI的PRV弱毒Bartha株在表达gI细胞系和空白载体转染的对照细胞系中的蚀斑形成单位(pfu)和组织细胞培养半数感染量(TCID50),结果显示：Bartha株在表达gI细胞系中的pfu和TCID\-\{50\}分别为对照细胞系的164%和200%。说明gI具有促进病毒增殖的功能。     

The gE and gI homologs from two alphaherpesviruses have conserved and divergent neuroinvasive properties.

The membrane glycoproteins gE and gI are encoded by pseudorabies virus (PRV), a neurotropic, broad-host-range alphaherpesvirus of swine. PRV gE and gI are required for anterograde spread to a restricted set of retinorecipient neurons in the brain after infection of the rat retina. A related alphaherpesvirus, encoding gE and gI homologs, is called bovine herpesvirus 1.1 (BHV-1.1). BHV-1.1 is a respiratory pathogen of highly restricted host range and, in contrast to PRV, is unable to propagate in or cause disease in rodents. We have shown previously that the BHV-1.1 gE and gI proteins are capable of complementing the virulence functions of PRV gE and gI in a rodent model (A. C. Knapp and L. W. Enquist, J. Virol. 71:2731-2739, 1997). We examined the ability of the BHV-1.1 gE and gI homologs to direct circuit-specific invasion of the rat central nervous system by PRV. Both complete open reading frames were cloned into a PRV mutant lacking the PRV gE and gI genes. Recombinant viruses were analyzed for the ability to invade the rat brain after infection of the retina. Surprisingly, in a portion of the animals tested, the BHV-1.1 gE and gI proteins functioned autonomously to promote spread of PRV to a subset of retinorecipient regions of the brain. First, the presence of BHV-1.1 gI alone, but not PRV gI alone, promoted viral invasion of the optic tectum. Second, expression of BHV-1.1 gE alone facilitated PRV infection of a subset of neurons in the hippocampus not normally infected by PRV. When both BHV-1.1 proteins were expressed in a coinfection, all retinorecipient regions of the rat brain were infected. Therefore, depending on the viral source, homologs of gE and gI differentially affect spread between synaptically connected neurons in the rat.     

插入单个loxP位点的伪狂犬病病毒上海株TK基因缺失株的构建

根据GenBank已发表的pEGFP－C1序列,设计并合成两对引物,PCR扩增出两端各含一loxP位点的GFP表达盒GFP-loxP。克隆于转移载体pSKLR获得pSKLR-GFP-loxP。基于同源重组原理, pSKLR-GFP-loxP与 PRV SH株基因组DNA共转染293T细胞,在BrdU 的筛选压力下,利用蚀斑法在TK-143细胞上筛选出表达GFP的TK基因缺失的重组毒株rPRV1。将表达Cre酶的质粒载体pPOG231与rPRV1基因组DNA共转染293T细胞,在Cre酶的作用下去除GFP表达盒以及一个loxP位点,筛选得到含单个loxP位点的重组病毒株rPRV2。PCR 扩增证实所获得的重组病毒TK缺失270bp,只有一个34bp的loxP位点,并且能在RK-13细胞上稳定传代。LD50试验表明rPrV2的毒力下降。     

Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins.

Transneuronal transport of pseudorabies virus (PRV) from the retina to visual centers that mediate visual discrimination and reflexes requires specific genes in the unique short region of the PRV genome. In contrast, these same viral genes are not required to infect retinorecipient areas of the brain involved in circadian rhythm regulation. In this report, we demonstrate that viral mutants carrying defined deletions of the genes encoding glycoprotein gI or gp63, or both, result in the same dramatic transport defect. Efficient export of either gI or gp63 from the endoplasmic reticulum to the Golgi apparatus in a fibroblast cell line requires the presence of both proteins. We also show that gI and gp63 physically interact, as demonstrated by pulse-chase and sucrose gradient sedimentation experiments. Complex formation is rapid compared with homodimerization of PRV glycoprotein gII. We suggest that gI and gp63 function in concert to affect neurotropism in the rat visual circuitry and that a heterodimer is likely to be the unit of function.     

Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins.

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.     

The attenuated pseudorabies virus strain Bartha fails to package the tegument proteins Us3 and VP22

The Bartha strain of pseudorabies virus has several recognized mutations, including a deletion in the unique short region encompassing the glycoprotein I (gI), gE, Us9, and Us2 genes and point mutations in the gC, gM, and UL21 genes. We have determined that Bartha has mutations in the serine/threonine kinase encoded by the Us3 gene relative to the wild-type Becker strain. Our analysis revealed that Becker virions contain the Us3 protein, whereas Bartha virions do not. To test whether the mutations in the Bartha Us3 protein were responsible for this observation, we constructed a recombinant Bartha strain, PRV632, which expresses the Becker Us3 protein. PRV632 failed to package Us3 into the tegument, indicating that mutations other than those in the Us3 primary amino acid sequence were responsible for the failure of Bartha to package its Us3 protein. A recombinant Becker strain, PRV634, which expresses the Bartha Us3 protein, was constructed to test whether it was capable of being packaged into virions. The Bartha Us3 protein was not incorporated into PRV634 virions efficiently, suggesting that the primary sequence of the Bartha Us3 protein affects packaging into the tegument. To determine whether the packaging of other tegument proteins was affected in the Bartha strain, we examined VP22. Whereas Becker packaged VP22 into virions, Bartha had a severe deficiency in VP22 incorporation. Analysis of VP22 expression in Bartha-infected cells revealed that Bartha VP22 had a slower mobility on sodium dodecyl sulfate-polyacrylamide gels, indicating either primary sequence differences and/or different posttranslational modifications relative to Becker VP22. Taken together, these data indicate that, while the primary sequence of the Us3 protein does affect its incorporation into the tegument, other factors are involved. Furthermore, our data suggest that one or more of the gI, gE, Us9, or Us2 genes influences the localization of the Us3 protein in infected cells, and this effect may be important for the proper incorporation of Us3 into virions.     

伪狂犬病病毒鄂A株TK－／gG－／LacZ＋突变株的构建

为了以国内地方分离株鄂A株为亲本构建伪狂犬病病毒双基因缺失株,采用外切酶Ⅲ和绿豆核酸酶酶切,构建了缺失主要毒力基因TK基因部分编码区的重组质粒pSTK1-4,进一步改造成为转移质粒pUCPB4。用HindⅢ将质粒pUCPB4线性化,然后与用EcoRI消化的伪狂犬病病毒鄂A株TK^-/LacZ^ 突变株基因组DNA共转染PK－15细胞,等完全病变后,收毒作空斑试验,PCR筛选TK缺失的重组病毒。重组病毒空斑纯化3次,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的无TK^-/LacZ^ 突变株污染的TK缺失的重组病毒,分别以TK缺失株病毒与鄂A野毒株为模板对TK基因进行PCR扩增,扩增产物经酶切分析和测序后发现：TK缺失重组病毒的TK基因缺失了205个碱基,XhoⅠ和SalⅠ位点消失,SmaⅠ酶切片段发生变化,两种病毒在PK－15细胞上形成空斑的大小和增殖 滴度无明显的差别。进一步提取TK缺失突变株基因组DNA,与含gG-LacZ的转移质粒pUSKZ通过磷酸钙法共转染PK－15细胞,待完全病变后,在X-gal存在下筛选蓝斑,将桃取的蓝斑纯化3次后,对纯化的重组病毒进行TK、LacZ扩增,结果既能扩增出较以鄂A野毒株为模板扩增的要小的TK基因片段,同时又能扩增出LacZ基因,证实所得到的重组病毒为TK^-/gG^-/LacZ^ 突变株。此双缺失突变株的构建成功,为在我国最终根除伪狂犬病提供了有用的工具。     

Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.

We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.     

表达猪繁殖与呼吸综合征病毒GP3基因的重组伪狂犬病病毒的构建

猪繁殖与呼吸综合征 (porcinereproductiveandrespiratorysyndrome ,PRRS)是引起怀孕母猪早产、流产、死胎及仔猪呼吸系统疾病的一种新发现的病毒性传染病[1] .该病毒的基因组为单股正链RNA ,约15kb ,含有 8个开放阅读框架 (ORFs) ,ORF1编码病毒非结构蛋白 (依赖RNA的RNA聚合酶 ) ,ORF2 ORF7编码病毒的结构蛋白 .其中ORF3含有 2 6 5个氨基酸 ,编码的GP3蛋白为高度糖基化的结构蛋白 ,有 7个糖基化位点 ,具有免疫原性[2 ,3 ] .目前 ,用于预防PRRS的疫苗主要是弱毒苗和灭活苗 ,虽然都有一定的免疫效果 ,但由于PRRS抗体依赖性…     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK-/gE-/GP5+expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV),based on the PRV genetically depleted vaccine strain TK-/gE-/LacZ+,scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene (the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK-/gE-/LacZ+,resulting in a PRV recombinant named TK-/gE-/GPSm+,which expressed the modified GPSm protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK-/gE-/GPSm+and TK-/gE-/GP5+expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK-/gE-/GPSm+against PRRSV were higher than that induced by TK-/gE-/GP5+.Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.     

Varicella-Zoster Virus Glycoprotein I Is Essential for Spread in Dorsal Root Ganglia and Facilitates Axonal Localization of Structural Virion Components in Neuronal Cultures

Neurons of the sensory ganglia are the major site of varicella-zoster virus (VZV) latency and may undergo productive infection during reactivation. Although the VZV glycoprotein E/glycoprotein I (gE/gI) complex is known to be critical for neurovirulence, few studies have assessed the roles of these proteins during infection of dorsal root ganglia (DRG) due to the high human specificity of the virus. Here, we show that the VZV glycoprotein I gene is an important neurotropic gene responsible for mediating the spread of virus in neuronal cultures and explanted DRG. Inoculation of differentiated SH-SY5Y neuronal cell cultures with a VZV gI gene deletion strain (VZV rOkaΔgI) showed a large reduction in the percentage of cells infected and significantly smaller plaque sizes in a comparison with cultures infected with the parental strain (VZV rOka). In contrast, VZV rOkaΔgI was not significantly attenuated in fibroblast cultures, demonstrating a cell type-specific role for VZV gI. Analysis of rOkaΔgI protein localization by immunofluorescent staining revealed aberrant localization of viral glycoprotein and capsid proteins, with little or no staining present in the axons of differentiated SH-SY5Y cells infected with rOkaΔgI, yet axonal vesicle trafficking was not impaired. Further studies utilizing explanted human DRG indicated that VZV gI is required for the spread of virus within DRG. These data demonstrate a role for VZV gI in the cell-to-cell spread of virus during productive replication in neuronal cells and a role in facilitating the access of virion components to axons.     

Transformation of Dendrobium orchid using particle bombardment of protocorms

Transformed dendrobium orchids (Dendrobium x Jaquelyn Thomas hybrids) were recovered from protocorms bombarded by particles coated with the plasmid pGA482GG/cpPRV4, which contains the plant expressible Nos-NPT II and papaya ringspot virus (PRV) coat protein (CP) genes. Approximately 280 protocorms from four crosses were bombarded and potentially transformed tissues were identified by growth and green color on half-strength Murashige and Skoog medium supplemented with 2% sucrose and 50–100 mg 1^–1 kanamycin sulfate. Kanamycin concentrations that prevented growth of nontransformed tissues could not be used for long-term selection because such levels suppressed the regeneration of potentially transformed tissues. PCR and restriction analysis 21 months after treatment found 13 of 13 plants from two crosses, which appeared kanamycin-tolerant, to contain the Nos-NPT II gene, while only one of these plants carried the vector-linked PRV CP-gene. These results support use of particle bombardment for transformation of this important ornamental monocot.     

表达猪繁殖与呼吸综合征病毒修饰型GP5m蛋白的重组伪狂犬病毒的构建及其在小鼠的免疫反应

猪伪狂犬病毒（PRV）是一种良好的兽用活病毒疫苗载体。但以PRV基因缺失疫苗株TK^-/gE^-/LacZ⁺为载体表达PRRSV GP5的重组病毒TK^-/gE^-/GP5⁺免疫实验动物后难以激发抗PRRSV的中和抗体。为了进一步增强这种重组病毒的免疫效力,用具有更好免疫原性的修饰的ORF5基因（ORF5m）代替天然ORF5基因,构建了表达PRRSV的修饰型GP5m蛋白的重组伪狂犬病毒TK^-/gE^-/GP5m⁺。经PCR、Southern blot、Western blot 证实构建正确,并能表达具有活性的GP5m蛋白。将TK^-/gE^-/GP5m⁺与TK^-/gE^-/GP5⁺分别免疫Balb/c小鼠,结果TK^-/gE^-/GP5m⁺免疫小鼠不仅产生了较高水平的抗PRRSV的中和抗体(3/6只达到了1∶16),而且在诱导PRRSV特异性细胞免疫方面也显著优于TK^-/gE^-/GP5⁺,表明TK^-/gE^-/GP5m⁺是一种极有希望的PRRSV和PRV二价基因工程候选疫苗。     

Murine gammaherpesvirus 68 lacking thymidine kinase shows severe attenuation of lytic cycle replication in vivo but still establishes latency

The lytic cycle functions of gammaherpesviruses have received relatively little attention to date, at least in part due to the lack of a convenient experimental model. The murine gammaherpesvirus 68 (MHV-68) now provides such a model and allows the roles of individual lytic cycle gammaherpesvirus proteins to be evaluated in vivo. We have used MHV-68 to determine the contribution of a gammaherpesvirus thymidine kinase (TK) to viral lytic replication and latency establishment. MHV-68 mutants with a disrupted TK gene grew normally in vitro but showed a severe attenuation of replication in the lungs after intranasal inoculation, with lytic titers at least 1,000-fold lower than those of wild-type and revertant viruses. Nevertheless, the establishment of latency by the TK-deficient mutants, while delayed, was not prevented by their lytic replication deficit. The viral TK clearly plays a crucial role in the capacity of MHV-68 to replicate efficiently in its natural host but does not seem to be essential to establish a persistent infection. The potential of TK-deficient mutants as gammaherpesvirus vaccines is discussed.     

猪伪狂犬病综合诊断及分离毒株gE和TK基因遗传变异分析

2012年以来,许多使用gE基因缺失活疫苗免疫过的猪场广泛性出现伪狂犬病毒(PRV)感染,gE抗体阳性率不断升高,伪狂犬典型病例不断增加。2018年3月鲁南地区几个种猪场先后发生疑似伪狂犬病疫情,怀孕母猪流产、产死胎和木乃伊胎,仔猪出现神经症状且死亡率高。通过对病死猪及死胚剖检进行初步诊断,取病料进一步进行病理组织学诊断及病毒分离鉴定。结果显示,病死猪均可见病毒性脑炎、肝细胞变性坏死及淋巴组织坏死等病理变化,在病变的神经元、肝细胞、扁桃体隐窝上皮细胞等细胞核内见红染包涵体。对分离到的4株PRV进行了gE和TK基因的序列测定及遗传变异分析发现,4株PRV的gE和TK核苷酸序列的同源性分别为98.8%~99.3%和98.9%~99.6%,与国内流行毒株其核苷酸序列的同源性分别99.1%~99.7%和98.6%~99.8%,与匈牙利和美国等流行毒株核苷酸序列的同源性分别为97.3%~97.8%和98.8%~99.5%,表明4株分离株高度同源,与国内PRV变异株处在同一分支,而与匈牙利和美国等毒株遗传距离较远。传统疫苗对PRV变异毒株不能提供有效保护,给猪场伪狂犬病的防控和净化工作带来了新的挑战。