Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.     

应用PCR检测鸡传染性喉气管炎病毒

根据传染性喉气管炎病毒(ILTV)TK基因序列，设计、合成1对引物，应用PCR技术对ILTV以色列疫苗株、河南分离株（ILTV-CG和ILTV-XY）进行PCR扩增，均能扩增出预期大小的目的片段，测序分析和酶切分析证实了PCR产物的特异性，而对其它禽病原体的扩增均为阴性。PCR检测ILTV DNA的最小检测量为21 pg。应用PCR检测人工接种后不同〖JP2〗天数采集的鸡的结膜拭子，接种后第2~5 d均能检测到ILTV。该方法可用于鸡传染性喉气管炎病的诊断和临诊样品检测。     

Efficacy of live virus vaccines against infectious laryngotracheitis assessed by polymerase chain reaction-restriction fragment length polymorphism

The efficacy of four different commercial live vaccines (vaccines A, B, C, and D) against the infectious laryngotracheitis virus (ILTV) was assessed in specific-pathogen-free (SPF) chickens. SPF chickens were vaccinated intraocularly at 6 wk old with ILTV live vaccines and were challenged intratracheally with the N91B01 strain of virulent Korean ILTV 2 wk after vaccination. The immunity against ILTV live vaccines was assessed by the incidence of latent infection by the challenge virus in the chickens' tracheas and trigeminal ganglia, the reisolation rate of the challenge virus, and the clinical signs in the chickens challenged with the N91B01 strain of ILTV. The latent infection in chickens was assessed by nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our data showed that the clinical signs and challenge virus isolation were negative in all chickens receiving four difference commercial ILTV live vaccines. The viral DNA of the vaccine strain, but not that of the challenge virus, was detected in chickens vaccinated with vaccine A by nested PCR-RFLP. The viral DNAs of both the vaccine and challenge strains were detected from chickens vaccinated with vaccines B, C, and D. This study showed that only vaccine A can protect chickens from latent infection with the field virulent ILTV. We speculate that the efficacy of infectious laryngotracheitis live vaccines to protect chickens from latent infection with virulent ILTVs can be assessed by nested PCR-RFLP analysis.     

Identification of the thymidine kinase gene of infectious laryngotracheitis virus.

An an initial step in the development of a recombinant poultry infectious laryngotracheitis virus (ILTV) vaccine, we report on the identification, cloning, and sequencing of a thymidine kinase (tk) gene from a virulent U.S. field isolate of ILTV. Degenerate oligonucleotide primers for the consensus nucleotide (ATP) binding site and the nucleoside (thymidine) binding site of other herpesvirus tk genes were used in the polymerase chain reaction (PCR) to amplify a fragment of ILTV DNA. The 344-base-pair (bp) amplified fragment was cloned into plasmid pKSII and used in Southern hybridizations to locate the ILTV tk gene on a 2.4-kb HindIII fragment. Upon cloning and sequencing this fragment, a 1089-bp open reading frame was identified, which is predicted to encode a protein demonstrating 27.9% amino acid homology to the herpes simplex virus type 1 (HSV-1) thymidine kinase protein. Analysis of the sequence revealed one region of difference from that reported for the Thorne strain of ILTV. In addition, the portion of the TK protein corresponding to the nucleotide binding domain is highly conserved among the avian herpesviruses.     

鸡传染性喉气管炎病毒WG株ICP4基因的鉴定及其在潜伏位点的表达

根据传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)SA-2株ICP4基因序列设计并合成3对引物,以ILTV中国王岗株(WG)DNA为模板扩增ICP4基因,并对其进行了序列测定。将ILTV WG株的ICP4基因及其推导的氨基酸序列,分别与ILTV SA-2株、BHV-1、EHV-1、EHV-4、MDV-1、MDV-2、HVT、PRV、VZV、HSV-1和HSV-2的ICP4基因及其推导的氨基酸序列比较后发现,ILTV毒株之间ICP4基因相对保守,核苷酸和氨基酸水平的同源性分别为99.7%和99.1%,但与其它α-疱疹病毒的ICP4基因的同源性则较低,低于3.0%。对潜伏感染鸡三叉神经节中病毒基因的检测显示,在人工感染ILTV WG株后第10 d～60 d内均能检测到ICP4特异RNA,而gB、gC、TK则未能检出。鉴于目前国内外对α-疱疹病毒潜伏感染相关基因以及ICP4基因序列和结构功能的研究,ILTV WG株ICP4基因的克隆和序列测定,以及病毒基因在潜伏感染鸡三叉神经节中的差异表达,为进一步研究ICP4基因的功能及确定潜伏感染相关基因奠定了基础。     

Molecular biology of avian infectious laryngotracheitis virus

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes an economically important chicken disease, which results in delayed growth, reduced egg production, and also frequently in death of the animals. After acute infection of the upper respiratory tract, the virus can establish latency in the central nervous system, and subsequent reactivations can lead to infection of naive chickens. For prevention of ILT, conventionally attenuated live vaccines are available. However, these vaccine strains are genetically not characterized, and reversions to a virulent phenotype occur. Although molecular analyses of ILTV are hampered by the lack of an optimal cell culture system, the complete nucleotide sequence of the ILTV genome has recently been elucidated, and several ILTV recombinants lacking nonessential, but virulence determining genes have been constructed. Animal trials indicated that genetically engineered stable gene deletion mutants are safe alternatives to the current vaccine strains. Furthermore, since live ILTV vaccines are suitable for fast and inexpensive mass administration, they are promising as vectors for immunogenic proteins of other chicken pathogens. Thus, immunization with ILTV recombinants expressing avian influenza virus hemagglutinin was shown to protect chickens against ILT and fowl plague. Using monospecific antisera and monoclonal antibodies several virion proteins of ILTV have been identified and characterized. Since they include immunogenic envelope glycoproteins, these results can contribute to the improvement of virus diagnostics, and to the development of marker vaccines.     

PCR扩增TK基因检测鸡传染性喉气管炎病毒的研究

根据已发表序列设计一对包含鸡传染性喉气管炎病毒（ILTV）TK基因全长核苷酸的1259bp引物，对2株ILTV强毒和1株ILTV疫苗毒进行PCR扩增，均能扩增出预期大小的目的片段，酶切分析证实了目的片段的特异性，而其它禽病原体的扩增均为阴性。PCR检测ILTV DNA的最小检测量为75pg。此方法检测人工接种鸡的气管棉拭样品，均能检测到ILTV，因此可用于临诊样品鸡传染性喉气管炎病的检测和诊断。     

犬瘟热病毒SH202003株全基因组序列分析

为了解上海地区犬瘟热病毒（Canine distemper virus，CDV）遗传变异情况，本研究采用首尾重叠的11对特异性引物，对CDV上海株SH202003进行RT-PCR扩增，将扩增片段进行反复测序，序列拼接后最终获得了SH202003株全基因组序列，应用Lasergene 7.0和Mega 6.0软件对全基因及H基因进行序列分析，并构建系统进化树。结果显示，SH202003株基因组全长为15 690 bp，编码6种结构蛋白（N、P、M、F、H和L），H和L基因间隔序列为CUA，L和5'端尾随序列为CAA，与Hebei株核苷酸和氨基酸相似性最高，达到98.6%和96.6%，与疫苗株核苷酸相似性在92.2%~94.3%，氨基酸相似性只有82.7%~87.0%；全基因进化树中，SH202003株与流行野毒株在同一分支，与疫苗株在不同的分支；H基因同样与Hebei株亲缘关系最近，核苷酸和氨基酸相似性分别为98.7%和99.5%，与疫苗株Snyder Hill、CDV3、Convac及Onderstepoort亲缘关系较远；SH202003株处于Asia-1型分支，属于Asia-1型强毒株；SH202003株具有9个潜在N-糖基化位点，与强毒株Hebei株一致。研究表明，上海株SH202003属于CDV强毒株，为Asia-1型，其H基因序列相对保守，具有9个潜在N-糖基化位点，但是全基因序列存在较多突变，与疫苗株的匹配度较差，可能是免疫犬依然发生犬瘟热的主要原因。     

鸡传染性喉气管炎病毒PCR诊断方法的建立与应用

根据GenBank登录的传染性喉气管炎病毒(ILTV)的TK基因序列设计并合成1对特异性引物,以ILTV疫苗株DNA为模板,建立了检测ILTV TK基因的PCR方法。应用该方法能从临床分离毒株和疫苗株中扩增到长为427 bp的目的片段;但不能从新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、禽呼肠孤病毒(ARV)、减蛋综合征病毒(EDSV)、H9亚型禽流感病毒(H9-AIV)、传染性支气管炎病毒(IBV)、大肠杆菌以及金黄色葡萄球菌等病原中扩增出阳性条带;敏感性试验表明其DNA最小检出量为4.9 ng;应用该方法和病毒分离法对2份临床病例和人工感染鸡的检测,两者符合率为100%。上述结果表明该PCR方法具有良好的特异性和敏感性,可用于传染性喉气管炎病毒鉴定和临床诊断。     

伪狂犬病病毒BJ/YT株毒力相关基因gE和TK序列分析

本试验对2012年从北京地区约克夏犬体内分离得到的伪狂犬病病毒(PRV)BJ/YT株主要毒力基因gE和TK的分子特征和进化关系进行了分析。结果显示,BJ/YT株gE基因与参考序列的核苷酸和氨基酸同源性分别为98.9%～100.0%和98.3%～100.0%;TK基因核苷酸和氨基酸同源性分别为99.2%～99.9%和99.0%～100.0%。BJ/YT株与同期河北猪源分离株进化关系较近,各毒株之间同源性高,并且存在一致的核苷酸突变位点;与其他参考序列比对分析结果显示,BJ/YT株主要毒力基因存在变异位点,但这些位点均不在已知的主要功能区和抗原表位区内。因此,从分子流行病学角度来看,BJ/YT毒株是近年北京及其周边地区PRV流行毒株,但gE和TK基因的变异对流行毒株的毒力无明显影响。     

Survival and immunization of raccoons after exposure to pseudorabies (Aujeszky's disease) virus gene-deleted vaccines

In a controlled experiment, 16 wild-trapped raccoons were exposed to 1 of 2 genetically modified live pseudorabies virus (PRV) vaccines used in swine. One vaccine had genes deleted for thymidine kinase (TK(-)) and glycoprotein G (gG(-)); the other had an additional deletion for glycoprotein E (gE(-)). These vaccines were administered orally and intranasally at four dose levels: 10(3), 10(4), 10(5), and 10(6) TCID(50). The 21 days survival rate was 37.5% for the gG(-)TK(-) vaccine; all of the survivors developed antibodies to PRV. All animals receiving the gG(-)gE(-)TK(-) vaccine survived; 75% (all except the lowest dose) developed anti-PRV antibodies. Survivors were challenged intranasally with a 3.2x10(3) TCID(50) dose of the virulent wildtype PRV Shope strain. Two of the remaining three gG(-)TK(-) vaccinated raccoons survived the challenge; for the gG(-)gE(-)TK(-) vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody titers were more likely to survive the challenge with the virulent PRV; there was a 100% mortality rate for raccoons lacking detectable anti-PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent virus.     

1株鸡传染性喉气管炎病毒的基因组特征与致病性

本研究旨在了解我国鸡传染性喉气管炎病毒(ILTV)流行毒株的分子特性.对1株分离自国内养殖场的鸡传染性喉气管炎病毒毒株BT株进行了部分基因序列(TK、ICP4、gB、UL32)测定分析,并研究了其对2周龄SPF鸡的致病性.遗传进化分析表明,BT株核苷酸序列与国内其他流行毒株相似性在97％以上,属于同一进化分支,未出现较...     

Characterization of western European field isolates and vaccine strains of avian infectious laryngotracheitis virus by restriction fragment length polymorphism and sequence analysis

Infectious laryngotracheitis is a dramatic disease of the upper respiratory tract in poultry caused by a herpesvirus. In this study we investigated the characteristics of western European field isolates of infectious laryngotracheitis virus (ILTV) to gain more information on their diversity. The examined 104 isolates, collected from acute outbreaks during the last 35 years, originated from eight different countries: Switzerland (48), Germany (21), Sweden (14), the United Kingdom (9), Italy (5), Belgium (4), Austria (2), and Norway (1). Two vaccines, a chicken embryo origin product and a tissue culture origin product, were included in the survey. Polymerase chain reaction (PCR) was performed to amplify a 2.1-kb DNA fragment of ILTV using primers generated for the thymidine kinase (TK) gene. After digestion of the resulting PCR products by restriction endonuclease HaeIII, restriction fragment length polymorphism analysis was carried out. PCR amplicons of three field isolates and both vaccine strains were selected for sequencing. Here 98 field isolates showed the same cleavage pattern and were identical to both vaccine strains (clone 1). They differed from five Swiss isolates with identical cleavage pattern (clone 2) and one Swedish isolate (clone 3). The present study demonstrated that at least three clones of ILTV have been circulating in western Europe during the last 35 years. The 104 isolates analyzed showed a high genetic similarity regarding the TK gene, and a large majority of the field isolates (98/104) were genetically related to the vaccine strains.     

鸡传染性喉气管炎病毒的分离和TK基因鉴定

从临床表现以呼吸困难、喉头出血为主要特征的某蛋鸡场患鸡喉头和气管等组织分离到一株病毒。该分离毒无血凝特性,应用检测ILTVTK基因的PCR能从分离毒中扩增出大小为427bp的特异性目的片段。测序结果与GenBank收录的ILTV不同毒株序列的同源性为100%。结果初步表明该分离毒为鸡传染性喉气管炎病毒(命名为ILTV-FJ)。     

63株IBV分离株M基因的遗传变异与系统进化分析

对1993－2010年从中国不同地区分离的63株传染性支气管炎病毒(infectious bronchitis virus,IBV)野毒株,采用RT-PCR方法克隆测定所分离野毒株的M基因核苷酸序列,并与GenBank中公布的部分国内外IBV毒株的M基因序列进行比较分析,研究中国IBV的分子流行病学特点和分子遗传变异规律。结果显示,所测毒株M基因具有6种不同长度的开放阅读框,这些长度的差异是由于5'端的核苷酸插入或缺失造成的;N端含有1~2个N-糖基化位点,其中1个糖基化位点"Asn-Cys-Thr"(NCT)是高度保守的。63个IBV分离株间氨基酸序列相似性为88.9%~100%,分离株与参考株间相似性为87.2%~100%。系统进化分析结果显示,本研究的63个IBV分离株可分为9个基因型,2005－2010年IBV中国流行株大部分与Mass型疫苗株处于不同的基因型,而且氨基酸序列相似性都小于94%。     

鸭瘟病毒转移质粒的构建

为了建立重组鸭瘟病毒技术,构建了鸭瘟病毒转移质粒。在对鸭瘟强毒和弱毒株TK基因进行测序分析后,将鸭瘟病毒TK-UL24DNA片段克隆于pUC18载体中,构建了质粒pTK;将PCR扩增的GFP真核表达盒插入pTK质粒的TK基因内部,获得转移载体质粒pTK-GFP。鸭瘟病毒TK-UL24测序分析表明鸭瘟强、弱毒株TK基因序列完全相同;转移载体携带Pcmv-GFP-SV40pA表达盒,测序验证其序列与源序列一致。pTK-GFP在脂质体介导下,转染鸭胚成纤维细胞和鸭肾细胞,在荧光显微镜下观察绿色荧光蛋白表达情况。质粒转染细胞后,绿色荧光蛋白得到了有效的表达,为进一步开展重组鸭瘟病毒的研究和构建具有遗传标记的鸭瘟疫苗奠定了基础。     

9株鸡毒支原体29 Ku多肽基因的克隆与序列分析

根据已发表的鸡毒支原体(MG)S6株29Ku多肽基因序列设计了1对引物,以9株(广西分离株5株、标准株4株)DNA为模板进行PCR扩增,均得到802bp的特异性片段,将9株MG PCR产物纯化后克隆到pMD18-T载体上,得到重组质粒.重组质粒经PCR法和EcorⅠ、SalⅠ双酶切等方法鉴定后,测定了9株29 Ku多肽基因序列,并在基因库中S6标准株的29 Ku多肽基因序列进行分析比较.结果表明,5株分离株与5株标准株29Ku多肽基因核苷酸序列同源性分别为94.4%～99.9%,推导的氨基酸同源性分别为89.7%～99.2%.从各毒株的进化分析表明,5个分离株与标准强毒株S6、A5969、K1501和PG31强毒株间遗传距离较近,而5个分离株与标准株F疫苗株间遗传距离则较远.     

Examination of the effect of a naturally occurring mutation in glycoprotein L on Marek's disease virus pathogenesis

We recently reported a comparison of glycoprotein-encoding genes of different Marek's disease virus pathotypes (MDVs). One mutation found predominantly in very virulent (vv)+MDVs was a 12-bp (four-amino acid) deletion in the glycoprotein L (gL)-encoding gene in four of 23 MDV strains examined (three were vv+MDVs and one was a vvMDV). This mutation was noted in the gL of the TK (615K) strain, but not in the RL (615J) strain of MDV. These strains have identical mutations in the meq gene characteristic of vv+MDVs but can be distinguished by the mutation in the gL-encoding gene. The TK strain was originally isolated from vaccinated chickens and appeared to confer or enhance horizontal transmission of the vaccine virus, herpesvirus of turkeys (HVT). Because the molecular basis for increased virulence of MDV field strains is unknown, we hypothesized that one mechanism might be by coreplication of MDV-1 strains with HVT and that it could be mediated by the mutation of gL, an essential component of the glycoprotein H/L complex. In this study, we compared the pathogenicity of TK (615K) and RL (615J) strains of MDV in the presence and absence of simultaneous HVT coinfection. MDV infections were monitored at the levels of viremia (for both MDV-1 and HVT), clinical signs of MD, tumor incidence, and mortality in 1) inoculated chickens, 2) chickens exposed at 1 day of age, 3) chickens exposed at 2 wk of age, and 4) chickens exposed to both TK/HVT- and RL/HVT-infected chickens at 6 wk of age. We found high incidences of clinical MD signs in all inoculated treatment groups and all chickens exposed to TK and RL viruses, regardless of the presence of HVT. The median time to death of chickens exposed to TK1HVT-infected chickens, however, was lower than the other treatment groups for contact-exposed chickens. Although this difference was not considered to be statistically significant to a rigorously interpreted degree because of the removal of chickens for sampling from the test groups, these data suggest that replication of the TK strain and HVT, when coadministered, might incrementally affect the virulence of MDV-1 strains. The strict correlation of this enhancement of virulence with the mutation in gL, however, requires additional experiments with genetically identical MDV background strains.     

Some characteristics of four attenuated vaccine virus strains and a virulent strain of Aujeszky's disease virus

In the present study four attenuated virus strains, used as vaccines, and a virulent strain of Aujeszky's disease virus (ADV) were compared with respect to their virulence in mice, their ability to induce virus-specified thymidine kinase (TK) in infected cells, and their cleavage profiles of viral DNA's after treatment with the restriction endonuclease KpnI. The survival time of mice inoculated with the B-KAL or the virulent NIA-3 strain was comparable, whereas the Bartha and BUK strains required significantly longer periods to kill mice. Mice were resistant to the MK-25 strain of ADV. The strains were assayed for TK phenotype by plaque autoradiography after 3H-thymidine labelling of infected cells. MK-25 proved to be the only strain defective in induction of TK in pig kidney cells. Restriction endonuclease analysis of viral DNA's revealed that each vaccine strain showed a characteristic fragment pattern that could easily be differentiated from that of other vaccine and field strains of ADV. The present results demonstrate that the mouse virulence test and the TK assay detect differences in biological properties of ADV strains, but that restriction endonuclease analysis is required for unambiguous identification of vaccine and field strains of ADV.