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.     

一种独特的疱疹病毒-鸡传染性喉气管炎病毒

作者对鸡传染性喉气管炎病毒（ILTV）从以下4个方面进行了综述：①ILTV宿主的高度特异性;②病毒感染细胞的特殊途径;③病毒基因组内的基因容量和排列;④ILTV与其它疱疹病毒同源序列比较而推测出它们之间的进化关系.上述几方面的论述都表明ILTV具有区别于一般疱疹病毒的独特的特点.     

Protection of chickens from Newcastle disease and infectious laryngotracheitis with a recombinant fowlpox virus co-expressing the F, HN genes of Newcastle disease virus and gB gene of infectious laryngotracheitis virus

A recombinant fowlpox virus (rFPV) coexpressing the Newcastle disease virus (NDV) fusion and hemagglutinin-neuraminidase genes and infectious laryngothracheitis virus (ILTV) glycoprotein B gene was constructed. This virus was then evaluated for its ability to protect specific-pathogen-free (SPF) chickens against clinical symptoms and death after challenge by virulent NDV and ILTV. SPF chickens were grouped and vaccinated with the rFPV and commercial NDV (La Sota) and ILTV attenuated live vaccine (Nobilis ILT), respectively. After challenge with NDV 10 days postvaccination, 70% of chickens vaccinated with rFPV were protected from death, whereas 100% of the commercial NDV-vaccinated chickens were protected from death. In contrast, 100% of the unvaccinated chickens died after challenge. After challenge with ILTV, both the rFPV and commercial ILTV-vaccinated chickens were completely protected from death and 70% of chickens were protected from respiratory signs. In comparison, 100% of the unvaccinated chickens developed severe respiratory disease and 10% of chickens died. The protective efficacy was also measured by the antibody responses and isolation of challenge viruses. Results showed that this rFPV could be a potential vaccine for preventing NDV and ILTV by a single immunization.     

传染性喉气管炎病毒田间分离株重要基因的序列分析

为了解我国田间传染性喉气管炎病毒(infectious laryngotracheitis virus,ILTV)的具体流行情况,利用序列测定和PCR-RFLP对2010-2014年分离的19株田间毒株进行分析,并将结果与6株疫苗株对比。序列分析发现,与CEO疫苗株相比,9株毒株的ICP4基因不但没有4个连续氨基酸(丙氨酸-丙氨酸-甘氨酸-天冬氨酸)的缺失,而且在201位有1个蛋氨酸的插入。TK基因序列分析发现252位氨基酸不能决定该毒株的毒力,而可能是由终止密码子下游24 bp是否存在典型的多聚腺苷酸信号(AATAAA)来决定,即存在时为强毒株。综合PCR-RFLP和序列分析结果发现,我国田间流行毒株中既存在有野毒株和疫苗株的相关株,也存在两者的重组毒株,提示我们在对ILTV进行防制时应当注意弱毒疫苗的使用。     

In vitro infection studies with infectious laryngotracheitis virus

Infectious laryngotracheitis (ILT) virus strains were studied for their ability to infect chicken macrophages, lymphocytes, and kidney cells in vitro. Although macrophages were as susceptible as chicken kidney cells to infection, replication of most virus strains in macrophages was markedly restricted. Only a few isolates induced progressive infections in macrophages, and even with these the donor of the macrophages influenced replication. Thus, it appears that both cell genotype and virus genotype may help determine the extent of restriction of virus replication. Macrophages were more susceptible to an attenuated vaccine strain of ILT virus than to virulent virus strains. Spleen lymphocytes, peripheral blood lymphocytes, thymocytes, bursal lymphocytes, buffy coat leukocytes, and activated T-cells were nearly or totally refractory to infection by ILT virus.     

Reproducibility of a virus-neutralization test for infectious laryngotracheitis virus

A virus-neutralization test for infectious laryngotracheitis virus was performed in microtiter plates using standard techniques. To assess the reproducibility of the test, 11 sera were each titrated repeatedly once a week for 8 weeks, and the results were compared to a standard. The standard used for reproducibility was that the 95% logarithmic confidence intervals of the mean calculated from three titrations of the same serum had to be smaller than the logarithmic distance "within" two microtiter plate wells. For the virus neutralization test to give reproducible results, such confidence intervals had to fall "within" two wells at least 75% of the time. Over the 8 weeks, percent reproducibility varied from 43.5% to 81.5%. The infectious laryngotracheitis virus-neutralization test did not meet our defined standard of reproducibility with positive antisera. Results with negative control sera were reproducible, however. Percent reproducibility varied from 31.8% to 93.8% for different sera tested, but it was not related to the titer of the sera.     

鸡传染性喉气管炎病毒适应性细胞的筛选

为了克服鸡传染性喉气管炎弱毒活疫苗产品制备中存在的缺陷,通过对BHK-21细胞、Vero细胞、DF1细胞、原代鸡胚成纤维细胞(CEF)、原代鸡胚肝细胞、原代鸡胚肾细胞等六种细胞进行病毒适应性培养,采用PCR和免疫荧光(IFA)跟踪检测的方法成功筛选出具有稳定病毒滴度的病毒适应性细胞——鸡胚肝细胞。然后采用新的克隆方法缩短了克隆时长并对筛选的鸡胚肝细胞进行克隆纯化,通过与原代鸡胚肝细胞进行比较,结果显示本试验所采用的克隆方法具有高几率的特点,并且能够克隆出高活性的鸡胚肝细胞,并能够繁殖较高效价的鸡传染性喉气管炎病毒,为解决鸡传染性喉气管炎弱毒疫苗在生产上存在的瓶颈问题提供了重要的基础条件。     

Differentiation of infectious laryngotracheitis virus strains using restriction endonucleases

Strains of infectious laryngotracheitis virus (ILTV) were examined using an indirect immunofluorescent test (IIF) and with restriction endonucleases for detecting intratypic differences. Electrophoretic analysis of ILTV DNA fragments cleaved with restriction endonuclease Hind 111 clearly distinguished between strains. The IIF test did not discriminate between strains. A molecular weight estimate of ILTV DNA was made by summation of restriction endonuclease fragments cleaved with BamH1 (102.1 X 10(6)) and Hind111 (97.35 X 10(6)). Differences between the estimates may indicate the presence of submolar fragments.     

禽传染性喉气管炎病毒gD蛋白单克隆抗体的制备

为制备抗鸡传染性喉气管炎病毒(ILTV)糖蛋白gD的单克隆抗体(MAb),本研究通过原核表达gD重组蛋白,纯化后免疫6周龄雌性BALB/c小鼠,经细胞融合筛选获得一株稳定分泌抗ILTV gD蛋白的杂交瘤细胞株,MAb亚型经鉴定为IgG1,轻链为κ链。Western blot结果显示,这株杂交瘤细胞分泌的MAb能够识别ILTV。ILTV gD蛋白的MAb的制备,为ILTV检测方法的建立奠定了基础。     

传染性喉气管炎病毒WG株Us区基因结构分析

根据GenBank中的传染性喉气管炎病毒(ILTV)全序列(NCBT登录号:NC-006623).利用Oligo6.2分析序列并设计6对引物,以ILTV WG株基因组DNA为模板,PCR扩增了长度为13.1 kb的区域,得到了完整的WG株的Us区序列,初步鉴定了WG株的Us区基因结构.将WG株的Us区序列分别与ILTV USDA株、BHV-1、CeHV-1、EHV-1、HSV-1、HSV-2、MDV、PrV、HvT、VZV相比较,ILTV WG株与USDA株同源性为99.2%,而与其他疱疹病毒之间的同源性较低,而且Us区大小也不一致;与已发表的ILTV USDA株Us区基因序列分别比较后发现,两者之间差异较大的基因分别为gJ基因和gD基因.其中,gJ基因在第1983个碱基处比USDA株多出30 bp,DNAStar预测这30 bp可能形成一个独立的抗原表位;gD基因的长度在不同的ILTV毒株之间差别较大,与其他疱疹病毒具有相似的结构特征.     

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.