山东半岛地区不同来源的禽I型副粘病毒毒力差异比较研究
摘要
本论文对山东半岛地区的4株禽Ⅰ型副粘病毒强毒株进行了分离,对其血清学、临床毒力表现和分子生物学方面进行了研究。
将4株地方强毒株经绒毛尿囊腔接种10日龄鸡胚,鸡胚接种后36-72h鸡胚全身出血,含病毒的尿囊液红细胞凝集效价(HA)为1∶160-1∶640,并且能被ND标准阳性血清所抑制,不能被AI标准阳性血清所抑制。人工发病实验结果表明3株鸡源禽Ⅰ型副粘病毒属于强毒株,鸽源禽Ⅰ型副粘病毒对鸡不发病,对鸽则表现为强毒株。
为了探讨山东半岛地区NDV毒力分布情况,将分离获得的其中3株NDV的鸡胚平均致死时间(Mean death time, MDT)、1日龄雏鸡脑内致病指数(Intracerebral pathogenicity index,ICPI)和6周龄鸡静脉内致病指数(Intravenous pathogenicity index,IVPI)进行测定。NDV-PL(蓬莱)株、NDV-ZY(招远)株、NDV-LZ(莱州)株的MDT分别为63.23、60、61.5,ICPI分别为0.9、1.83、1.5,IVPI分别为2.35、2.74、2.61,结果表明3株NDV中,NDV-LZ株的各项指标均为强毒力病毒,NDV-ZY株和NDV-PL株除ICPI结果显示为中等毒力病毒外,其余结果均显示为强毒力病毒,表明这3株新城疫病毒毒株对鸡有较强的致病力。
利用自己设计的引物和参考已发表的引物,通过一步法RT-PCR扩增出三个毒株的479bp的片段和两个毒株的497bp的片段,这两个目的片段含有F蛋白的酶切裂解位点。将目的片段在72℃加腺苷酸尾后,克隆至pGEM-T Easy载体中,构建了相应的重组质粒,经酶切与PCR鉴定后,送交测序并贮存基因。4株cDNA测序结果表明,各毒株的酶切裂解位点都符合强毒株的酶切裂解位点的序列,表明我们所分离的毒株都属于强毒株。
针对F基因479bp和497bp片段,通过DNASIS软件构建了相应的系统发育树,系统发育分析表明山东半岛地区禽Ⅰ型副粘病毒与欧美及中东地区基因群明显分离,但鸽瘟病毒与东北地区禽Ⅰ型副粘病毒的同源性很高。
Studies were carried out to characterize an avian paramyxovirus isolated strains of NDV-PL, NDV-LZ, PPMV-Ⅰ, NDV-ZY. The four strains of virulent Newcastle Disease Virus(NDV)from peninsular Shandong province were isolated through chicken embryo inoculation and the harvested infectious allantoic fluid was tested by HA assay. The result attained was 1:160-1:640 and the HA activity could especially inhibited with antiserum against NDV.
In order to study the distribution of the NDV virulence, MDT (Mean death time), ICPI (Intracerebral pathogenicity index) and IVPI (Intravenous pathogenicity index) were measured for the three strains virus.The MDT result of the strain of NDV-PL、NDV-ZY、NDV-LZ were 61.5、60、63.23.The ICPI result of the strain of NDV-PL、NDV-ZY、NDV-LZ were 0.9、1.83、1.5, and The IVPI result of the strain of NDV-PL、NDV-ZY、NDV-LZ were 2.35、2.61、2.74. The result confirmed that the three strains were virulence stains except the ICPI of NDV-LZ indicating middle virulent strains.
The fusion protein is a major pathogenic and immunogenic protein of NDV. One step RT-PCR was employed to amplify a cDNA fragment of F gene of NDV-PL、NDV-LZ、NDV-La Sota、PPMV-Ⅰ、NDV-ZY strains using two pair of specific primers. The products were directly cloned into the T-T window of pGEM-T easy vector and sequenced.The sequence result revealed that the F gene fragment of all strains contained 497bp and 480bp coding for 163 amino acid and 160 amino acid. The nucleotide sequence and predicted amino acid sequence of F gene of the isolate showed 74.3%-97.0%、74.8%-95.5% homology respectively with other NDV strains. It was found that the predicted amino acid sequence of the cleavage site sequence 112Arg Arg Gln Lys Arg Phe117 matching that of virulent avian paramyxovirus-Ⅰstrain.
A phylogenetic tree was constructed and the result indicated that there discrepancy between NDV isolated from Shandong peninsular andⅡgenotype in Westen countries,but NDV from South China were of the same lineage as those from Taiwan, belonging to the novel genotypeⅦ.
将4株地方强毒株经绒毛尿囊腔接种10日龄鸡胚,鸡胚接种后36-72h鸡胚全身出血,含病毒的尿囊液红细胞凝集效价(HA)为1∶160-1∶640,并且能被ND标准阳性血清所抑制,不能被AI标准阳性血清所抑制。人工发病实验结果表明3株鸡源禽Ⅰ型副粘病毒属于强毒株,鸽源禽Ⅰ型副粘病毒对鸡不发病,对鸽则表现为强毒株。
为了探讨山东半岛地区NDV毒力分布情况,将分离获得的其中3株NDV的鸡胚平均致死时间(Mean death time, MDT)、1日龄雏鸡脑内致病指数(Intracerebral pathogenicity index,ICPI)和6周龄鸡静脉内致病指数(Intravenous pathogenicity index,IVPI)进行测定。NDV-PL(蓬莱)株、NDV-ZY(招远)株、NDV-LZ(莱州)株的MDT分别为63.23、60、61.5,ICPI分别为0.9、1.83、1.5,IVPI分别为2.35、2.74、2.61,结果表明3株NDV中,NDV-LZ株的各项指标均为强毒力病毒,NDV-ZY株和NDV-PL株除ICPI结果显示为中等毒力病毒外,其余结果均显示为强毒力病毒,表明这3株新城疫病毒毒株对鸡有较强的致病力。
利用自己设计的引物和参考已发表的引物,通过一步法RT-PCR扩增出三个毒株的479bp的片段和两个毒株的497bp的片段,这两个目的片段含有F蛋白的酶切裂解位点。将目的片段在72℃加腺苷酸尾后,克隆至pGEM-T Easy载体中,构建了相应的重组质粒,经酶切与PCR鉴定后,送交测序并贮存基因。4株cDNA测序结果表明,各毒株的酶切裂解位点都符合强毒株的酶切裂解位点的序列,表明我们所分离的毒株都属于强毒株。
针对F基因479bp和497bp片段,通过DNASIS软件构建了相应的系统发育树,系统发育分析表明山东半岛地区禽Ⅰ型副粘病毒与欧美及中东地区基因群明显分离,但鸽瘟病毒与东北地区禽Ⅰ型副粘病毒的同源性很高。
Studies were carried out to characterize an avian paramyxovirus isolated strains of NDV-PL, NDV-LZ, PPMV-Ⅰ, NDV-ZY. The four strains of virulent Newcastle Disease Virus(NDV)from peninsular Shandong province were isolated through chicken embryo inoculation and the harvested infectious allantoic fluid was tested by HA assay. The result attained was 1:160-1:640 and the HA activity could especially inhibited with antiserum against NDV.
In order to study the distribution of the NDV virulence, MDT (Mean death time), ICPI (Intracerebral pathogenicity index) and IVPI (Intravenous pathogenicity index) were measured for the three strains virus.The MDT result of the strain of NDV-PL、NDV-ZY、NDV-LZ were 61.5、60、63.23.The ICPI result of the strain of NDV-PL、NDV-ZY、NDV-LZ were 0.9、1.83、1.5, and The IVPI result of the strain of NDV-PL、NDV-ZY、NDV-LZ were 2.35、2.61、2.74. The result confirmed that the three strains were virulence stains except the ICPI of NDV-LZ indicating middle virulent strains.
The fusion protein is a major pathogenic and immunogenic protein of NDV. One step RT-PCR was employed to amplify a cDNA fragment of F gene of NDV-PL、NDV-LZ、NDV-La Sota、PPMV-Ⅰ、NDV-ZY strains using two pair of specific primers. The products were directly cloned into the T-T window of pGEM-T easy vector and sequenced.The sequence result revealed that the F gene fragment of all strains contained 497bp and 480bp coding for 163 amino acid and 160 amino acid. The nucleotide sequence and predicted amino acid sequence of F gene of the isolate showed 74.3%-97.0%、74.8%-95.5% homology respectively with other NDV strains. It was found that the predicted amino acid sequence of the cleavage site sequence 112Arg Arg Gln Lys Arg Phe117 matching that of virulent avian paramyxovirus-Ⅰstrain.
A phylogenetic tree was constructed and the result indicated that there discrepancy between NDV isolated from Shandong peninsular andⅡgenotype in Westen countries,but NDV from South China were of the same lineage as those from Taiwan, belonging to the novel genotypeⅦ.
引文
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[2]张鹤晓,高志强,赖平安,等.新城疫病毒通用型实时RT- PCR检测方法的建立与应用[J] .动物医学进展, 2005, 26 (11) : 53～56.
[3]Chambers P., Miller N S., Bingham R W. et al. Molecular cloning of complementary DNA to NDV, and nucleotide sequence analysis of the junction between the genes encoding the HN and the large protein. Virol, 1986, 67: 475～486.
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