带有REV-LTR的MDV重组野毒株生物学特性的研究
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摘要
近几年来,经MDV疫苗CVI988/Rispens疫苗免疫后仍有些鸡群显示较高的肿瘤发生率。因此,国内外学者怀疑是否出现了能突破该疫苗保护作用的致病性更强的MDV野毒株。
张志等2001年从广西某个发生肿瘤的鸡群中分离到一株带有REV-LTR的天然MDV野毒株GX0101。为了显示免疫失败是否与该流行株的致病性增强有关,张志(2004)和庄国庆(2006)分别在SPF鸡中做了人工感染试验,表明GX0101对鸡的致病性高于强毒GA。本研究在经HVT免疫的SPF鸡的攻毒试验中,GX0101株的致死率(28.6%)和致肿瘤率(7.1%)均低于超强毒参考株Md5的致死率(63.1%)和致肿瘤率(19.0%),但是,利用MDV特异性核酸探针对同罩饲养的对照鸡的羽毛囊DNA检测表明,与GX0101攻毒鸡同罩饲养的未经免疫未攻毒的对照鸡,从攻毒后第28d就有6/15的比例从羽毛囊中检出MDV,而与vvMd5接种鸡同一隔离罩的对照鸡,在35d时才在2/14的个体中检出MDV,即GX0101的横向传播能力大于超强毒株。在SPF鸡试验中这一结果表明,MDV的致病性不一定与横向传播力相平行。由此推测,显著增高的横向传播能力可能就是这一整合进REV-LTR的重组病毒株能在鸡群中逐渐流行开来的选择性竞争优势之一。
试验还进一步比较了斑点杂交和琼脂扩散试验在动物实验中的应用,实验结果表明本试验证明斑点杂交试验在攻毒后第10d即可从羽毛囊中检测到病毒,而琼脂扩散试验在攻毒后第14d检测到病毒,斑点杂交的最高检出率为100%,而琼脂扩散试验的最高检出率为80%,斑点杂交的检出率也高于与琼脂扩散试验。斑点杂交的检测灵敏度达1pg DNA水平。可以对大量的样品进行快速,简便,准确的检测,可作为临床MDV检测的有效手段;因此,斑点杂交在实际生产中有很广泛的应用。
为了模仿并跟踪反转录病毒与MDV病毒在鸡体内自然发生的基因重组,用PCR来追踪鸡体内二种病毒发生重组的动态。根据MDV基因组上易插入REV的LTR的高频位点的序列合成7条引物,根据REV的LTR合成4条引物,由此交叉组成28对引物,同样设计ALV-MDV之间30对引物来扩增重组片段,从中分离筛选整合进反转录病毒基因序列的重组MDV。此次试验MDV与REV或ALV人工共感染,在连续传到第17代时,利用PCR还没有扩增检测有重组片段。这说明二种不同病毒在体内发生重组的机率确实非常小。但是能从鸡群中比较容易分离到重组野毒株,GX0101还说明这是经过长期的选择而产生的。
In recent years, some fowls disclose higher tumorigenesis for chickens viccinated against Marek's disease CVI988/Rispens .so scholar of exterior and interior suspected whether or not occurrence the higher pathogenicity of wild strain MDV which breakthrough this vaccine of protection.
Zhangzhi isolated A recombinant Marek's disease virus field strain with REV-LTR of GX0101 from fowl group which tumorigenic in 2001. In order to disclose the failure of immune whether or not concerned with the pathogenicity enhance of this popular strain .Zhangzhi(2004)and Zhuang guoqing(2006) did artificial infextation experiment, indicated the pathogenicity of GX0101 highter than the GA,this research adopt mortality and tumor rates of GX0101 were 28.6% and 7.1% for SPF chickens viccinated against Marek's disease virus. Mortality and tumor rates of vvMd5 were 63.1% and 19.0% in the same viccinated SPF chickens. Mortality and tumor rates of GX0101 were lower than that of vvMd5. However, horizontal transmission ability of GX0101 was stronger than that of Md5. After 28 days challenged by GX0101, MDV was detected in 6 of 15 samples. After 35 days challenged by vvMd5, MDV was detected in 2 of 14 samples. Horizontal transmission ability of MDV isolates was not necessarily parallel to their pathogenicity.It is suggesting that the increased horizontal transmission may be one of selective competitive advantages. Such advantages may help recombinant viruses with the LTR-insert become more and more popular gradually in chicken flocks.
This expenment compared the application in animal experiment between dot blot hybridization and agar diffusion reactionanimal experiment, the result indicated the virus can be detected from feather by dot blot hybridization after 10 days challenged, after 14days challenged can be detected by agar diffusion reaction, the highest detection rate of dot blot hybridization is 100%, but the the highest detection rate of agar diffusion reaction is 80%. The detection rate of dot blot hybridization was highter than the agar diffusion reaction. The sensitivity of dot blot hybridization was 1pg DNA, the detection of dot blot hybridization was fast, simple ,precise to a devil of sample. the dot blot hybridization can be trate as a utility means in detect MDV. Accordingly, dot blot hybridization has widely use in practical parturition.
In order to imitate and pursuit retrovirus with marek's disease virus abiogenetic gene recombination in chincken 's vivo. To amplify and clone the integrated REV LTR with MDV sequence at the junction,4 primersfrom REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hotpoint were synthesized and 28(4x7)pairs of primers(one from REV and another from MDVfor each pair)were used in PCR while the genomic DNA of both strains were used as the emplates. And we designed 30 pairs (one from REV and another from MDVfor each pair)were used in PCR while the genomic DNA of both strains were used as the emplates.of primers recombinated MDV which integrated the gene order of retrovirus.this expenment artificial coinfection with MDV and REV or ALV, when continue the 17th generation , can not isolate the re- combinate virus , the probability of two virus recombinated was very tiny, But,it is easily to isolate recombinated wild strain ,this expenment demonstrate the GX0101 under the long-standing selection.
This research disclose , the horizontal transmission of recombinant Marek's disease virus field strain with REV-LTR of GX0101 highter than the rMD5 ,
张志等2001年从广西某个发生肿瘤的鸡群中分离到一株带有REV-LTR的天然MDV野毒株GX0101。为了显示免疫失败是否与该流行株的致病性增强有关,张志(2004)和庄国庆(2006)分别在SPF鸡中做了人工感染试验,表明GX0101对鸡的致病性高于强毒GA。本研究在经HVT免疫的SPF鸡的攻毒试验中,GX0101株的致死率(28.6%)和致肿瘤率(7.1%)均低于超强毒参考株Md5的致死率(63.1%)和致肿瘤率(19.0%),但是,利用MDV特异性核酸探针对同罩饲养的对照鸡的羽毛囊DNA检测表明,与GX0101攻毒鸡同罩饲养的未经免疫未攻毒的对照鸡,从攻毒后第28d就有6/15的比例从羽毛囊中检出MDV,而与vvMd5接种鸡同一隔离罩的对照鸡,在35d时才在2/14的个体中检出MDV,即GX0101的横向传播能力大于超强毒株。在SPF鸡试验中这一结果表明,MDV的致病性不一定与横向传播力相平行。由此推测,显著增高的横向传播能力可能就是这一整合进REV-LTR的重组病毒株能在鸡群中逐渐流行开来的选择性竞争优势之一。
试验还进一步比较了斑点杂交和琼脂扩散试验在动物实验中的应用,实验结果表明本试验证明斑点杂交试验在攻毒后第10d即可从羽毛囊中检测到病毒,而琼脂扩散试验在攻毒后第14d检测到病毒,斑点杂交的最高检出率为100%,而琼脂扩散试验的最高检出率为80%,斑点杂交的检出率也高于与琼脂扩散试验。斑点杂交的检测灵敏度达1pg DNA水平。可以对大量的样品进行快速,简便,准确的检测,可作为临床MDV检测的有效手段;因此,斑点杂交在实际生产中有很广泛的应用。
为了模仿并跟踪反转录病毒与MDV病毒在鸡体内自然发生的基因重组,用PCR来追踪鸡体内二种病毒发生重组的动态。根据MDV基因组上易插入REV的LTR的高频位点的序列合成7条引物,根据REV的LTR合成4条引物,由此交叉组成28对引物,同样设计ALV-MDV之间30对引物来扩增重组片段,从中分离筛选整合进反转录病毒基因序列的重组MDV。此次试验MDV与REV或ALV人工共感染,在连续传到第17代时,利用PCR还没有扩增检测有重组片段。这说明二种不同病毒在体内发生重组的机率确实非常小。但是能从鸡群中比较容易分离到重组野毒株,GX0101还说明这是经过长期的选择而产生的。
In recent years, some fowls disclose higher tumorigenesis for chickens viccinated against Marek's disease CVI988/Rispens .so scholar of exterior and interior suspected whether or not occurrence the higher pathogenicity of wild strain MDV which breakthrough this vaccine of protection.
Zhangzhi isolated A recombinant Marek's disease virus field strain with REV-LTR of GX0101 from fowl group which tumorigenic in 2001. In order to disclose the failure of immune whether or not concerned with the pathogenicity enhance of this popular strain .Zhangzhi(2004)and Zhuang guoqing(2006) did artificial infextation experiment, indicated the pathogenicity of GX0101 highter than the GA,this research adopt mortality and tumor rates of GX0101 were 28.6% and 7.1% for SPF chickens viccinated against Marek's disease virus. Mortality and tumor rates of vvMd5 were 63.1% and 19.0% in the same viccinated SPF chickens. Mortality and tumor rates of GX0101 were lower than that of vvMd5. However, horizontal transmission ability of GX0101 was stronger than that of Md5. After 28 days challenged by GX0101, MDV was detected in 6 of 15 samples. After 35 days challenged by vvMd5, MDV was detected in 2 of 14 samples. Horizontal transmission ability of MDV isolates was not necessarily parallel to their pathogenicity.It is suggesting that the increased horizontal transmission may be one of selective competitive advantages. Such advantages may help recombinant viruses with the LTR-insert become more and more popular gradually in chicken flocks.
This expenment compared the application in animal experiment between dot blot hybridization and agar diffusion reactionanimal experiment, the result indicated the virus can be detected from feather by dot blot hybridization after 10 days challenged, after 14days challenged can be detected by agar diffusion reaction, the highest detection rate of dot blot hybridization is 100%, but the the highest detection rate of agar diffusion reaction is 80%. The detection rate of dot blot hybridization was highter than the agar diffusion reaction. The sensitivity of dot blot hybridization was 1pg DNA, the detection of dot blot hybridization was fast, simple ,precise to a devil of sample. the dot blot hybridization can be trate as a utility means in detect MDV. Accordingly, dot blot hybridization has widely use in practical parturition.
In order to imitate and pursuit retrovirus with marek's disease virus abiogenetic gene recombination in chincken 's vivo. To amplify and clone the integrated REV LTR with MDV sequence at the junction,4 primersfrom REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hotpoint were synthesized and 28(4x7)pairs of primers(one from REV and another from MDVfor each pair)were used in PCR while the genomic DNA of both strains were used as the emplates. And we designed 30 pairs (one from REV and another from MDVfor each pair)were used in PCR while the genomic DNA of both strains were used as the emplates.of primers recombinated MDV which integrated the gene order of retrovirus.this expenment artificial coinfection with MDV and REV or ALV, when continue the 17th generation , can not isolate the re- combinate virus , the probability of two virus recombinated was very tiny, But,it is easily to isolate recombinated wild strain ,this expenment demonstrate the GX0101 under the long-standing selection.
This research disclose , the horizontal transmission of recombinant Marek's disease virus field strain with REV-LTR of GX0101 highter than the rMD5 ,
引文
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