广西猪圆环病毒2型分子流行病学调查及分离鉴定

发布时间：2018-06-15 10:45

本文选题：猪圆环病毒2型 + 全基因扩增　；参考：《广西大学》2015年硕士论文

【摘要】：猪圆环病毒2型(Porcine circo virus type 2, PCV2)于1998年被发现以来,现如今已成为一种广泛流行于世界各地区的疾病病原之一。它严重危害猪群,破坏猪的淋巴系统,使猪的淋巴细胞减少,进而造成猪的免疫力下降,处于亚健康状态,容易受其他病原或者外界环境因素的影响而导致发病。因此,PCV2在全球范围内已造成了巨大经济损失并且还在制约着世界养猪行业的发展。为了解广西PCV2的流行情况,本研究对2012年8月至2015年1月期间采集的广西不同地区的284样品淋巴结及肺脏组织,采用PCR方法进行PCV2检测,共检测出144份阳性样品,阳性率达50.7%；其中春、夏、秋、冬四季的PCV2阳性率分别为42.4%(36/85)、91.5%(43/47)、38.7%(43/111)和53.7%(22/41)。说明了广西猪群中普遍存在PCV2感染现象,并且在夏季流行较为严重。为进一步了解PCV2在广西流行的主要基因型。本研究选取检测为阳性的部分样品进行PCV2全基因的克隆,成功克隆出45株PCV2全基因序列。遗传进化分析结果发现,其中6株属于PCV2a型,39株属于PCV2b型,其中14株属于PCV2b-1A/B亚群,25株属于PCV2b-1C亚群。说明了广西部分地区PCV2b流行广泛,其中又以PCV2b-1C亚群流行为主。结合本实验所保存的24株PCV2全基因进行分析,充分说明了广西PCV2亚群流行发展的变化规律,是由PCV2b-lA/B亚群向PCV2b-1C亚群转变。本研究得到6株PCV2a与中国吉林疫苗株HM038034及匈牙利株AY256459共同处于PCV2a的分支上。所得14株PCV2b-lA/B与中国广东株EF421971、中国安徽株GU450327、中国浙江株AY691169、中国河南株FJ440338、中国辽宁株HM776448及中国北京株EF524530处于同一分支上,而不处于中国浙江疫苗株AY686764、中国山西疫苗株HM641752和荷兰株AF201897所在的分支上。本研究所得25株PCV2b-1C中,有23株与中国黑龙江株HM038017、中国湖北疫苗株FJ870971、中国上海疫苗株AY686763、中国湖北株AY291317及中国甘肃株FJ948168处于同一分支。另外2株PCV2b-1C与中国湖北株AY035820、印度尼西亚株EU302139和越南株JX506730及JX099782处于同一分支,并且为PCV2a与PCV2b-1C ORF2的重组株。45株PCV2全基因核苷酸的同源性达94.2-99.9%,45株PCV2各基因型之间ORF1核苷酸具有很高的同源性为96.5-100%,而ORF2的核苷酸同源性为88.9-99.9%。可以看出ORF1较ORF2保守,PCV2三个亚型的主要差异表现在ORF2。45株PCV2 ORF1氨基酸同源性为97.5-100%之间,而45株PCV2 ORF2氨基酸同源性为87.2-100%之间。根据PCV2 ORF2推导出的氨基酸序列比对发现,ORF2氨基酸序列中存在3个主要的变异度较高区域：53-91 aa、121-151 aa和185-215 aa,并且包含着3个Cap蛋白的抗原表位：69-83 aa、117-131 aa和193-207 aa, Cap蛋白主要的氨基酸差异位点也在其中。PCV2各基因型主要在8、53、57、59、68、77、86-91、121、130、131、133、134、151、169、185、187、190-191、206、210、215、232、234的氨基酸位点上表现不同,D77、TNKISI86-91、P131、S133、I187、S190和K232是PCV2a区别于PCV2b的特点,F8、I53、N68、T121、N134、D215、K234是PCV2b中1C亚群区别于1A/B亚群的特点。为将广西流行的PCV2毒株分离培养出来,本研究采用PK-15细胞带毒传代的方法,从144份PCV2阳性样品中选取部分样品在PK-15细胞中进行PCV2的分离,细胞带毒传代至10代时,采用PCR的鉴定方法,成功分离并鉴定出三株PCV2分离株GXGG-4-2013、GXNN-2-2014和GXCZ-2014。三株PCV2分离株同属PCV2b型,其中GXGG-4-2013、 GXNN-2-2014属于PCV2b-1C亚群,GXCZ-2014属于PCV2b-1A/B亚群。GXGG-4-2013株经过80代的带毒传代培养,每隔十代扩增分离病毒的全基因,8个代次的全基因核苷酸序列同源性很高在99.5-100%之间,说明其核苷酸保持相对稳定,未出现很大的变异。本研究分析广西部分地区PCV2的流行情况及PCV2分子流行特点,进而为广西PCV2的防控提供理论参考。分离出的3株PCV2可以为以后研究PCV2分离株的生物特性提供材料。
[Abstract]:Since its discovery in 1998, the porcine circovirus 2 (Porcine Circo virus type 2, PCV2) has now become one of the disease pathogens that are widely prevalent in all regions of the world. It seriously endangering pigs, destroying the lymphatic system of pigs, reducing the lymphocytes of pigs, and thus forming a pig's immune decline, being in a subhealthy state and easy to suffer from it. Therefore, PCV2 has caused huge economic losses worldwide and is still restricting the development of the world's pig industry. To understand the prevalence of PCV2 in Guangxi, 284 samples of lymph nodes and lungs in different regions of Guangxi collected from August 2012 to January 2015 A total of 144 positive samples were detected by PCV2 by PCR method, and the positive rate was 50.7%. The positive rates of PCV2 in spring, summer, autumn and winter were 42.4% (36/85), 91.5% (43/47), 38.7% (43/111) and 53.7% (22/41). It indicated that the prevalence of PCV2 infection in the Guangxi pigs was more serious in summer. To understand the main genotypes of PCV2 in Guangxi, this study selected the positive part of the sample to clone PCV2 whole gene and successfully cloned 45 PCV2 whole gene sequences. The results of genetic evolution analysis found that 6 of them belong to PCV2 A and 39 belong to PCV2 B type, of which 14 belong to PCV2b-1A/B subgroup and 25 belong to PCV2b-1C subgroup. It is clear that the prevalence of PCV2b is widespread in parts of Guangxi, among which PCV2b-1C subgroup is the main epidemic. Combined with the analysis of the 24 PCV2 whole genes preserved in this experiment, the variation of the epidemic development of the Guangxi PCV2 subgroup is fully explained, which is transformed from the PCV2b-lA/B subgroup to the PCV2b-1C subgroup. This study obtains 6 PCV2a and Jilin vaccine strain HM03803 in China. 4 and the Hungarian strain AY256459 are in the branch of PCV2a. The 14 strains of PCV2b-lA/B are on the same branch as China Guangdong strain EF421971, China Anhui strain GU450327, China Zhejiang strain AY691169, China Henan strain FJ440338, China Liaoning strain HM776448 and Beijing strain in China, but not in Zhejiang vaccine strain AY686764, China Shanxi. On the branch of the vaccine strain HM641752 and the Holland strain AF201897, 23 of the 25 PCV2b-1C strains obtained from this study were in the same branch of the Chinese Heilongjiang strain HM038017, the Hubei vaccine strain FJ870971 in China, the Shanghai vaccine strain of China, the Hubei strain AY291317 in China and the Gansu strain FJ948168 of China, and the other 2 strains of PCV2b-1C and Hubei strain AY035820 of China. The Indonesia strain EU302139 and the Vietnamese strain JX506730 and JX099782 are in the same branch, and the homology of the PCV2 full gene nucleotides of the recombinant strain.45 strain of PCV2a and PCV2b-1C ORF2 is 94.2-99.9%. The ORF1 nucleotide of the 45 PCV2 genotypes has a high homology for 96.5-100%. It is found that ORF1 is more conservative than ORF2, and the main difference between the three PCV2 subtypes is that the PCV2 ORF1 amino acid homology of the ORF2.45 strain is 97.5-100%, and the 45 PCV2 ORF2 amino acid homology is between 87.2-100%. According to the amino acid sequence alignment derived from PCV2 ORF2, there are 3 major regions of high variability in the amino acid sequence: 53-91 AA, 121-151 AA and 185-215 AA, and contain 3 Cap protein epitopes: 69-83 AA, 117-131 AA and 193-207 AA, and the main amino acid difference loci of Cap protein are also in the amino acid site of.PCV2, which are mainly in the amino acid site of 8,53,57,59,68,77,86-91121130131133134151169185187190-191206210215232234. D77, TNKISI86-91, P131, S133, I187, S190 and K232 are the characteristics that differ from PCV2b in PCV2a, F8, I53, N68, etc. When the samples were separated from the PK-15 cells for the separation of PCV2 and the cells were transmitted to the 10 generation, three PCV2 isolates, GXGG-4-2013, GXNN-2-2014 and GXCZ-2014., were isolated and identified by PCR. GXGG-4-2013 and GXNN-2-2014 belonged to the PCV2b-1C subgroup. After 80 generations of 4-2013 generations, the whole gene of the virus was amplified and separated every ten generations, and the nucleotide sequence of the 8 generations was highly homologous to 99.5-100%, indicating that the nucleotides remained relatively stable, and there was no large variation. This study analyzed the prevalence of PCV2 and the epidemic characteristics of PCV2 molecules in parts of Guangxi. It provides a theoretical reference for the prevention and control of PCV2 in Guangxi. 3 strains of PCV2 isolated can provide materials for further study of the biological characteristics of PCV2 isolates.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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