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一株猪源H5亚型禽流感病毒的序列分析及致病性试验

发布时间:2019-05-19 20:00
【摘要】:本研究从2014年6月至2015年10月采集华南地区某些养猪场育肥猪和屠宰场待宰猪的猪鼻拭子500份和猪肺210份,将病料无菌处理后接种于9~11d龄鸡胚尿囊腔,将接种鸡胚在无菌条件下收获鸡胚液,1%的鸡红细胞测定是否有血凝活性(HA实验),有血凝活性的尿囊液用H1、H3、H5、H6、H7和H9亚型流感标准阳性血清、NDV标准阳性血清进行血凝抑制试验(HI实验)。然后进行序列测定,分离到1株猪源H5N6亚型禽流感病毒,命名为A/Avian/Huanan/C135/2015(H5N6)简称C135。测定C135毒株的全基因序列,遗传演化分析结果表明:NA基因ORF框长度为1380个核苷酸,颈部缺失33个核苷酸,该基因上不存在E119V、R152K、H275Y、R293K、N295S等耐药位点突变。HA基因全长为1776个核苷酸,含有一个完整ORF框,包含1704个核苷酸,可推导编码567个氨基酸,其5’非编码区和3’非编码区分别含有28个和44个核苷酸,未发现核苷酸的插入和缺失;裂解位点为RRRKR↓G,符合高致病性病毒的基因特点。N端糖基化位点中,26位、27位、39位、181位、302位、499位和558位存在糖基化位点。M基因经过分析后发现:H5N6亚型AIV的全长1027个核苷酸,完整的ORF框由两部分构成,M1长度为759个核苷酸,M2长度为294个核苷酸,推导能编码97个氨基酸。PB1基因系列分析发现:全长为2341bp个核苷酸,包含完整ORF框,长度为2274个核苷酸,推导编码757个氨基酸,其中5’非编码区和3’非编码区分别含有24个和43个核苷酸,无核苷酸的插入和缺失;病毒的13位均突变为P,678位没有发生突变均为S。PB2基因由2341bp个核苷酸组成,完整的ORF框长度为2280bp核苷酸,推导可编码759个氨基酸,其5’非编码区和3’非编码区分别含有27个和34个核苷酸。PB2基因分析发现,627位为E,没有发生突变;与毒力紧密相关的701位依然为D,这些基因未发生突变。PA基因片段长度为2233个核苷酸,含有一个长度为2151个核苷酸的完整ORF框,推导能编码716个氨基酸,其5’非编码区和3’非编码区分别含有24个和58个核苷酸,未发现氨基酸的插入或缺失,但发生了N615K突变,突变为K;与病毒在细胞中生长有关的638位为R、与复制有关的539位为K、与聚合酶活性相关的510位为H,与致病性相关的224位与383位分别为S和D,没有发生突变。NP基因长度为1565个核苷酸,含有一个完整的长度为1497个核苷酸的ORF框,推导可编码498个氨基酸,未发现核苷酸的插入和缺失。其5’非编码区和3’非编码区分别含有45个和23个核苷酸。毒株的位点分析发现,影响试验动物肺脏病毒滴度相关的479位,存在L479F突变;319位没有发生突变,仍为N。NS基因ORF框分为两个部分,ORF1从27~704位核苷酸,是一个连续的片段,包含678个核苷酸,NS1推导可编码225个氨基酸,ORF2又包含不连续的两部分,一部分是从27位~56位核苷酸,一部分是从514位~849位核苷酸,共有366个核苷酸,位于5’端的ORF1与ORF2有重叠30个核苷酸部分,间隔457个核苷酸后的336个核苷酸则是NS2第二部分OFR框,NS2推导可编码121个氨基酸,位点分析发现:C135的42位突变位S,92位突变位E,103位突变位非致病性K,106位突变位V,149位均为G;在NS1的末端为ESEV残基。为进一步了解猪源禽流感病毒对动物的致病性试验,流感病毒C135以106EID50剂量干冰麻醉后,滴鼻的方式感染12~14g的雌性BALB/c小鼠;以105EID50剂量,通过滴鼻点眼的方式感染4~6周龄的SPF鸡。结果表明,攻毒组小鼠死亡率达到了75%。小鼠脑中没有检测到病毒,肺脏中病毒滴度较高为4.75~6.5log_(10)EID_(50)/mL,说明病毒可以在小鼠肺脏内增殖,但不具备入脑的能力;期间观察到小鼠出现身体消瘦、被毛凌乱、神经症状等情况,解剖发现内脏出现了病理变化,试验表明该毒株对小鼠有强致病性。病毒能够使全部SPF鸡在感染后2d内死亡,在脑、心、肝、脾、肺、肾6个组织脏器中检测到较高滴度病毒为4.91~7.08log_(10)EID_(50)/mL;同居组的脑、心、肝、脾、肺、肾6个组织脏器中未检测到病毒,表明该病毒对SPF鸡具有高致死率。
[Abstract]:In this study,500 parts of pig nasal swabs and 210 parts of pig lung were collected from June 2014 to October 2015 in some pig farms in South China. the chicken embryo liquid is harvested under the aseptic condition,1 percent of the chicken red blood cells are tested for hemagglutination activity (HA test), and the blood bag liquid with the blood clotting activity is used for the H1, H3, H5, H6, H7 and H9 subtype influenza standard positive serum, The hemagglutination inhibition test (HI test) was performed in the NV standard positive serum. The sequencing was then carried out to isolate the H5N1 avian influenza virus, named A/ Aian/ Huanan/ C135/2015 (H5N6) as C135. The results of the genetic evolution of the whole gene sequence of the C135 strain show that the length of the ORF of the NA gene is 1380 nucleotides, and the neck is missing 33 nucleotides, and there is no mutation of the resistance sites such as E119V, R152K, H275Y, R293K and N295S. The total length of the HA gene is 1776 nucleotides, which contains a complete ORF box containing 1704 nucleotides, and can be derived to encode 567 amino acids. The 5 'non-coding region and the 3' non-coding region contain 28 and 44 nucleotides, respectively, and the insertion and deletion of the core acid is not found; the cleavage site is RRRKR-G, And the method accords with the gene characteristic of the high-pathogenicity virus. In the N-terminal glycosylation site, the glycosylation sites were present in 26,27,39,181,302,499 and 558 sites. After the M gene was analyzed, it was found that the total length of the AIV of the H5N6 subtype AIV was 1027 nucleotides, the complete ORF frame was composed of two parts, the length of the M1 was 759 nucleotides, and the length of the M2 was 294 nucleotides, and the deduced amino acids can be encoded. The results showed that the total length of the PB1 gene was 2341 bp. The total length was 2341 bp. The total length was 2274 nucleotides, and it was deduced that 757 amino acids were encoded. The 5 'non-coding region and the 3' non-coding region respectively contain 24 and 43 nucleonic acid, and the insertion and deletion of the non-nuclear-acid-free acid. The 13-position mutation of the virus is P and 678, and no mutation is found in the S. The PB2 gene is composed of 2341 bp nucleotides, the length of the complete ORF is 2280 bp, and the deduced amino acid can be encoded. The 5 'non-coding region and the non-coding region of the 3' non-coding region respectively contain 27 and 34 nucleotides. The PB2 gene analysis found that the 627-bit was E and there was no mutation; the 701-position closely related to the virulence was still D, and the genes did not mutate. The length of the fragment of the PA gene is 2233 nucleotides, and contains a complete ORF box with a length of 2151 nucleotides, which can encode 716 amino acids. The 5 'non-coding region and the 3' non-coding region contain 24 and 58 nucleotides, respectively, and the insertion or deletion of the amino acid is not found, but the N615K mutation occurs, The mutation was K; the 638-position related to the growth of the virus in the cell was R, the 539-bit related to the replication was K, the 510-bit related to the polymerase activity was H, the 224-and 383-bits associated with the pathogenicity were S and D, respectively, and no mutation occurred. The length of NP gene is 1565 nucleotides, which contains an ORF frame of 1497 nucleotides with length of 1497. It is deduced that 498 amino acids can be encoded without the insertion and deletion of the nucleotides. The 5 'non-coding region and the 3' non-coding region contain 45 and 23 nucleotides, respectively. The site analysis of the strain found that there were 479 sites related to the titer of the lung virus of the test animals, and the L479F mutation was present; there was no mutation in 319 bits, and the ORF of the N. NS gene was divided into two parts, and the ORF1 from 27 to 704 was a continuous fragment containing 678 nucleotides. The NS1 derivation can encode 225 amino acids, and the ORF2 also contains two parts which are not continuous, a part of which is from 27 to 56 nucleotides, a part of which is from 514 to 849, and a total of 366 nucleotides, and the ORF1 and ORF2 located at the 5 'end have 30 nucleotides that overlap each other, The results of the analysis showed that the S,92-bit mutation, the non-pathogenic K, the 106-bit mutant and the 149-position of the mutation in the 42-position mutant of C135 were G. The ESEV residue is at the end of NS1. In order to further understand the pathogenic test of the avian influenza virus on the animals, the influenza virus C135 was infected with 12 to 14 g of female BALB/ c mice in a nasal way after the 106 EID50 dose of dry ice was anesthetized, and the SPF chickens of 4 to 6 weeks of age were infected by a drop-in-eye method at a dose of 105 EID50. The results showed that the mortality of mice in the attack group reached 75%. No virus was detected in the brain of the mouse, and the virus titer in the lung was 4.75-6.5 log _ (10) EID _ (50)/ mL, indicating that the virus could proliferate in the lung of the mouse, but did not have the ability to enter the brain. During the period, the mice were observed to be emaciated, The test indicated that the strain was highly pathogenic to the mice. the virus can cause all SPF chickens to die within 2 days after infection, and the high-titer virus is detected to be 4.91-7.08 log _ (10) EID _ (50)/ mL in the organs of the brain, the heart, the liver, the spleen, the lung and the kidney; and the viruses are not detected in the brain, the heart, the liver, the spleen, the lung and the kidney of the cohabiting group, Indicating that the virus has high toxicity to the SPF chicken.
【学位授予单位】:华南农业大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:S852.65

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