一株豬源H5亞型禽流感病毒的序列分析及致病性試驗(yàn)
發(fā)布時(shí)間：2019-05-19 20:00
【摘要】：本研究從2014年6月至2015年10月采集華南地區(qū)某些養(yǎng)豬場(chǎng)育肥豬和屠宰場(chǎng)待宰豬的豬鼻拭子500份和豬肺210份,將病料無菌處理后接種于9~11d齡雞胚尿囊腔,將接種雞胚在無菌條件下收獲雞胚液,1%的雞紅細(xì)胞測(cè)定是否有血凝活性(HA實(shí)驗(yàn)),有血凝活性的尿囊液用H1、H3、H5、H6、H7和H9亞型流感標(biāo)準(zhǔn)陽性血清、NDV標(biāo)準(zhǔn)陽性血清進(jìn)行血凝抑制試驗(yàn)(HI實(shí)驗(yàn))。然后進(jìn)行序列測(cè)定,分離到1株豬源H5N6亞型禽流感病毒,命名為A/Avian/Huanan/C135/2015(H5N6)簡(jiǎn)稱C135。測(cè)定C135毒株的全基因序列,遺傳演化分析結(jié)果表明:NA基因ORF框長(zhǎng)度為1380個(gè)核苷酸,頸部缺失33個(gè)核苷酸,該基因上不存在E119V、R152K、H275Y、R293K、N295S等耐藥位點(diǎn)突變。HA基因全長(zhǎng)為1776個(gè)核苷酸,含有一個(gè)完整ORF框,包含1704個(gè)核苷酸,可推導(dǎo)編碼567個(gè)氨基酸,其5’非編碼區(qū)和3’非編碼區(qū)分別含有28個(gè)和44個(gè)核苷酸,未發(fā)現(xiàn)核苷酸的插入和缺失;裂解位點(diǎn)為RRRKR↓G,符合高致病性病毒的基因特點(diǎn)。N端糖基化位點(diǎn)中,26位、27位、39位、181位、302位、499位和558位存在糖基化位點(diǎn)。M基因經(jīng)過分析后發(fā)現(xiàn):H5N6亞型AIV的全長(zhǎng)1027個(gè)核苷酸,完整的ORF框由兩部分構(gòu)成,M1長(zhǎng)度為759個(gè)核苷酸,M2長(zhǎng)度為294個(gè)核苷酸,推導(dǎo)能編碼97個(gè)氨基酸。PB1基因系列分析發(fā)現(xiàn):全長(zhǎng)為2341bp個(gè)核苷酸,包含完整ORF框,長(zhǎng)度為2274個(gè)核苷酸,推導(dǎo)編碼757個(gè)氨基酸,其中5’非編碼區(qū)和3’非編碼區(qū)分別含有24個(gè)和43個(gè)核苷酸,無核苷酸的插入和缺失;病毒的13位均突變?yōu)镻,678位沒有發(fā)生突變均為S。PB2基因由2341bp個(gè)核苷酸組成,完整的ORF框長(zhǎng)度為2280bp核苷酸,推導(dǎo)可編碼759個(gè)氨基酸,其5’非編碼區(qū)和3’非編碼區(qū)分別含有27個(gè)和34個(gè)核苷酸。PB2基因分析發(fā)現(xiàn),627位為E,沒有發(fā)生突變;與毒力緊密相關(guān)的701位依然為D,這些基因未發(fā)生突變。PA基因片段長(zhǎng)度為2233個(gè)核苷酸,含有一個(gè)長(zhǎng)度為2151個(gè)核苷酸的完整ORF框,推導(dǎo)能編碼716個(gè)氨基酸,其5’非編碼區(qū)和3’非編碼區(qū)分別含有24個(gè)和58個(gè)核苷酸,未發(fā)現(xiàn)氨基酸的插入或缺失,但發(fā)生了N615K突變,突變?yōu)镵;與病毒在細(xì)胞中生長(zhǎng)有關(guān)的638位為R、與復(fù)制有關(guān)的539位為K、與聚合酶活性相關(guān)的510位為H,與致病性相關(guān)的224位與383位分別為S和D,沒有發(fā)生突變。NP基因長(zhǎng)度為1565個(gè)核苷酸,含有一個(gè)完整的長(zhǎng)度為1497個(gè)核苷酸的ORF框,推導(dǎo)可編碼498個(gè)氨基酸,未發(fā)現(xiàn)核苷酸的插入和缺失。其5’非編碼區(qū)和3’非編碼區(qū)分別含有45個(gè)和23個(gè)核苷酸。毒株的位點(diǎn)分析發(fā)現(xiàn),影響試驗(yàn)動(dòng)物肺臟病毒滴度相關(guān)的479位,存在L479F突變;319位沒有發(fā)生突變,仍為N。NS基因ORF框分為兩個(gè)部分,ORF1從27~704位核苷酸,是一個(gè)連續(xù)的片段,包含678個(gè)核苷酸,NS1推導(dǎo)可編碼225個(gè)氨基酸,ORF2又包含不連續(xù)的兩部分,一部分是從27位~56位核苷酸,一部分是從514位~849位核苷酸,共有366個(gè)核苷酸,位于5’端的ORF1與ORF2有重疊30個(gè)核苷酸部分,間隔457個(gè)核苷酸后的336個(gè)核苷酸則是NS2第二部分OFR框,NS2推導(dǎo)可編碼121個(gè)氨基酸,位點(diǎn)分析發(fā)現(xiàn):C135的42位突變位S,92位突變位E,103位突變位非致病性K,106位突變位V,149位均為G;在NS1的末端為ESEV殘基。為進(jìn)一步了解豬源禽流感病毒對(duì)動(dòng)物的致病性試驗(yàn),流感病毒C135以106EID50劑量干冰麻醉后,滴鼻的方式感染12~14g的雌性BALB/c小鼠;以105EID50劑量,通過滴鼻點(diǎn)眼的方式感染4~6周齡的SPF雞。結(jié)果表明,攻毒組小鼠死亡率達(dá)到了75%。小鼠腦中沒有檢測(cè)到病毒,肺臟中病毒滴度較高為4.75~6.5log_(10)EID_(50)/mL,說明病毒可以在小鼠肺臟內(nèi)增殖,但不具備入腦的能力;期間觀察到小鼠出現(xiàn)身體消瘦、被毛凌亂、神經(jīng)癥狀等情況,解剖發(fā)現(xiàn)內(nèi)臟出現(xiàn)了病理變化,試驗(yàn)表明該毒株對(duì)小鼠有強(qiáng)致病性。病毒能夠使全部SPF雞在感染后2d內(nèi)死亡,在腦、心、肝、脾、肺、腎6個(gè)組織臟器中檢測(cè)到較高滴度病毒為4.91~7.08log_(10)EID_(50)/mL;同居組的腦、心、肝、脾、肺、腎6個(gè)組織臟器中未檢測(cè)到病毒,表明該病毒對(duì)SPF雞具有高致死率。
[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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65
本文編號(hào)：2481028
[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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65
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