本文關(guān)鍵詞： H9N2亞型AIV 分離鑒定 相對(duì)熒光定量PCR 雛鴨 排毒 病毒載量　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：H9N2亞型AIV在鴨群中很少引起發(fā)病,但家鴨被認(rèn)為是AIV的巨大儲(chǔ)存庫,在一定程度上影響著禽流感病毒的發(fā)生和傳播。目前在我國許多地區(qū)的鴨群中分離和檢測(cè)到H9N2亞型AIV,對(duì)于它的潛在危害已經(jīng)受到越來越多的關(guān)注。本研究包括以下三個(gè)部分:1.H9亞型AIV的分離鑒定、生物學(xué)特性研究以及全基因序列分析本試驗(yàn)從山東濟(jì)寧送檢的疑似H9N2亞型AIV感染的病鴨氣管,肺臟等組織中分離得到1株H9亞型禽流感病毒,命名為JN1株。通過血凝血抑、EID50、IVPI、MDT和抗原相關(guān)系數(shù)等的測(cè)定分析和比較JN1株的致病力。結(jié)果表明,JN1株的EID50為10-7.54/0.2mL,MDT為72h,IVPI為0.26。JN1株和H9N2標(biāo)準(zhǔn)株抗原相關(guān)指數(shù)為0.470.5,抗原相關(guān)性較差,說明,JN1株已在抗原性上發(fā)生改變。根據(jù)H9N2亞型AIV在GenBank中的序列,設(shè)計(jì)引物擴(kuò)增JN1株的全基因組序列;利用MEGA5.1軟件對(duì)其8個(gè)基因各自繪制系統(tǒng)發(fā)育樹并進(jìn)行分析。系統(tǒng)發(fā)育分析表明,JN1株的HA與DK/HK/Y280/97在同一分支上,NA、M、NP、PA、PB1、PB2基因與SH/F/98同源性較高,NS基因則與鴨源H5N1亞型AIV NS基因同源性高達(dá)99%。HA的裂解位點(diǎn)為PSRSSR↓GL,存在6個(gè)潛在糖基化位點(diǎn),在234位的氨基酸殘基為L,NA基因頸部存在缺失。2.H9N2亞型AIV病毒熒光定量PCR快速檢測(cè)方法的建立與應(yīng)用根據(jù)JN1株的HA基因,在其保守區(qū)域設(shè)計(jì)合成1對(duì)特異性引物,同時(shí)設(shè)計(jì)1對(duì)擴(kuò)增內(nèi)參基因β-actin的引物,擴(kuò)增片段分別為139 bp,119 bp。以陽性重組質(zhì)粒作為標(biāo)準(zhǔn)品做標(biāo)準(zhǔn)曲線,建立檢測(cè)H9N2亞型AIV的相對(duì)熒光定量RT-PCR方法。結(jié)果表明,HA基因和β-actin基因標(biāo)準(zhǔn)曲線線性關(guān)系R值在0.99以上,表現(xiàn)出良好的線性關(guān)系;熒光定量PCR最小檢出模板濃度約為10 Copies/μL,比RT-PCR靈敏100倍以上;特異性良好;HA基因和β-actin基因的批內(nèi)、批間變異系數(shù)均小于1%。3.H9N2亞型AIV的排毒規(guī)律及組織中病毒載量的變化將純化的H9N2亞型AIV通過點(diǎn)眼滴鼻感染3周齡健康雛鴨,107.84EID50/只。攻毒后,每隔2天采集咽拭子、泄殖腔棉拭子以及血液,并隨機(jī)剖殺3只。利用建立的檢測(cè)方法檢測(cè)病毒的排毒規(guī)律及組織中病毒相對(duì)表達(dá)量,同時(shí)測(cè)定血清HI抗體效價(jià)。結(jié)果顯示:血清HI試驗(yàn)結(jié)果顯示,雛鴨接種JN1株之后抗體效價(jià)一直維持在較低水平,雛鴨感染H9N2亞型AIV后,第2天即可檢測(cè)到排毒,在檢測(cè)過程中,出現(xiàn)了兩個(gè)排毒高峰,無論是泄殖腔棉拭子還是咽拭子,均在第3天達(dá)到排毒高峰,在第11天排毒量降到最低,第13天突然升高,后降低。在排毒過程中,咽拭子排毒量要高于泄殖腔棉拭子。人工感染第2天即可在各個(gè)組織中檢測(cè)到H9N2亞型AIV,感染后7-11天在各組織中的相對(duì)表達(dá)量較高。H9N2亞型AIV相對(duì)表達(dá)量最高的器官為氣管、肝臟、胰腺和腺胃,相對(duì)表達(dá)量在0.4~0.6之間,表達(dá)量較低的為肺臟、脾臟、腎臟和法氏囊,相對(duì)表達(dá)量在0.2~0.4之間。本試驗(yàn)分離到的鴨源H9N2亞型AIV,為低毒力株,但對(duì)鴨有一定的致病性。利用建立的HA基因的相對(duì)熒光定量RT-PCR方法對(duì)雛鴨感染H9N2亞型AIV后排毒規(guī)律和組織病毒載量測(cè)定時(shí)發(fā)現(xiàn),在實(shí)驗(yàn)室條件下,3周齡雛鴨人工感染H9N2亞型AIV后2-17d均有排毒,2-7d排毒量較多。感染后7-11天組織含毒量較高。
[Abstract]:H9N2 subtype AIV rarely cause disease in ducks, but the duck is considered to be a huge repository of AIV, to a certain extent affect the occurrence and spread of avian influenza virus. At present in many areas of our country the ducks in the separation and detection of H9N2 subtype AIV, the potential harm it has been more attention. This study includes the following three parts: the isolation and identification of 1.H9 subtype AIV, biological characteristics and genome sequence analysis of the test from the suspected disease duck tracheal H9N2 subtype AIV infection in Shandong from Jining, 1 strains of H9 subtype avian influenza virus isolated from lung tissue, named JN1 the blood clotting inhibition. Strains EID50, IVPI, MDT, and the correlation coefficient of determination of antigen analysis and comparison of the pathogenicity of JN1 strain. The results showed that the JN1 strain of EID50 10-7.54/0.2mL, MDT 72h, IVPI 0.26.JN1 strain and the H9N2 strain related antigen index is 0.47 0.5, the antigen poor correlation shows that JN1 strains have been changed. According to the antigenicity of H9N2 subtype AIV in GenBank sequence, amplified genome sequence of JN1 strain primers; phylogenetic tree and analyze the 8 genes each rendering system using MEGA5.1 software. The system development analysis showed that HA and DK/HK/Y280/97 JN1 were in the same branch, NA, M, NP, PA, PB1, PB2 gene and SH/F/98 NS gene homology, with duck origin H5N1 subtype AIV NS gene homologous to the cleavage site of up to 99%.HA for PSRSSR: GL, there are 6 potential glycosylation sites in amino acid residue based on the 234 for the L, the rapid detection method of fluorescence quantitative PCR deletion of.2.H9N2 subtype AIV virus NA gene exists in the neck of the establishment and application of the HA gene of JN1 strain in the conservative region, 1 pairs of primers were designed for amplification of 1, while the design of reference gene beta -actin primers, amplified fragment Some were 139 BP, 119 bp. positive recombinant plasmid as a standard for the standard curve, the relative fluorescence quantitative RT-PCR method was established to detect H9N2 subtype AIV gene. The results showed that the standard curve of HA gene and beta -actin linear relationship between the R value above 0.99, showing a good linear relationship between the fluorescence quantitative PCR minimum detection template; the concentration of Copies/ is about 10 L, 100 times more sensitive than RT-PCR or above; good specificity; HA gene and beta -actin gene in batch, viral shedding pattern and interassay coefficients of variation were less than 1%.3.H9N2 AIV subtype in the quantity change of the purified H9N2 subtype AIV through oral nasal infection 3 weeks old healthy ducklings, only 107.84EID50/. After infection, every 2 days, throat swab, cloacal swabs and blood, and killed randomly 3 rats. Expression of virus excretion and tissue using the detection method of virus detection, measured at the same time Set the HI titers in serum. The results showed that the serum HI test results showed that the ducklings inoculated with JN1 strain after antibody titer remained at a low level, Ducklings Infected with H9N2 subtype AIV after second days can be detected in the detection process, detoxification, detoxification appeared two peak, either the cloacal swab or pharynx swab, reached the peak in the third day detox, detox in eleventh days to a minimum, thirteenth days suddenly increased, and then decreased. In the process of detoxification, detoxification of throat swab was higher than the cloacal swab. Artificial infection second days in all tissues detected by H9N2 subtype AIV infection 7-11 days in different tissues relative higher expression of.H9N2 subtype AIV relative expression of the highest organ of the trachea, liver, pancreas and stomach, the relative expression between 0.4~0.6, the low expression level in lung, spleen, kidney and bursa, relative expression in 0.2~0.4 . in the test was duck origin H9N2 subtype AIV, but for the low virulent strains, there is a certain pathogenicity of duck. The relative fluorescence quantitative RT-PCR method for HA gene based discovery of excretion of virus load test and tissue Ducklings Infected with H9N2 subtype AIV, under the condition of laboratory, 3 week old Ducklings Infected with H9N2 subtype AIV 2-17d were 2-7d after detoxification, detoxification was more higher. 7-11 days after infection of virus content in tissue.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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