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  本文選題：高致病性豬繁殖與呼吸綜合征病毒　切入點：TJM-F92株　出處：《中國農(nóng)業(yè)科學院》2015年博士論文　論文類型：學位論文

【摘要】：豬繁殖與呼吸綜合征(porcine reproductive and respiratory syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一種以母豬繁殖障礙、仔豬和育肥豬呼吸道疾病為主要特征的傳染病,給養(yǎng)豬業(yè)帶來了巨大經(jīng)濟損失。免疫疫苗是控制PRRS的有效方法。一般情況下滅活疫苗具有良好的安全性,但不能提供有效保護。PRRSV活疫苗可刺激機體產(chǎn)生細胞免疫與體液免疫應(yīng)答,但活疫苗存在毒力返強的風險,并且由于PRRSV具有免疫抑制作用,通過傳統(tǒng)方法制備的PRRSV活疫苗一般很難改變其免疫抑制性,接種此類活疫苗會對機體造成免疫抑制,并且會干擾同期免疫的其他活疫苗的免疫應(yīng)答。高致病性豬繁殖與呼吸綜合征病毒TJM-F92疫苗株(HP-PRRSV TJM-F92)是一株在Nsp2基因存在不連續(xù)30個氨基酸(aa)缺失的同時還存在一段連續(xù)缺失120個aa的弱毒疫苗株。本研究通過將PRRSV TJM-F92株基礎(chǔ)種子接種PRRS健康易感仔豬,連續(xù)在豬體內(nèi)傳代五代來驗證該疫苗株是否存在毒力返強風險。并開展了PRRSV TJM-F92株對豬瘟兔化弱毒株(CSFV C株)及偽狂犬Bartha-K61疫苗株(PRV Bartha-K61株)的免疫干擾研究。將PRRSV TJM-F92株基礎(chǔ)種子免疫接種PRRS抗原、抗體陰性健康易感豬,采集病毒血癥載毒高峰期(接種病毒后第5日~第9日)的血清樣品,繼代接種易感豬進行5代毒力返強試驗,并對返強第5代分離病毒進行Nsp2基因序列分析,檢驗PRRSV TJM-F92株的安全性及Nsp2基因存在的120個aa在易感動物體內(nèi)的穩(wěn)定性。試驗結(jié)果表明,每代易感豬均未見臨床異常。大體剖檢結(jié)果顯示,免疫組與對照組試驗豬肺臟、脾臟、腎臟、肝臟、心臟、扁桃體和淋巴結(jié)眼觀無明顯變化。對肺臟進行病理組織學檢測,結(jié)果顯示,肺泡結(jié)構(gòu)正常、清晰,支細管周圍有少量淋巴細胞,免疫組與對照組無差異,未見由間質(zhì)性肺炎引起的損傷。將PRRSV TJM F92經(jīng)豬體內(nèi)連續(xù)傳代五代后,在TJM-F92株Nsp2區(qū)域存在的120個氨基酸的缺失仍然存在,說明該缺失可在豬體內(nèi)穩(wěn)定遺傳。試驗結(jié)果表明PRRSV TJM-F92疫苗株無毒力返強風險。本研究通過開展高劑量PRRSV TJM-F92株對低劑量CSFV C株或低劑量PRV Bartha-K61株同時免疫與間隔免疫干擾研究,高致病性豬繁殖與呼吸綜合征、偽狂犬二聯(lián)活疫苗對豬的免疫學功能影響試驗,分別從體液免疫、細胞免疫、免疫攻毒及病理學變化等方面比較了毒種聯(lián)合免疫組、毒種間隔免疫組與各毒種單免組在免疫及攻毒后的各項免疫學指標變化情況及攻毒保護情況。結(jié)果,高劑量PRRSV TJM-F92株對低劑量CSFV C株或低劑量PRV Bartha-K61株同時免疫、間隔7日免疫或間隔14日免疫,PRRSV TJM F92株對CSFV C株、PRV Bartha-K61株抗體水平無免疫干擾作用,與CSFV C株、PRV Bartha-K61株單免組抗體水平無顯著差異。攻擊CSFV強毒或PRV強毒后,各毒種聯(lián)合免疫組、毒種間隔免疫組試驗動物在臨床癥狀、體溫、攻毒保護率、病理學變化等方面與各毒種單免組試驗動物無顯著差異,各免疫組仔豬全部建活并得到有效保護。對4~6周齡PRRS與PR健康易感仔豬免疫高致病性豬繁殖與呼吸綜合征、偽狂犬二聯(lián)活疫苗,通過流式細胞儀對試驗動物外周淋巴血液中免疫細胞進行檢測,同時通過檢測抗體效價,統(tǒng)計攻毒保護率,進行病理組織學檢測,并與PRRSV單苗免疫組與PRV單苗免疫組進行比較,結(jié)果表明,PRRSV+PRV二聯(lián)苗、PRV單苗組、PRRSV單苗組免疫仔豬后抗體產(chǎn)生情況良好,可刺激機體產(chǎn)生細胞免疫應(yīng)答,未引起T細胞比例降低,攻毒保護率均為5/5。上述試驗結(jié)果表明,PRRSV TJM-F92株疫苗對CSFV C株或PRV Bartha-K61株等活疫苗毒株無免疫抑制作用。綜上所述,PRRSV TJM-F92株不存在毒力返強風險,進一步證明其為一株非常安全的疫苗毒株。免疫干擾試驗結(jié)果證實PRRSV TJM-F92株對CSFV C株、PRV Bartha-K61株等活疫苗毒株無免疫抑制作用,為制備PRRSV與CSFV二聯(lián)活疫苗、PRRSV與PRV二聯(lián)活疫苗或以PRRSV TJM-F92株為基礎(chǔ)的多聯(lián)活疫苗奠定了基礎(chǔ)。
[Abstract]:Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS) is composed of porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV) a in sows piglets and fattening pigs caused by the disease of respiratory tract infectious disease characteristic, has brought huge economic losses to the pig industry. Vaccination is an effective way to control the PRRS. Generally the inactivated vaccine has good safety, but can not provide effective protection of.PRRSV vaccine can induce cellular immune and humoral immune response, but there is a risk of vaccine virulence, and because PRRSV has immunosuppressive effect by traditional preparation method the PRRSV vaccine is difficult to change the immune suppression, the vaccine inoculation can cause immune suppression on the body, and may interfere with the immune period Other immune responses. The vaccine of highly pathogenic porcine reproductive and respiratory syndrome virus vaccine strain TJM-F92 (HP-PRRSV TJM-F92) is a plant in the Nsp2 gene are not continuous 30 amino acids (AA) there is also a lack of continuous loss of 120 AA attenuated vaccine strains. In this study, through the PRRSV TJM-F92 strain based seed inoculation PRRS susceptible healthy piglets, pigs in continuous five generations to verify the existence of vaccine strain virulence return risk. And carry out a PRRSV TJM-F92 strain of hog cholera Lapinised virus (CSFV strain C) and Pseudorabies Vaccine Strain Bartha-K61 (PRV strain Bartha-K61) immune PRRSV interference study. TJM-F92 strain of foundation seed inoculated with PRRS antigen and antibody negative healthy susceptible pigs, collecting viremia viral peak (fifth ~ ninth days after virus inoculation) serum samples, subinoculation susceptible pigs were the 5 generation of virulence test, and on the back The fifth generation of isolated virus were Nsp2 gene sequence analysis, 120 aa safety inspection of PRRSV and Nsp2 gene of strain TJM-F92 in susceptible stability within the object. The experimental results show that each generation of susceptible pigs showed no clinical abnormalities. Gross necropsy results showed that immunization group and control group test of pig lungs, spleen the liver, kidney, heart, eye view, tonsil and lymph node without significant change. Histopathological detection of lung showed normal alveolar structure, clear, there is a small amount of lymphocytes around the straw, immune group and control group had no difference, no interstitial pneumonia caused by the injury. The PRRSV TJM F92 by pigs after five passages, still exist in the deletion of 120 amino acids of TJM-F92 strain Nsp2 region, indicating that the loss of genetic stability in pigs. The test results show that the PRRSV TJM-F92 vaccine strain avirulent. The risk return Through the study of high dose of PRRSV TJM-F92 strain of CSFV low dose or low dose of PRV C strain Bartha-K61 strain and Study on immunity and interval immune interference, highly pathogenic porcine reproductive and respiratory syndrome, pseudorabies two combined live vaccine against porcine immune function tests, separately from humoral immunity, cellular immunity, immunity and the pathological changes were compared with virus immune group, virus immune group and the interval virus free single group in the changes of immunological indexes of the immunity and infection after andchallenge protection. As a result, the high dose of PRRSV TJM-F92 strain and C strain is immune to the low dose CSFV or low dose of PRV Bartha-K61 strain the 7 day interval, or immune 14 days interval immunization, PRRSV TJM of CSFV F92 strain C strain, no immune interference effects of PRV Bartha-K61 strain and CSFV antibody level, C strain, PRV Bartha-K61 strain free single group antibody level had no significant difference. CSFV attack Virulent or virulent PRV after the virus immunization group, virus interval immunization group of experimental animal in clinical symptoms, body temperature, protective rate, pathological changes and other aspects of the virus free single group of experimental animal had no significant difference, the immune group were all built and have been effectively protected. To live 4~6 week old PRRS PR health and susceptibility of highly pathogenic porcine reproductive and respiratory syndrome immune piglets Pseudorabies Vaccine, two, were detected by flow cytometry in experimental animal peripheral lymphoid blood immune cells and detected by antibody titers, statistical protective rate, histopathological detection, and compared with single PRRSV vaccine group and PRV vaccine group. The results showed that two PRRSV+PRV vaccine, PRV vaccine group, PRRSV vaccine group immunized piglets antibody production in good condition, can induce cellular immune response, did not cause the proportion of T cells decreased, attack Virus protection rate was 5/5. the results showed that PRRSV TJM-F92 vaccine had no inhibitory effect on immune CSFV C strain or PRV Bartha-K61 strain live vaccine strains. In summary, the PRRSV TJM-F92 strain does not exist virulence return risk, further proved to be a very safe vaccine virus strains. The results show that PRRSV TJM-F92 interference immunity strains of CSFV C strain, Bartha-K61 strain PRV without immunosuppressive effect of live vaccine, preparation of PRRSV and CSFV two combined live vaccine for PRRSV, and PRV two or PRRSV TJM-F92 vaccine strain based multiple live vaccine.

【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S855.3

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