高致病性猪繁殖与呼吸综合征病毒TJM-F92疫苗株毒力返强及其对其他疫苗株的免疫干扰研究

发布时间：2018-03-17 00:37

本文选题：高致病性猪繁殖与呼吸综合征病毒　切入点：TJM-F92株　出处：《中国农业科学院》2015年博士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种以母猪繁殖障碍、仔猪和育肥猪呼吸道疾病为主要特征的传染病,给养猪业带来了巨大经济损失。免疫疫苗是控制PRRS的有效方法。一般情况下灭活疫苗具有良好的安全性,但不能提供有效保护。PRRSV活疫苗可刺激机体产生细胞免疫与体液免疫应答,但活疫苗存在毒力返强的风险,并且由于PRRSV具有免疫抑制作用,通过传统方法制备的PRRSV活疫苗一般很难改变其免疫抑制性,接种此类活疫苗会对机体造成免疫抑制,并且会干扰同期免疫的其他活疫苗的免疫应答。高致病性猪繁殖与呼吸综合征病毒TJM-F92疫苗株(HP-PRRSV TJM-F92)是一株在Nsp2基因存在不连续30个氨基酸(aa)缺失的同时还存在一段连续缺失120个aa的弱毒疫苗株。本研究通过将PRRSV TJM-F92株基础种子接种PRRS健康易感仔猪,连续在猪体内传代五代来验证该疫苗株是否存在毒力返强风险。并开展了PRRSV TJM-F92株对猪瘟兔化弱毒株(CSFV C株)及伪狂犬Bartha-K61疫苗株(PRV Bartha-K61株)的免疫干扰研究。将PRRSV TJM-F92株基础种子免疫接种PRRS抗原、抗体阴性健康易感猪,采集病毒血症载毒高峰期(接种病毒后第5日~第9日)的血清样品,继代接种易感猪进行5代毒力返强试验,并对返强第5代分离病毒进行Nsp2基因序列分析,检验PRRSV TJM-F92株的安全性及Nsp2基因存在的120个aa在易感动物体内的稳定性。试验结果表明,每代易感猪均未见临床异常。大体剖检结果显示,免疫组与对照组试验猪肺脏、脾脏、肾脏、肝脏、心脏、扁桃体和淋巴结眼观无明显变化。对肺脏进行病理组织学检测,结果显示,肺泡结构正常、清晰,支细管周围有少量淋巴细胞,免疫组与对照组无差异,未见由间质性肺炎引起的损伤。将PRRSV TJM F92经猪体内连续传代五代后,在TJM-F92株Nsp2区域存在的120个氨基酸的缺失仍然存在,说明该缺失可在猪体内稳定遗传。试验结果表明PRRSV TJM-F92疫苗株无毒力返强风险。本研究通过开展高剂量PRRSV TJM-F92株对低剂量CSFV C株或低剂量PRV Bartha-K61株同时免疫与间隔免疫干扰研究,高致病性猪繁殖与呼吸综合征、伪狂犬二联活疫苗对猪的免疫学功能影响试验,分别从体液免疫、细胞免疫、免疫攻毒及病理学变化等方面比较了毒种联合免疫组、毒种间隔免疫组与各毒种单免组在免疫及攻毒后的各项免疫学指标变化情况及攻毒保护情况。结果,高剂量PRRSV TJM-F92株对低剂量CSFV C株或低剂量PRV Bartha-K61株同时免疫、间隔7日免疫或间隔14日免疫,PRRSV TJM F92株对CSFV C株、PRV Bartha-K61株抗体水平无免疫干扰作用,与CSFV C株、PRV Bartha-K61株单免组抗体水平无显著差异。攻击CSFV强毒或PRV强毒后,各毒种联合免疫组、毒种间隔免疫组试验动物在临床症状、体温、攻毒保护率、病理学变化等方面与各毒种单免组试验动物无显著差异,各免疫组仔猪全部建活并得到有效保护。对4~6周龄PRRS与PR健康易感仔猪免疫高致病性猪繁殖与呼吸综合征、伪狂犬二联活疫苗,通过流式细胞仪对试验动物外周淋巴血液中免疫细胞进行检测,同时通过检测抗体效价,统计攻毒保护率,进行病理组织学检测,并与PRRSV单苗免疫组与PRV单苗免疫组进行比较,结果表明,PRRSV+PRV二联苗、PRV单苗组、PRRSV单苗组免疫仔猪后抗体产生情况良好,可刺激机体产生细胞免疫应答,未引起T细胞比例降低,攻毒保护率均为5/5。上述试验结果表明,PRRSV TJM-F92株疫苗对CSFV C株或PRV Bartha-K61株等活疫苗毒株无免疫抑制作用。综上所述,PRRSV TJM-F92株不存在毒力返强风险,进一步证明其为一株非常安全的疫苗毒株。免疫干扰试验结果证实PRRSV TJM-F92株对CSFV C株、PRV Bartha-K61株等活疫苗毒株无免疫抑制作用,为制备PRRSV与CSFV二联活疫苗、PRRSV与PRV二联活疫苗或以PRRSV TJM-F92株为基础的多联活疫苗奠定了基础。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S855.3

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