猪圆环病毒2型SH毒株增殖条件的优化与免疫原性稳定性研究
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摘要
猪圆环病毒(porcine circovirus,PCV)有2种基因型,即PCV1和PCV2,其中PCV1不引起感染猪发病,而PCV2是引起断奶仔猪多系统衰弱综合症(Postweaning multisystemic wasting syndrome,PMWS)的主要病原,它还与猪繁殖障碍(reproductive failure)、猪皮炎与肾病综合症(porcine dermatitis andnephropathy syndrome,PDNS)和呼吸道疾病综合症(porcine respiratory diseasesyndrome,PRDs)有关,这些疾病被通称为PCV2相关性疾病(PCVD)。目前,PCVD给世界养猪业带来了巨大的损失,但我国尚无商品化PCV2疫苗。
PK-15细胞通常被用来体外增殖PCV2,但PCV2在其上增殖滴度较低且不稳定,用300mM的D-氨基葡萄糖处理病毒可以增加PCV2复制,同时会使细胞发生退行性变化。为了获得高滴度病毒,本研究采用逐步降低细胞维持液中D-氨基葡萄糖的浓度,以筛选刺激病毒增殖的最佳浓度,并且通过优化IPMA试验条件,用于测定PCV2在PK-15细胞上的增殖情况。结果为,在维持液中加入1-2mMD-氨基葡萄糖既能有效的刺激病毒的复制,又不影响细胞的活性和传代,接种PCV2后37℃培养72h病毒滴度最高。将PCV2连续传代至第50代,收获F20和F50病毒液,其TCID50达到10~(6.25)/ml。
为了进一步研究该病毒的免疫原性是否稳定,我们将F20和F50的两个代次的病毒液用0.2%的甲醛37℃作用16h,然后取病毒液接种PK-15细胞,盲传3代,用PCR方法检测灭活效果,然后制成两个不同代次的油乳剂灭活疫苗。取23头30日龄PCV2阴性普通断奶仔猪,随机分为4组,第1,2,3组每组6头,第四组5头。第1组和第2组分别免疫两个不同代次的疫苗,第3组为非免疫攻毒组;第4组为空白对照组。免疫组每头猪肌肉注射2ml灭活疫苗,首免后第21天加强免疫1次。首免后每周采血,测定ELISA抗体。除空白对照组外,其余猪在第42天攻毒PCV2,攻毒后4、7和11、19天用钥匙孔血蓝蛋白(KLH)和巯基乙酸培养基进行免疫刺激,隔离饲养观察,30天后全部宰杀。攻毒后通过ELISA抗体、攻毒后临床表现及体温变化、剖解时的病理变化、相对日增重、病毒血症及排毒情况等指标评价疫苗效果。结果为,两个代次的疫苗均能产生较好的免疫应答,抗体水平相差不大。攻毒后,两个免疫组的猪均无明显临床症状,而非免疫攻毒组有明显的异常临床表现,增重缓慢,攻毒对照猪平均体温高于免疫猪,空白对照猪在整个攻毒期中体温始终保持正常。攻毒后免疫猪及空白对照猪平均体重均升高,而攻毒对照猪平均体重有所降低,且两免疫组和空白对照组之间相对日增重差异不显著。病毒血症检测表明,攻毒21天后未在免疫猪血清中检测到PCV2,而在4头攻毒对照猪血清中都检测到了PCV2。并且两个免疫组的猪的剖检病理变化和病理组织学变化都明显比非免疫攻毒组要轻。以上结果表明,PCV2-SH株具有稳定的免疫原性,可以提供有效免疫保护作用。
上述研究结果表明,采用D-氨基葡萄糖可以明显提高PCV2滴度,PCV2 SH毒株在PK15细胞上连续传代,病毒滴度稳定,并具有较好免疫原性,从而为PCV2灭活疫苗研制奠定了重要基础。
Porcine circovirus(PCV) is one kind of smallest viruses we have known by now, which has two gene types,PCV1 and PCV2.PCV1 can be detected in most of PK15 cells.Porcine circovirus 2(PCV2) is the main causative agent of postweaning multisystemic wasting syndrome(PMWS),porcine dermatitis nephropathy syndrome (PDNS),reproductive failure and porcine respiratory disease complex(PRDC) etc.We called these PCVD and PCVD caused huge loss in many nations and regions in the world.
Currently,cultivation of PCV2 virus in vitro has been carried out in PK15 in laboratories world-wide.However,the virus titers,expressed as TCID50,obtained from PK15 cell cultures were low.The 300 mM D-Glucosamine could be used to enhance the replication of PCV2 in PK15 cells,but it could harm to the cells.In order to acquire the virus with high titres,in this study,the concentration of D-Glucosamine in the cultures was optimized,and the titers of virus were detected with IPMA which reaction condition was selected.The results showed that maximum virus titers were acquired when PCV2-SH strain was cultured in PK15 cell with RMPI-1640 containing 1-2 mM D(+)-Glucosamine at 37℃for 72 h.Moreover,PCV2-SH strain was passaged serially until 50 passage in PK15 cells.The virus titres of F20 and F50 were all 10~(6.25) TCID_(50)/ml which detected by IPMA.
To study on the stability of immunogenicity of PCV2 SH strain,F20 and F50 passage viruses were inactivated with 0.2%formaldehyde under 37℃.The inactivation effect was estimated by PCR after passaged on PK-15 cell by three times. Then the inactivate vaccines were individually made with the F20 and F50 passage virus and the immunogenicities of the viruses were evaluated in pigs.Twenty-three healthy piglets were assigned to four groups(group 1,2,3 had six pigs and group 4 five),F20 vaccined,F50 vaccined,PCV2 challenged control and empty control group. Group 1 and Group 2 were vaccinated individually with 2 ml killed vaccine and boosted 3 weeks later.Twenty-one days later,Group 1,2 and 3 were all challenged with 3×10~(6.25)TCID_(50) of PCV2-SH.The pigs were weighed at the days of challenge and the end of the experiment.All groups were isolated and killed at 35 days after PCV2 challeng.PCV2-specific antibody were detected at every week after the first vaccination by indirect ELISA.The efficacy of inactivated vaccines were evaluated by seroconversion,clinical signs(fever),gross lesions,growth parameters and viremia.The results showed that the higher ELISA antibody titers of two vaccines could be detected in piglets after second immunization.After PCV2 challenged,the vaccinated and control groups had no clinical signs,challenged groups had increased rectal temperature and showed growth retardation..Viremia were detected in 4 piglets of challenged groups after PCV2 challenge,but no viremia in all piglets of vaccinated groups at 21 days.Gross leision and histopathological findings in all piglets of challenged groups were more severe than that of vaccinated groups.It demonstrated that PCV2 strain had stable immunogencity,and could provid protetive efficity.The PCV2 SH strain might be an candidate virus for PCV2 inactivated vaccine.The study laid the foundation of the reseach in killed vaccine against the disease associated with PCV2.
PK-15细胞通常被用来体外增殖PCV2,但PCV2在其上增殖滴度较低且不稳定,用300mM的D-氨基葡萄糖处理病毒可以增加PCV2复制,同时会使细胞发生退行性变化。为了获得高滴度病毒,本研究采用逐步降低细胞维持液中D-氨基葡萄糖的浓度,以筛选刺激病毒增殖的最佳浓度,并且通过优化IPMA试验条件,用于测定PCV2在PK-15细胞上的增殖情况。结果为,在维持液中加入1-2mMD-氨基葡萄糖既能有效的刺激病毒的复制,又不影响细胞的活性和传代,接种PCV2后37℃培养72h病毒滴度最高。将PCV2连续传代至第50代,收获F20和F50病毒液,其TCID50达到10~(6.25)/ml。
为了进一步研究该病毒的免疫原性是否稳定,我们将F20和F50的两个代次的病毒液用0.2%的甲醛37℃作用16h,然后取病毒液接种PK-15细胞,盲传3代,用PCR方法检测灭活效果,然后制成两个不同代次的油乳剂灭活疫苗。取23头30日龄PCV2阴性普通断奶仔猪,随机分为4组,第1,2,3组每组6头,第四组5头。第1组和第2组分别免疫两个不同代次的疫苗,第3组为非免疫攻毒组;第4组为空白对照组。免疫组每头猪肌肉注射2ml灭活疫苗,首免后第21天加强免疫1次。首免后每周采血,测定ELISA抗体。除空白对照组外,其余猪在第42天攻毒PCV2,攻毒后4、7和11、19天用钥匙孔血蓝蛋白(KLH)和巯基乙酸培养基进行免疫刺激,隔离饲养观察,30天后全部宰杀。攻毒后通过ELISA抗体、攻毒后临床表现及体温变化、剖解时的病理变化、相对日增重、病毒血症及排毒情况等指标评价疫苗效果。结果为,两个代次的疫苗均能产生较好的免疫应答,抗体水平相差不大。攻毒后,两个免疫组的猪均无明显临床症状,而非免疫攻毒组有明显的异常临床表现,增重缓慢,攻毒对照猪平均体温高于免疫猪,空白对照猪在整个攻毒期中体温始终保持正常。攻毒后免疫猪及空白对照猪平均体重均升高,而攻毒对照猪平均体重有所降低,且两免疫组和空白对照组之间相对日增重差异不显著。病毒血症检测表明,攻毒21天后未在免疫猪血清中检测到PCV2,而在4头攻毒对照猪血清中都检测到了PCV2。并且两个免疫组的猪的剖检病理变化和病理组织学变化都明显比非免疫攻毒组要轻。以上结果表明,PCV2-SH株具有稳定的免疫原性,可以提供有效免疫保护作用。
上述研究结果表明,采用D-氨基葡萄糖可以明显提高PCV2滴度,PCV2 SH毒株在PK15细胞上连续传代,病毒滴度稳定,并具有较好免疫原性,从而为PCV2灭活疫苗研制奠定了重要基础。
Porcine circovirus(PCV) is one kind of smallest viruses we have known by now, which has two gene types,PCV1 and PCV2.PCV1 can be detected in most of PK15 cells.Porcine circovirus 2(PCV2) is the main causative agent of postweaning multisystemic wasting syndrome(PMWS),porcine dermatitis nephropathy syndrome (PDNS),reproductive failure and porcine respiratory disease complex(PRDC) etc.We called these PCVD and PCVD caused huge loss in many nations and regions in the world.
Currently,cultivation of PCV2 virus in vitro has been carried out in PK15 in laboratories world-wide.However,the virus titers,expressed as TCID50,obtained from PK15 cell cultures were low.The 300 mM D-Glucosamine could be used to enhance the replication of PCV2 in PK15 cells,but it could harm to the cells.In order to acquire the virus with high titres,in this study,the concentration of D-Glucosamine in the cultures was optimized,and the titers of virus were detected with IPMA which reaction condition was selected.The results showed that maximum virus titers were acquired when PCV2-SH strain was cultured in PK15 cell with RMPI-1640 containing 1-2 mM D(+)-Glucosamine at 37℃for 72 h.Moreover,PCV2-SH strain was passaged serially until 50 passage in PK15 cells.The virus titres of F20 and F50 were all 10~(6.25) TCID_(50)/ml which detected by IPMA.
To study on the stability of immunogenicity of PCV2 SH strain,F20 and F50 passage viruses were inactivated with 0.2%formaldehyde under 37℃.The inactivation effect was estimated by PCR after passaged on PK-15 cell by three times. Then the inactivate vaccines were individually made with the F20 and F50 passage virus and the immunogenicities of the viruses were evaluated in pigs.Twenty-three healthy piglets were assigned to four groups(group 1,2,3 had six pigs and group 4 five),F20 vaccined,F50 vaccined,PCV2 challenged control and empty control group. Group 1 and Group 2 were vaccinated individually with 2 ml killed vaccine and boosted 3 weeks later.Twenty-one days later,Group 1,2 and 3 were all challenged with 3×10~(6.25)TCID_(50) of PCV2-SH.The pigs were weighed at the days of challenge and the end of the experiment.All groups were isolated and killed at 35 days after PCV2 challeng.PCV2-specific antibody were detected at every week after the first vaccination by indirect ELISA.The efficacy of inactivated vaccines were evaluated by seroconversion,clinical signs(fever),gross lesions,growth parameters and viremia.The results showed that the higher ELISA antibody titers of two vaccines could be detected in piglets after second immunization.After PCV2 challenged,the vaccinated and control groups had no clinical signs,challenged groups had increased rectal temperature and showed growth retardation..Viremia were detected in 4 piglets of challenged groups after PCV2 challenge,but no viremia in all piglets of vaccinated groups at 21 days.Gross leision and histopathological findings in all piglets of challenged groups were more severe than that of vaccinated groups.It demonstrated that PCV2 strain had stable immunogencity,and could provid protetive efficity.The PCV2 SH strain might be an candidate virus for PCV2 inactivated vaccine.The study laid the foundation of the reseach in killed vaccine against the disease associated with PCV2.
引文
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