甲型肝炎病毒在太平洋牡蛎中的富集及消减规律

Bioaccumulation and depuration of hepatitis A virus in Pacific oyster

目的探讨总甲型肝炎病毒(hepatitis A virus,HAV)和活HAV在太平洋牡蛎消化腺中富集和净化消减动态规律,以及在牡蛎不同组织中的分布。方法通过人工暂养太平洋牡蛎和人为HAV污染养殖水体,并自染毒24 h后净化养殖的方式,建立牡蛎体内HAV富集和净化衰减模型。采用免疫沉淀提取结合一步法实时荧光定量RT-PCR(quantitative real-time reverse transcriptase polymerase chain reaction,qRT-PCR)法,分别检测0~264 h不同时间点的养殖水体、牡蛎消化腺以及牡蛎其他组织的总HAV含量;采用qRT-PCR和叠氮溴化丙锭-定量RT-PCR(propidium monoazide-quantitative reverse transcriptase-polymerase chain reaction,PMA-qRT-PCR)同时检测0、3、6、9、12、24、48、72、96、120、144、168、192、216、240和264 h牡蛎消化腺总HAV和活HAV含量,并对两者进行相关性分析;对0、3和24 h的染毒牡蛎进行免疫组化检测,评价各组织的组化动态改变。结果在24 h内,养殖水体的总HAV含量从0 h的5.544 Lgcopies/g下降至4.428 Lgcopies/g;但净化养殖后1 d(48 h),水体仍检出2.757 Lgcopies/g,并可持续检出至216 h。而牡蛎消化腺的总HAV在染毒3 h后即迅速升高至5.810 Lgcopies/g,12 h达最高点(6.152 Lgcopies/g),为养殖水体的29.7倍。净化养殖后,牡蛎消化腺中总HAV仍持续高水平,至72 h后才缓慢下降,264 h仍为4.398 Lgcopies/g。与总HAV的变化规律一致,活HAV在3 h即达3.432 LgCCID_(50)/g,至12 h达最高点(3.929 LgCCID_(50)/g),72 h后缓慢下降,264 h为2.678 LgCCID_(50)/g。全部检测时间点总HAV和活HAV含量的相关系数(R2)达0.8116,相关性良好,10 CCID50/g活HAV约相当于81 copies/g的总HAV。染毒后24 h,消化腺、鳃、唇瓣、外套膜和闭壳肌中均可检出不同含量的总HAV,其中,消化腺最高(P<0.05),唇瓣最低;免疫组化显示,染毒后3 h唇瓣中即可见强阳性HAV颗粒,提示在污染早期,唇瓣可能较消化腺更加敏感。结论太平洋牡蛎受污染后3 h即可在消化腺中检出高水平的HAV,12 h达最高点。净化养殖不能有效降低牡蛎体内的HAV水平,且在消化腺内11 d仍维持良好的病毒活性,并可持续排毒,对环境造成二次污染。我国应进一步加强牡蛎等可生食贝类与HAV的相关基础、监测和管理策略研究,为加强我国后甲肝疫苗时代的HAV预防和控制做好源头控制,更好地保障我国的食品安全。

Objective To investigate the dynamics of accumulation and depuration of hepatitis A virus(HAV)in Pacific oysters as well as the distribution of HAV in different tissues of oyster.Methods Accumulation and depuration models of HAV in oyster were established by artificially feeding oyster in live HAV-contaminated water which was replaced with clean water 24 h after infection.The total HAV contents in cultured seawater,oyster digestive glands and other tissues at different time points ranging from 0 to 264 h were determined by immunoprecipitation combined with one-step quantitative real-time reverse transcriptase polymerase chain reaction(qRT-PCR),while the total HAV and live HAV contents in oyster digestive glands 0,3,6,9,12,24,48,72,96,120,144,168,192,216,240 and 264 h after infection by qRT-PCR and propidium monoazide(PMA)-qRT-PCR,and the correlation of results was analyzed.The tissues of oysters0,3 and 24 h after infection were subjected to immunohistochemical assay to evaluate the dynamic change.Results The total HAV content in the cultured seawater slowly decreased from the 5.544 Lgcopies/g at 0 h to 4.428 Lgcopies/g at24 h,which was 2.757 Lgcopies/g at 48 h(one day after replacement with clean water)and was continuously positive up to 216 h.However,the total HAV contents in oyster digestive glands increased rapidly to 5.810 Lgcopies/g 3 h and reached a peak value of 6.152 Lgcopies/g 12 h after infection,which was 29.7 times of that in contaminated seawater.After replacement with clean water,the total HAV contents in oyster digestive glands was still relatively high until 72 h,which decreased slowly and was still 4.398 Lgcopies/g 264 h after infection.The live HAV content increased to 3.432 LgCCID_(50)/g at 3 h and reached a peak value of 3.929 LgCCID_(50)/g at 12 h,and then slowly decreased at 72 h and was 2.678 LgCCID_(50)/g at 264 h,which was consistent with the total RNA content.The correlation coefficient(R2)between the total and live HAV contents was 0.8116,indicating a good correlation.A portion of 10 CCID50/g live HAV was approximately equivalent to 81 copies/g total HAV.Total HAV at different concentrations were detected in digestive gland,branchia,labellum,mantle and adductor muscle at 24 h after infection,which was the highest in digestive glands(P<0.05)and the lowest in labellum.Immunohistochemical assay showed strong positive HAV particles in labellum 3 h after infection,suggesting that labellum might be more sensitive than digestive glands at the early stage of contamination.Conclusion High levels of HAV may be detected in the gland of Pacific oysters 3 h after contamination,which reached the highest point at 12 h.Depuration cannot effectively decrease the HAV level in oysters,and the activity of HAV may be maintained in glands for 11 d and can sustainably detoxify virus,resulting in secondary pollution.Therefore,relative researches should be strengthened to improve the prevention and control of HAV from source in post-vaccine era,including further studies on monitoring and managing HAV-related edible shellfish.