Department of Agriculture and Natural Resources, Delaware State University, Dover, Delaware 19901, USA.
Appl Environ Microbiol. 2011 Dec;77(23):8360-9. doi: 10.1128/AEM.06887-11. Epub 2011 Sep 23.
The goal of this study was to determine how enteric viruses persist within shellfish tissues. Several lines of novel evidence show that phagocytic blood cells (hemocytes) of Eastern oysters (Crassostrea virginica) play an important role in the retention of virus particles. Our results demonstrated an association of virus contamination with hemocytes but not with hemolymph. Live oysters contaminated overnight with hepatitis A virus (HAV) and murine norovirus (MNV) had 56% and 80% of extractable virus associated with hemocytes, respectively. Transfer of HAV-contaminated hemocytes to naïve (virus-free) oysters resulted in naïve oyster meat testing HAV positive for up to 3 weeks. Acid tolerance of HAV, MNV, poliovirus (PV), and feline calicivirus (FCV) correlated with the ability of each virus to persist within oysters. Using reverse transcription-PCR (RT-PCR) to evaluate persistence of these viruses in oysters, we showed that HAV persisted the longest (>21 days) and was most acid resistant, MNV and PV were less tolerant of acidic pH, persisting for up to 12 days and 1 day, respectively, and FCV did not persist (<1 day) within oysters and was not acid tolerant. This suggests that the ability of a virus to tolerate the acidic conditions typical of phagolysosomal vesicles within hemocytes plays a role in determining virus persistence in shellfish. Evaluating oyster and hemocyte homogenates and live contaminated oysters as a prelude to developing improved viral RNA extraction methods, we found that viruses were extracted more expediently from hemocytes than from whole shellfish tissues and gave similar RT-PCR detection sensitivities.
本研究旨在确定肠道病毒如何在贝类组织中持续存在。有几条新的证据表明，东方牡蛎（Crassostrea virginica）的吞噬血细胞（血球）在保留病毒颗粒方面发挥着重要作用。我们的结果表明，病毒污染与血球有关，但与血淋巴无关。经 overnight 污染甲型肝炎病毒（HAV）和鼠诺如病毒（MNV）的活牡蛎中，分别有 56%和 80%的可提取病毒与血球相关。将 HAV 污染的血球转移到无感染（无病毒）的牡蛎中，导致无感染的牡蛎肉在长达 3 周的时间内检测出 HAV 呈阳性。HAV、MNV、脊髓灰质炎病毒（PV）和猫杯状病毒（FCV）的耐酸性与每种病毒在牡蛎中持续存在的能力相关。我们使用逆转录-PCR（RT-PCR）评估这些病毒在牡蛎中的持续存在情况，表明 HAV 持续时间最长（>21 天），耐酸性最强，MNV 和 PV 对酸性 pH 的耐受性较低，分别持续长达 12 天和 1 天，而 FCV 则在牡蛎中无法持续存在（<1 天），也不耐酸性。这表明病毒耐受存在于血球内吞噬溶酶体囊泡内的酸性条件的能力在决定病毒在贝类中的持续存在中起作用。在开发改进的病毒 RNA 提取方法之前，我们评估了牡蛎和血球匀浆以及受污染的活牡蛎，发现病毒从血球中提取比从整个贝类组织中提取更迅速，并且具有相似的 RT-PCR 检测灵敏度。
