Evaluation of a Porcine Gastric Mucin and RNase A Assay for the Discrimination of Infectious and Non-infectious GI.1 and GII.4 Norovirus Following Thermal, Ethanol, or Levulinic Acid Plus Sodium Dodecyl Sulfate Treatments
Evaluation of a Porcine Gastric Mucin and RNase A Assay for the Discrimination of Infectious and Non-infectious GI.1 and GII.4 Norovirus Following Thermal, Ethanol, or Levulinic Acid Plus Sodium Dodecyl Sulfate Treatments
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DOI：
10.1007/s12560-015-9219-z
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发表时间：
2016-03-01
影响因子：
3.4
通讯作者：
Cannon, Jennifer L.
中科院分区：
文献类型：
--
作者：
Afolayan, Olamide T.;Webb, Cathy C.;Cannon, Jennifer L.
人类诺如病毒（NoV）是全球食源性疾病的主要来源。由于人NoV不能在体外培养，因此需要区分感染性与非感染性NoV的方法。本研究的目的是评估NoV基因型GI.1和GII.4与猪胃粘蛋白（PGM）中表达的组织血型抗原的结合，作为在热处理后检测感染性病毒的替代物。（99 ℃/5分钟），70%乙醇或0.5%乙酰丙酸（LV）加0.01或0.1%十二烷基硫酸钠（SDS）消毒剂处理，并确定与PGM结合的GI.1和GII.4的检测限。将处理的和对照的病毒样品加到用1 μ g/ml PGM包被的96孔板上，然后用RNA酶A（5 ng/μ l）处理以降解暴露的RNA。对于GI. 1和GII. 4，热处理后每ml的平均对数基因组拷贝数（gc/ml）减少和定量循环（Cq）值的相对差异（RD）分别为1.77/5.62和1.71/7.25（RNA酶A）以及1.73/5.50和1.56/6.58（无RNA酶A）。用70% EtOH处理NoV分别导致gc/ml和Cq值的平均RD的0.05/0.16（GI. 1）和3.54/10.19（GII. 4）log降低。LV（0.5%）与0.1% SDS联合给药使GI.1和GII.4 NoV的降幅更大，获得的Cq值的平均RD分别为8.97和8.13，高于0.5% LV/0.01% SDS。PGM结合后的病毒回收率随着GII.4 > GI.1而变化。PGM结合是在衣壳破坏后鉴定感染性和非感染性NoV的有希望的替代物，然而，结果取决于病毒株和灭活方法。
Human noroviruses (NoVs) are a major source of foodborne illnesses worldwide. Since human NoVs cannot be cultured in vitro, methods that discriminate infectious from non-infectious NoVs are needed. The purpose of this study was to evaluate binding of NoV genotypes GI.1 and GII.4 to histo-blood group antigens expressed in porcine gastric mucin (PGM) as a surrogate for detecting infectious virus following thermal (99 degrees C/5 min), 70 % ethanol or 0.5 % levulinic acid (LV) plus 0.01 or 0.1 % sodium dodecyl sulfate (SDS) sanitizer treatments and to determine the limit of detection of GI.1 and GII.4 binding to PGM. Treated and control virus samples were applied to 96-well plates coated with 1 mu g/ml PGM followed by RNase A (5 ng/mu l) treatment for degradation of exposed RNA. Average log genome copies per ml (gc/ml) reductions and relative differences (RD) in quantification cycle (Cq) values after thermal treatment were 1.77/5.62 and 1.71/7.25 (RNase A) and 1.73/5.50 and 1.56/6.58 (no RNase A) for GI.1 and GII.4, respectively. Treatment of NoVs with 70 % EtOH resulted in 0.05/0.16 (GI.1) and 3.54/10.19 (GII.4) log reductions in gc/ml and average RD in Cq value, respectively. LV (0.5 %) combined with 0.1 % SDS provided a greater decrease of GI.1 and GII.4 NoVs with 8.97 and 8.13 average RD in Cq values obtained, respectively than 0.5 % LV/0.01 % SDS. Virus recovery after PGM binding was variable with GII.4 > GI.1. PGM binding is a promising surrogate for identifying infectious and non-infectious NoVs after capsid destruction, however, results vary depending on virus strain and inactivation method.