2014-2015年中国部分地区蜜蜂慢性麻痹病病毒流行病学调查及系统发育分析

【目的】前期笔者课题组对北京地区蜂群进行了常见病毒感染情况的调查，发现蜜蜂慢性麻痹病毒（Chronic bee paralysis virus，CBPV）在病蜂群中的检出率显著高于在健康蜂群中的检出率，据此推测可借助病蜂群对CBPV进行研究。鉴于目前国内CBPV流行病学数据的匮乏，论文旨在了解中国主要养蜂区病蜂群中CBPV的分布及遗传变异情况，以期为该病的防控提供依据。【方法】2014-2015年间从四川、安徽、浙江、河南、山东、山西、黑龙江、北京8个主要养蜂省（市）共采集到136份病蜂样品，采用RT-PCR法检测CBPV的感染情况。每份样品随机选取10只蜜蜂，取其头部，利用Trizol法提取样品的总RNA；通过凝胶电泳及NanodropND-2000对总RNA的质量进行定性与定量检测。以样品总RNA为模板合成cDNA第一条链，而后以cDNA为模板扩增CBPV的特异性片段。所有扩增片段均进行琼脂糖凝胶电泳，对部分阳性样品进行测序与序列分析；通过扩增片段与阳性片段的比对确定每份样品中CBPV的检出情况。为研究CBPV中国分离株的系统发育特性，以样品cDNA为模板，扩增全部阳性样品的RdRP基因（RNA-dependent RNA polymerase）特异性片段，而后进行测序与序列分析。将序列提交至GenBank获取基因登录号。对获得的分离株的RdRP基因与GenBank上发表的相应序列进行比较分析，利用CLUSTAL W软件对全部参考序列及本研究获得的分离株的对应序列进行同源性比对，应用软件MEGA 6.0 中邻接法（neighbor-joining）对所有序列绘制系统进化树。【结果】蜜蜂头部总RNA质量检测结果显示，所有样品的总RNA相对完整，且浓度处于（980.5±37.1）ng·L^-1范围内，A₂₆₀/A₂₈₀ 在1.92-2.24。CBPV感染率检测结果显示，136份病蜂样本中共检出120份CBPV阳性样品，阳性率高达88.2%。全部阳性样品的电泳结果可见与预期大小一致的特异性片段，其序列与靶序列同源性达到99%以上。RdRp基因特异性片段的序列分析表明，本研究共获得8个CBPV中国分离株，其序列与多种其他地区的CBPV的相应序列同源性达到97%以上。中国分离株GenBank登录号分别为KT374042-KT374049。对其系统进化树分析后可知，世界流行的CBPV分离株可分为5个进化分支：两个欧洲分支（European clade 1、 European clade 2），两个中日混合分支（Chinese -Japanese clade 1、Chinese-Japanese clade 2）和一个美国分支（US clade）。获得的8个中国分离株均存在于同一进化分支上，且与之前得到的6个江苏分离株以及4个日本分离株共同形成两个混合分支。【结论】进一步证实了中国病蜂群中普遍存在CBPV感染；系统进化关系表明，目前国内CBPV分离株之间的遗传关系无明显地域性特征，与日本分离株亲缘关系较近。有关CBPV的监测工作亟需加强，且其预警作用需引起重视。

Epidemiological Survey and Molecular Phylogenetic Analysis of Chronic bee paralysis virus in China from 2014 to 2015

【Objective】 The presence of several common honey bee viruses in colonies located in Beijing were investigated in the past years. The Chronic bee paralysis virus (CBPV) was found that its occurrence in diseased colonies was significantly higher than in healthy ones. CBPV was supposed to be investigated in diseased colonies. Since the data on CBPV are little, the objective of this study is to investigate the occurrence and variation of CBPV from China so as to provide a theoretical basis for the prevention of CBPV disease. 【Method】 A total of 136 samples from diseased colonies in Sichuan, Anhui, Zhejiang, Henan, Shandong, Shanxi, Heilongjiang and Beijing in 2014-2015 were collected and detected for CBPV by RT-PCR. Ten heads of bees were randomly collected from each sample. Total RNA was extracted using the TRIZOL method. RNA quality was qualitatively and quantitatively determined by electrophoresis and NanodropND-2000. The first chain of cDNA was synthesized using total RNA as template. The specific fragments of CBPV were amplified using cDNA as template. All amplified bands were performed by agarose gel electrophoresis. Parts of the positive PCR products were sequenced and analyzed. The occurrence of CBPV from each sample was confirmed by comparing the amplified bands with the positive ones. To analyze the phylogeny of Chinese CBPV isolates, RdRP genes of all CBPV positive samples were amplified using cDNA as template, then sequenced and analyzed. The gene sequences were submitted to GenBank for accession numbers. RdRP genes obtained from this study were analyzed and compared with those available on GenBank. All reference sequences and the sequences from this study were aligned using the software of CLUSTAL W. The phylogenetic tree of CBPV was conducted using the neighbor-joining method of MEGA 6.0. 【Result】 RNA integrity from honey bee heads of all samples was relatively preserved. The RNA yield was (980.5±37.1)ng·mL^-1 and A₂₆₀/A₂₈₀ was 1.92-2.24. A total of 120 samples out of 136 were positive for CBPV and the positive rate was 88.2%. A target band was amplified from each positive sample. The nucleotide sequences of positive PCR products shared ＞99% sequence identity with target sequences. RdRP sequence analysis revealed that in total 8 CBPV isolates were obtained in this study, and the nucleotide sequences shared ＞97% sequence identity with those from other countries. Accession numbers for Chinese isolates of CBPV were KT374042-KT374049. There are 5 branches for the worldwide CBPV isolates including two European clades (European clade 1 and European clade 2), two Chinese-Japanese clades (Chinese-Japanese clade 1 and Chinese-Japanese clade 2), and one US clade. All 8 CBPV isolates in this study belongs to the same branch, and had been grouped with 4 Japanese isolates and 6 previous Jiangsu isolates into two mixed Chinese-Japanese clades. 【Conclusion】 CBPV is probably widely spreaded in diseased Apis mellifera colonies in China, and the genetic relationship of prevalent CBPV isolates in China exist no obvious geographical characteristics, seem to be closely related to Japan strains. Intensive CBPV surveillance should be urgently carried out, and put more attention to its function of early warning.