广东省阿卡斑病毒和OYAV的检测及分子特征研究

Detection and Molecular Characterization of the Akabane Virus and Oya Virus in Guangdong Province,China

阿卡斑病毒(Akabane virus,AKV)和Oya病毒(Oya virus,OYAV)均属于泛布尼亚病毒科、正布尼亚病毒属的两种病毒,可引起动物疾病,目前广东省尚不清楚是否存在这两种病毒。为了解这两种病毒在广东省的存在状况及分子特征,2018年4月至7月,分别在广东省的佛山、信宜、河源和阳江四个地市利用人诱法和诱蚊灯法采集吸血昆虫标本。采集的标本分类后,按20~50只/批,蠓虫按200~500只/批分装,液氮保存运输。标本破碎离心后,上清用于接种细胞和病毒核酸提取。Real-time RT-PCR检测AKV和OYAV阳性的样本用于S基因的扩增、测序和系统进化分析。本次研究共采集各类吸血昆虫421批次,Real-time RT-PCR检测AKV阳性10份,均来自蠓虫标本,RT-PCR检测AKV阳性5份,其中4份成功测序;Real-time RT-PCR检测OYAV阳性15份,其中蠓虫13份、中华按蚊和三带喙库蚊各1份,RT-PCR检测OYAV阳性6份,其中4份成功测序。病毒分离结果均为阴性。进化分析结果显示,所有序列共分为5个血清群,本次研究的8条序列均位于Simbu群,其中4个新毒株与OYAV代表株NIV86209的核苷酸同源性最高可达96.87%,氨基酸同源性最高为100%;另外4株新病毒与AKV代表株GXLCH02的核苷酸同源性最高达98.60%,氨基酸同源性最高为99.57%。本研究结果揭示,AKV和OYAV在广东省的虫媒中分布广泛,主要以蠓虫分布为主,来自蠓虫中的新OYAV和AKV与其它媒介或宿主中的AKV和OYAV高度同源,未显示明显媒介差异的分子特征。

The Akabane virus(AKV) and Oya virus(OYAV) belong to the Peribunyaviridae family and Orthobunyavirus genus,respectively. They can cause diseases in humans and animals. The distribution of AKV and OYAV in Guangdong Province,China,is unknown. To ascertain the molecular characteristics and distribution of AKV and OYAV in Guangdong Province,China,insect samples were collected using lamp-traps and bait methods in the cities of Foshan,Xinyi,Heyuan and Yangjiang,China from April to July 2018. Using20–50 mosquitoes or 200–500 midges as pools,collected samples were transferred in liquid nitrogen. Then,all samples were broken and centrifuged. The supernatant was inoculated on C6/36 cell lines and used as nucleotide extractions. AKV and OYAV were detected using real-time reverse transcription-polymerase chain reaction(RT-PCR) with commercial kits. Positive samples were used for amplification,sequencing and phylogenetic analyses of the S gene. A total of 421 pools of insect samples were collected. Ten pools of midge samples were positive for AKV according to real-time RT-PCR,five of them were positive for RT-PCR amplification,and four PCR products were sequenced. Fifteen pools were positive for OYAV according to realtime RT-PCR(thirteen pools of midges, one pool of Anopheles sinensis and one pool of Culex tritaeniorhynchus),six of thirteen pools were positive for RT-PCR amplification,and four of six pools were sequenced. All viral isolation was negative. Phylogenetic analyses showed that all sequences could be divided into five serogroups. Eight sequences were located in the Simbu group,and four new sequences shared 96.87%and 100% identification with the representative strain(NIV86209)at nucleotide and amino-acid levels. The other four new sequences shared 98.60% and 99.57% identification with the representative strain(GXLCH02)at nucleotide and amino-acid levels. In conclusion,AKV and OYAV have wide distribution in Guangdong Province, China, and midges are dominant vectors. New OYAV and AKV from midges share high identification with other vectors or hosts. Dramatic differences were not observed for molecular characteristics from different types of vectors.