一株新正黏病毒(龙川病毒)的鉴定及分子特征研究
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摘要
2013年在中国广东省开展了虫媒病毒分布及其分子特征的研究,在广东省龙川县采集的致倦库蚊中分离到一株病毒,该病毒可以引起C6/36细胞的病理性效应。为了对该病毒进行鉴定,并对其分子特征进行研究,将病毒培养物用氯化铯进行梯度离心,负染电镜观察病毒的形态,提取并单引物等温扩增(SPIA)病毒RNA,利用Ion Torrent平台进行深度测序,获得的序列用CLC Genomic Wokbench 9.0软件进行拼接,本地BLAST进行分类比对。用MEGA软件登录GenBank,下载正黏病毒科代表株序列并绘制进化树,同时用ClustalW2软件与新毒株进行氨基酸同源性比对和编码框序列的分析。电镜观察结果显示该病毒呈球形,直径大小约100nm;深度测序共获得6个完整开放阅读框(Open reading fragment,ORF)的节段序列,5个节段(PB1、PB2、PA、NA、HA)的氨基酸序列与正黏病毒科的Quaranjavirus属病毒同源关系在26.38%～67.23%之间,其中PB1的同源性最高,PB2的同源性最低,未发现与其它病毒有同源关系。本研究在中国首次从蚊虫中分离并鉴定了一株新的虫媒病毒,属于正黏病毒科Quaranjavirus属的家族成员,命名为龙川病毒,这是中国国内首次报道从致倦库蚊中分离到龙川病毒。
The distribution and molecular characterization of arbovirus were performed in Guangdong Province,China,in 2013. A strain of virus was isolated from Culex pipiens fatigans in Longchuan County. The virus resulted in a cytopathogenic effect in C6/36 lines. To enable the identification and molecular characterization of the virus,viral particles were purified using density gradient centrifugation employing cesium chloride. The viral shape was observed using negative-staining methods under electron cryomicroscopy. RNA was amplified by single-primer isothermal amplification and deep sequencing was carried out on the Ion Torrent platform. The generated sequences were subjected to quality control and classified by BLAST. CLC Genomic Workbench was used to assemble the sequences. Representative sequences from the Orthomyxoviridae family were downloaded from GenBank. Phylogenetic trees were constructed using MEGA. Amino-acid and nucleotide sequences were compared among new and representative strains using ClustalW2. Transmission electron microscopy showed that these novel viral particles were spheres and were 100 nm in diameter. Six segments with complete openreading frames were obtained by deep sequencing. Amino acid identification of 26.38%～67.23% among five segments(PB1,PB2,PA,NA and HA)was obtained with representatives from the Quaranjavirus genus of the Orthomyxoviridae family. PB1 and PB2 had the highest and lowest identification,respectively. A closer relation was not found with the other viruses. In summary, we identified a novel arbovirus within the Orthomyxoviridae family from Longchuan County. The virus was named the"Longchuan virus". This is the first report of isolation of the Longchuan virus from Culex pipiens fatigans in China.
The distribution and molecular characterization of arbovirus were performed in Guangdong Province,China,in 2013. A strain of virus was isolated from Culex pipiens fatigans in Longchuan County. The virus resulted in a cytopathogenic effect in C6/36 lines. To enable the identification and molecular characterization of the virus,viral particles were purified using density gradient centrifugation employing cesium chloride. The viral shape was observed using negative-staining methods under electron cryomicroscopy. RNA was amplified by single-primer isothermal amplification and deep sequencing was carried out on the Ion Torrent platform. The generated sequences were subjected to quality control and classified by BLAST. CLC Genomic Workbench was used to assemble the sequences. Representative sequences from the Orthomyxoviridae family were downloaded from GenBank. Phylogenetic trees were constructed using MEGA. Amino-acid and nucleotide sequences were compared among new and representative strains using ClustalW2. Transmission electron microscopy showed that these novel viral particles were spheres and were 100 nm in diameter. Six segments with complete openreading frames were obtained by deep sequencing. Amino acid identification of 26.38%～67.23% among five segments(PB1,PB2,PA,NA and HA)was obtained with representatives from the Quaranjavirus genus of the Orthomyxoviridae family. PB1 and PB2 had the highest and lowest identification,respectively. A closer relation was not found with the other viruses. In summary, we identified a novel arbovirus within the Orthomyxoviridae family from Longchuan County. The virus was named the"Longchuan virus". This is the first report of isolation of the Longchuan virus from Culex pipiens fatigans in China.
引文
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[13]Allison A B,Ballard J R,Tesh R B,Brown J D,Ruder M G,Keel M K,Munk B A,Mickley R M,Gibbs S E,Travassos da Rosa A P,Ellis J C,Ip H S,Shearn-Bochsler V I,Rogers M B,Ghedin E,Holmes E C,Parrish C R,Dwyer C.Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus[J].J Virol,2015,89(2):1389-1403.
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[3]前田宁子,王静舒,胡宜.流感病毒的分型及基因组结构[J].日本医学介绍,2004,25(9):387-389.
[4]Hause B M,Ducatez M,Collin E A,Ran Z,Liu R,Sheng Z,Armien A,Kaplan B,Chakravarty S,Hoppe A D,Webby R J,Simonson R R,Li F.Isolation of a novel swine influenza virus from Oklahoma in 2011which is distantly related to human influenza C viruses[J/OL].PLoS Pathog,2013,9:e1003176.
[5]曾晓旭,王大燕,舒跃龙.正黏病毒科索戈托病毒研究进展[J].病毒学报,2013,29(5):555-558.DOI:10.13242/j.cnki.bingduxuebao.002445
[6]Presti R M,Zhao G,Beatty W L,Mihindukulasuriya KA,da Rosa A P,Popov V L,Tesh R B,Virgin H W,Wang D.Quaranfil,Johnston Atoll,and Lake Chad viruses are novel members of the family orthomyxoviridae[J].J Virol,2009,83(22):11599-11606.
[7]Fourrier,M;Heuser,S;Munro,E;Snow,M.Characterization and comparison of the full 3′and 5′untranslated genomic regions of diverse isolates ofinfectious salmon anaemia virus by using a rapid and universal method[J].J Virol Methods,2011,174(1-2):136-143.
[8]Clouthier S C,Rector T,Brown N E,Anderson E D.Genomic organization of infectious salmon anaemia virus[J].J Gen Virol,2002,83(Pt 2):421-428.
[9]Peng R,Zhang S,Cui Y,Shi Y,Gao G F,Qi J.Structures of human-infecting Thogotovirus fusogens support a common ancestor with insect baculovirus[J/OL].Proc Natl Acad Sci U S A,2017,114(42):E8905-E8912.
[10]Shi M,Neville P,Nicholson J,Eden J S,Imrie A,Holmes E C.High resolution meta-transcriptomics reveals the ecological dynamics of mosquito-associated RNA viruses in Western Australia[J/OL].J Virol,2017,91(17):e00680-17.
[11]Shi M,Lin X D,Tian J H,Chen LJ,Chen X,Li C X,Qin X C,Li J,Cao J P,Eden J S,Buchmann J,Wang W,Xu J,Holmes E C,Zhang Y Z.Redefining the invertebrate RNA virosphere[J].Nature,2016,540(7634):539-543.
[12]Lvov D K,Alkhovsky S V,Shchelkanov M Y,Shchetinin A M,Deryabin P G,Aristova V A,Gitelman A K,Samokhvalov E I,Botikov A G.Taxonomic status of the Tyulek virus(TLKV)(Orthomyxoviridae,Quaranjavirus,Quaranfil group)isolated from the ticks Argas vulgaris Filippova,1961(Argasidae)from the birds burrow nest biotopes in the Kyrgyzstan[J].Vopr Virusol,2014,59(2):28-32.
[13]Allison A B,Ballard J R,Tesh R B,Brown J D,Ruder M G,Keel M K,Munk B A,Mickley R M,Gibbs S E,Travassos da Rosa A P,Ellis J C,Ip H S,Shearn-Bochsler V I,Rogers M B,Ghedin E,Holmes E C,Parrish C R,Dwyer C.Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus[J].J Virol,2015,89(2):1389-1403.