广东省三株蚊媒黄病毒的鉴定和系统进化分析
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摘要
为了解广东省蚊媒病毒的种类和分子特征,本研究对从蚊子中分离的三株黄病毒提取病毒核酸并进行二代测序,经序列拼接和比对,基因组序列中缺失的gap通过RT-PCR进行扩增填补。用MEGA软件登录GenBank下载黄病毒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。结果显示利用二代测序技术获得了三株病毒基因组序列的96.35%～98.26%,并用RT-PCR成功补齐所有gap,序列比对显示其中一株与黄斑库蚊病毒核苷酸和氨基酸同源性98.51%,两株病毒与广平病毒的核苷酸同源性分别为97.30%和89.78%。三株病毒都属于黄病毒家族中的未分类黄病毒或昆虫特异性黄病毒,编码区序列中C蛋白的同源性最低,NS5的同源性最高。本研究发现的三株蚊媒黄病毒中,库蚊黄斑病毒在国内为首次鉴定;两株广平病毒核苷酸同源性存在较大差异,可能存在不同基因亚型。
We wished to ascertain the species and molecular characteristics of arboviruses in Guangdong Province,China. RNA from three strains of flaviviruses were extracted by next-generation sequencing(NGS). All reads were assembled using de novo sequencing and local BLAST. Gaps were filled by reverse transcription-polymerase chain reaction(RT-PCR)amplification. Representative sequences from the Flavivirus genus were downloaded from GenBank and phylogenetic trees were constructed using MEGA. Sequences of amino acids and nucleotides were compared among new strains and representatives using ClustalW2. Results showed that three viruses from Culex pipiens fatigans and Culex tritaeniorhynchus obtained 96.35%～98.26% genomic sequences using NGS,and that these gaps were filled using RT-PCR. One of them shared 98.51% identification with the Culex theileri flavivirus at the nucleotide level. The other two virus strains shared 97.30% and 89.78%identification with the Quang Binh virus at the nucleotide level,respectively. They belonged to non-classified flaviviruses. The C protein shared the lowest identification among their encoding sequences,but NS5 shared the highest identification. We concluded that these three new virus strains were identified using NGS,and that the Culex theileri flavivirus is the first to be reported in China;two strains of Quang Binh viruses might belong to two subtypes because of the big difference between their nucleotide sequences.
We wished to ascertain the species and molecular characteristics of arboviruses in Guangdong Province,China. RNA from three strains of flaviviruses were extracted by next-generation sequencing(NGS). All reads were assembled using de novo sequencing and local BLAST. Gaps were filled by reverse transcription-polymerase chain reaction(RT-PCR)amplification. Representative sequences from the Flavivirus genus were downloaded from GenBank and phylogenetic trees were constructed using MEGA. Sequences of amino acids and nucleotides were compared among new strains and representatives using ClustalW2. Results showed that three viruses from Culex pipiens fatigans and Culex tritaeniorhynchus obtained 96.35%～98.26% genomic sequences using NGS,and that these gaps were filled using RT-PCR. One of them shared 98.51% identification with the Culex theileri flavivirus at the nucleotide level. The other two virus strains shared 97.30% and 89.78%identification with the Quang Binh virus at the nucleotide level,respectively. They belonged to non-classified flaviviruses. The C protein shared the lowest identification among their encoding sequences,but NS5 shared the highest identification. We concluded that these three new virus strains were identified using NGS,and that the Culex theileri flavivirus is the first to be reported in China;two strains of Quang Binh viruses might belong to two subtypes because of the big difference between their nucleotide sequences.
引文
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[12]梁文凯,荆春霞,王环宇.库蚊黄病毒的研究进展[J].中华实验和临床病毒学杂志,2015,29(2):188-190.
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[14]Parreira R,Cook S,Lopes?,de Matos A P,de Al-meida A P,Piedade J,Esteves A. Genetic characteriza-tion of an insect-specific flavivirus isolated from Culex theileri mosquitoes collected in southern Portugal[J].Virus Res,2012,167(2):152-161.
[15]Cook S, Moureau G, Harbach R E, Mukwaya L,Goodger K,Ssenfuka F,Gould E,Holmes E C,de Lamballerie X. Isolation of a novel species of flavivirus and a new strain of Culex flavivirus(Flaviviridae)from a natural mosquito population in Uganda[J]. J Gen Vi-rol,2009,90(Pt 11):2669-2678.
[16]Tyler S,Boiling B G,Blair C D,Brault A C,Pabbaraju K,Armijos M V,Clark D C,Calisher C H,Drebot M A.Distribution and phylogenetic comparisons of a novel mosquito flavivirus sequence present in Culex tarsalis Mosquitoes from western Canada with viruses isolated in California and Colorado[J]. Am J Trop Med Hyg,2011,85:162-168.
[17]Kyaw Kyaw A,Tun M M N,Buerano C C,Nabeshi-ma T,Sakaguchi M,Ando T,Inoue S,Mya Y Y,Hayasaka D,Thu H M,Thant K Z,Morita K. Isola-tion and genomic characterization of Culex flaviviruses from mosquitoes in Myammar[J].Virus Res,2018,2(247):120-124.
[18]郭凌云,李勤静,刘钢,杨永弘.二代测序技术在临床微生物领域中的应用进展[J].中华儿科杂志,2018,56(5):396-399.
[19]Ergünay K,Litzba N,Brinkmann A,Günay F,Kar S,?ter K,?rsten S,Sar?kaya Y,Alten B,Nitsche A,Linton Y M. Isolation and genomic characterization of Culex theileri flaviviruses in field-collected mosquitoes from Turkey[J]. Infect Genet Evol,2016,46:138-147.
[20]Crabtree M B,Nga P T,Miller B R. Isolation and char-acterization of a new mosquito flavivirus,Quang Binh vi-rus,from Vietnam[J]. Arch Virol,2009,154(5):857-860.
[2] Kuno G. Host range speci?city of?aviviruses:correla-tion with in vitro replication[J]. J Med Entomol,2007,44:93–101.
[3] Gritsun T S, Gould E A. Origin and evolution of3′UTR of?aviviruses:long direct repeats as a basis for the formation of secondary structures and their signi?cance for virus transmission[J]. Adv Virus Res,2007,69:203–248.
[4] Calzolari M,Zé-ZéL,Vázquez A,Sánchez Seco M P,Amaro F,Dottori M. Insect-specific flaviviruses,a worldwide widespread group of viruses only detected in insects[J]. Infect Genet Evol,2016,40:381-388.
[5] Vázquez A,Sánchez-Seco M P,Palacios G,Molero F,Reyes N,Ruiz S,Aranda C,Marqués E,Escosa R,Moreno J,Figuerola J,Tenorio A. Novel flavivirus-es detected in different species of mosquitoes in Spain[J]. Vector Borne Zoonotic Dis,2012,12(3):223-229.
[6]安淑一,刘家松,任毅,王作虪,韩悦,丁俊,郭军巧.辽宁发现库蚊黄病毒[J].病毒学报,2012,28(5):511-516.DOI:10.13242/j.cnki.bingduxuebao.002313
[7]刘然,郑旸,党荣理,张桂林,孙响,刘晓明,吴晓燕,李裕昌,杨银辉.利用深度测序技术发现新疆库蚊黄病毒[J].中国国情卫生检验杂志,2014,7:513-516.
[8] Huanyu W,Haiyan W,Shihong F,Guifang L,Hong L, Xiaoyan G, Lizhi S, Rayner S, Aiqiang X,Guodong L. Isolation and identification of a distinct strain of Culex Flavivirus from mosquitoes collected in Mainland China[J]. Virol J,2012,9:73.
[9] Blitvich B J,Firth A E. Insect-specific flaviviruses:a systematic review of their discovery,host range,mode of transmission,superinfection exclusion potential and genomic organization[J]. Viruses,2015,10;7(4):1927-1959.
[10]冯云,李鸿斌,朱进,章域震,杨卫红,范建华,梁国栋,张海林.首次从我国蚊虫中分离到类似广平病毒[J].病毒学报,2014,30(1):57-61. DOI:10.13242/j.cnki.bingduxuebao.000007
[11]Cammisa-Parks H,Cisar L A.,Kane A,Stollar V. The complete nucleotide sequence of cell fusing agent(CFA):homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses[J]. Virology,1992,189:511–524.
[12]梁文凯,荆春霞,王环宇.库蚊黄病毒的研究进展[J].中华实验和临床病毒学杂志,2015,29(2):188-190.
[13]Fang Y,Zhang Y,Zhou Z B,Shi W Q,Xia S,Li Y Y,Wu J T,Liu Q,Lin G Y. Co-circulation of aedes fla-vivirus,culex flavivirus,and quang binh virus in Shang-hai,China[J]. Infect Dis Poverty,2018,7(1):75.
[14]Parreira R,Cook S,Lopes?,de Matos A P,de Al-meida A P,Piedade J,Esteves A. Genetic characteriza-tion of an insect-specific flavivirus isolated from Culex theileri mosquitoes collected in southern Portugal[J].Virus Res,2012,167(2):152-161.
[15]Cook S, Moureau G, Harbach R E, Mukwaya L,Goodger K,Ssenfuka F,Gould E,Holmes E C,de Lamballerie X. Isolation of a novel species of flavivirus and a new strain of Culex flavivirus(Flaviviridae)from a natural mosquito population in Uganda[J]. J Gen Vi-rol,2009,90(Pt 11):2669-2678.
[16]Tyler S,Boiling B G,Blair C D,Brault A C,Pabbaraju K,Armijos M V,Clark D C,Calisher C H,Drebot M A.Distribution and phylogenetic comparisons of a novel mosquito flavivirus sequence present in Culex tarsalis Mosquitoes from western Canada with viruses isolated in California and Colorado[J]. Am J Trop Med Hyg,2011,85:162-168.
[17]Kyaw Kyaw A,Tun M M N,Buerano C C,Nabeshi-ma T,Sakaguchi M,Ando T,Inoue S,Mya Y Y,Hayasaka D,Thu H M,Thant K Z,Morita K. Isola-tion and genomic characterization of Culex flaviviruses from mosquitoes in Myammar[J].Virus Res,2018,2(247):120-124.
[18]郭凌云,李勤静,刘钢,杨永弘.二代测序技术在临床微生物领域中的应用进展[J].中华儿科杂志,2018,56(5):396-399.
[19]Ergünay K,Litzba N,Brinkmann A,Günay F,Kar S,?ter K,?rsten S,Sar?kaya Y,Alten B,Nitsche A,Linton Y M. Isolation and genomic characterization of Culex theileri flaviviruses in field-collected mosquitoes from Turkey[J]. Infect Genet Evol,2016,46:138-147.
[20]Crabtree M B,Nga P T,Miller B R. Isolation and char-acterization of a new mosquito flavivirus,Quang Binh vi-rus,from Vietnam[J]. Arch Virol,2009,154(5):857-860.