2012-2018年青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征
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摘要
目的了解2012-2018年青岛市人群A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征。方法收集2012年4月-2018年3月间青岛市A(H1N1)pdm09毒株397份,逆转录聚合酶链反应(RT-PCR)扩增神经氨酸酶(Neuraminidase,NA)和血凝素(Hemagglutinin,HA)基因全长,序列测定后进行耐药位点和氨基酸变异及进化分析。结果 5株发生了H275Y突变,为奥司他韦耐药株;另有4株S247N突变,可能为奥司他韦耐药株。2012-2018年H275Y突变株检出率依次为2.8%、2.0%、0.0%、1.1%、0.0%和0.7%。NA和HA进化树显示,2012-2013年青岛H275Y突变株与美国耐药株A/Tennessee/03/2013更接近,2013-2014年青岛H275Y突变株与国内株和日本札幌耐药株更接近,这两个年度耐药株的毒株起源可能有所不同。突变株在酶活性位点、抗原决定簇、受体结合位点及其他功能位点(如HA位点D222、Q223和NA位点V241I、N369K和N386K)的转变与野生敏感株一致。结论青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株明显增加且流行起源不同,但并未取得比野生株更强的流行能力。奥司他韦仍可作为流感预防和治疗的有效手段。
Objective To understand the characteristics of oseltamivir-resistant influenza A(H1 N1)pdm09 virus isolated from patients in Qingdao during the 2012-2018 influenza season.Methods A total of 397 throat swabs were collected with influenza A(H1 N1)pdm09 viruses in Qingdao from April 2012 to March 2018.The mutations of gene and amino acid locus were analyzed through the whole genome sequencing of hemagglutinin(HA)and neuraminidase(NA)by reverse transcriptase-polymerase chain reaction(RT-PCR).Results Five influenza A(H1 N1)pdm09 viruses with H275 Y substitution in the NA protein were resistant to oseltamivir,and 4 viruses with S247 N substitution were presumably oseltamivir-resistant.The annual detection rates for H275 Y were 2.8%,2.0%,0.0%,1.1%,0.0% and 0.7% from 2012 to 2018,respectively.NA and HA phylogenetic trees showed that H275 Y viruses in Qingdao were genetically similar to A/Tennessee/03/2013 in the United States in 2012-2013,but H275 Y viruses in 2013-2014 were more similar to those from mainland China and Sapporo cluster.This suggested that the origin of drug-resistant mutants in these two periods may be different.The mutations at enzyme activity sites,antigenic epitopes,receptor binding sites and other functional sites(such as D222,Q223 in HA and V241 I,N369 K and N386 K in NA)were consistent with that of wild-sensitive strains.Conclusion The oseltamivir-resistant strains of influenza A(H1 N1)pdm09 virus in Qingdao increase significantly and share different origins,but they do not achieve a stronger epidemic capacity than wild strains.Oseltamivir can also be used as an effective means of prevention and treatment.
Objective To understand the characteristics of oseltamivir-resistant influenza A(H1 N1)pdm09 virus isolated from patients in Qingdao during the 2012-2018 influenza season.Methods A total of 397 throat swabs were collected with influenza A(H1 N1)pdm09 viruses in Qingdao from April 2012 to March 2018.The mutations of gene and amino acid locus were analyzed through the whole genome sequencing of hemagglutinin(HA)and neuraminidase(NA)by reverse transcriptase-polymerase chain reaction(RT-PCR).Results Five influenza A(H1 N1)pdm09 viruses with H275 Y substitution in the NA protein were resistant to oseltamivir,and 4 viruses with S247 N substitution were presumably oseltamivir-resistant.The annual detection rates for H275 Y were 2.8%,2.0%,0.0%,1.1%,0.0% and 0.7% from 2012 to 2018,respectively.NA and HA phylogenetic trees showed that H275 Y viruses in Qingdao were genetically similar to A/Tennessee/03/2013 in the United States in 2012-2013,but H275 Y viruses in 2013-2014 were more similar to those from mainland China and Sapporo cluster.This suggested that the origin of drug-resistant mutants in these two periods may be different.The mutations at enzyme activity sites,antigenic epitopes,receptor binding sites and other functional sites(such as D222,Q223 in HA and V241 I,N369 K and N386 K in NA)were consistent with that of wild-sensitive strains.Conclusion The oseltamivir-resistant strains of influenza A(H1 N1)pdm09 virus in Qingdao increase significantly and share different origins,but they do not achieve a stronger epidemic capacity than wild strains.Oseltamivir can also be used as an effective means of prevention and treatment.
引文
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[3] Lackenby A,Besselaar TG,Daniels RS,et al.Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals,2016-2017[J].Antiviral Research,2018,157:38-46.
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[6] Hoffmann E,Stech J,Guan Y,et al.Universal primer set for the full-length amplification of all influenza A viruses[J].Archives of Virology,2001,146(12):2275-2289.
[7] Hui-Ling Y,Erich H,Garry T,et al.Importance of neuraminidase Active-Site residues to the neuraminidase inhibitor resistance of influenza viruses[J].Journal of Virology,2006,80(17):8787-8795.
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[9] Igarashi M,Ito K,Yoshida R,et al.Predicting the antigenic structure of the pandemic(H1N1)2009 influenza virus hemagglutinin[J].PLOS One,2010,5(1):e8553.
[10] Mir MA,Lal RB,Sullender W,et al.Genetic diversity of HA1 domain of hemagglutinin gene of pandemic influenza H1N1pdm09 viruses in New Delhi,India[J].Journal of Medical Virology,2012,84(3):386-393.
[11] Hurt AC,Chotpitayasunondh T,Cox NJ,et al.Antiviral resistance during the 2009 influenza A H1N1 pandemic:public health,laboratory,and clinical perspectives[J].Lancet Infectious Diseases,2012,12(3):240-248.
[12] Hurt AC,Hardie K,Wilson NJ,et al.Characteristics of a widespread community cluster of H275Y Oseltamivir-Resistant A(H1N1)pdm09 influenza in Australia[J].Journal of Infectious Diseases,2012,206(2):148-157.
[13] Takashita E,Kiso M,Fujisaki S,et al.Characterization of a large cluster of influenza A(H1N1)pdm09 virus cross-resistant to oseltamivir and peramivir during the 2013-2014 influenza season in Japan[J].Antimicrobial Agents and Chemotherapy,2015,59(5):2607-2617.
[14] Okomo-Adhiambo M,Fry AM,Su S,et al.Oseltamivir-resistant influenza A(H1N1)pdm09 viruses,United States,2013-14[J].Emerging Infectious Disease,2015,21(1):136-141.
[15] Huang W,Li X,Cheng Y,et al.Characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 virus during the 2013-2014 influenza season in Mainland China[J].Virology Journal,2015,12:96.
[16] 杨婷婷,黄维娟,刘晓琳,等.2005至2011年青岛地区甲型流行性感冒病毒耐药性和基因进化分析[J].中华传染病学杂志,2014,32(1):7-12.
[17] Hurt AC,Lee RT,Leang SK,et al.Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation[J].Eurosurveillance,2011,16(23):2-7.
[2] World Health Organization.WHO guidelines for pharmacological management of pandemic(H1N1)2009 influenza and other influenza viruses[EB/OL].[2019-02-28].https://www.who.int/csr/resources/publications/swineflu/h1n1_use_antivirals_20090820/en/.
[3] Lackenby A,Besselaar TG,Daniels RS,et al.Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals,2016-2017[J].Antiviral Research,2018,157:38-46.
[4] Meetings of the WHO working group on surveillance of influenza antiviral susceptibility-Geneva,November 2011 and June 2012[J].Weekly Epidemiology Record,2012,87(39):369-374.
[5] Summary of neuraminidase amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors[EB/OL].[2019-02-08].http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/NAI_Reduced_Susceptibility_Marker_Table_WHO.pdf?ua=1.
[6] Hoffmann E,Stech J,Guan Y,et al.Universal primer set for the full-length amplification of all influenza A viruses[J].Archives of Virology,2001,146(12):2275-2289.
[7] Hui-Ling Y,Erich H,Garry T,et al.Importance of neuraminidase Active-Site residues to the neuraminidase inhibitor resistance of influenza viruses[J].Journal of Virology,2006,80(17):8787-8795.
[8] Maurer-Stroh S,Ma JM,Lee RT,et al.Mapping the sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative to drug and antibody binding sites[J].Biology Direct,2009,4:18.
[9] Igarashi M,Ito K,Yoshida R,et al.Predicting the antigenic structure of the pandemic(H1N1)2009 influenza virus hemagglutinin[J].PLOS One,2010,5(1):e8553.
[10] Mir MA,Lal RB,Sullender W,et al.Genetic diversity of HA1 domain of hemagglutinin gene of pandemic influenza H1N1pdm09 viruses in New Delhi,India[J].Journal of Medical Virology,2012,84(3):386-393.
[11] Hurt AC,Chotpitayasunondh T,Cox NJ,et al.Antiviral resistance during the 2009 influenza A H1N1 pandemic:public health,laboratory,and clinical perspectives[J].Lancet Infectious Diseases,2012,12(3):240-248.
[12] Hurt AC,Hardie K,Wilson NJ,et al.Characteristics of a widespread community cluster of H275Y Oseltamivir-Resistant A(H1N1)pdm09 influenza in Australia[J].Journal of Infectious Diseases,2012,206(2):148-157.
[13] Takashita E,Kiso M,Fujisaki S,et al.Characterization of a large cluster of influenza A(H1N1)pdm09 virus cross-resistant to oseltamivir and peramivir during the 2013-2014 influenza season in Japan[J].Antimicrobial Agents and Chemotherapy,2015,59(5):2607-2617.
[14] Okomo-Adhiambo M,Fry AM,Su S,et al.Oseltamivir-resistant influenza A(H1N1)pdm09 viruses,United States,2013-14[J].Emerging Infectious Disease,2015,21(1):136-141.
[15] Huang W,Li X,Cheng Y,et al.Characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 virus during the 2013-2014 influenza season in Mainland China[J].Virology Journal,2015,12:96.
[16] 杨婷婷,黄维娟,刘晓琳,等.2005至2011年青岛地区甲型流行性感冒病毒耐药性和基因进化分析[J].中华传染病学杂志,2014,32(1):7-12.
[17] Hurt AC,Lee RT,Leang SK,et al.Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation[J].Eurosurveillance,2011,16(23):2-7.