| BackgroundA novel virus,namely,avian influenza A virus H7N9,occurred in East China on March 30th,2013,which could induce severe respiratory tract infection in human.Altogether 133 cases were infected with avian influenza virus(AIV)H7N9 from March to May 2015,which mainly distributed in Yangtze River Delta area in East China.Subsequently,human infection with AIV H7N9 in China had been remarkably paused,with only one case of infection was reported in Guangdong Province on August 10th.At the end of September 2015 A total of 679 cases were reported including 271 death cases.183 cases of patients were reported in Guangdong Province from 2013 to 2015.AIV spread across the country rapidly,which had become a threat to public health.The National Health and Family Planning Committee and Chinese Center for Disease Control and Prevention immediately published Diagnosis and Treatment Scheme for Human Infection with Avian Influenza H7N9(2014).Early detection,diagnosis and medication were underlined in the guideline,especially the early application of neuraminidase inhibitors.Neuraminidase(NA)is a kind of glycoprotein of influenza virus surface,coding by 6th gene,the enzymatic lysis terminal of which is connected to sialic acid receptor,which contributes to the release of progeny virus particles by the infected cells.NA inhibitors(NAIs)commonly used in clinic in China include peramivir,oseltamivir,zanamivir,and so on.Such drugs prevent the release of new virus particles on surface of the infected cells,thus achieving the effects of preventing and controlling influenza virus.At present,a vast majority of human infection with AIV H5N1 is sensitive to NAIs.But according to Diagnosis and Treatment Scheme for Human Infection with Avian Influenza H7N9(2014),do clinician seize the best time of NAIs treatment?What about the actual therapeutic effect of NAIs when it face with AIV H7N9?Will it cause AIV H7N9 to resist drug resistance while use NAIs heavily?To answer these questions scientifically,it is necessary to carry out a retrospective study on application of NAIs in patients with AIV H7N9 in Guangdong Province from 2013 to 2015,as well as to study the sensitivity and its molecular characteristics of human infection with AIV H7N9 in Guangdong Province to NAIs.This research proposed to collect epidemiological and clinical data of 183 cases of human infection with AIV H7N9 in Guangdong Province from 2013 to 2015 from the National Avian Influenza Surveillance Network Database;analyzed the time duration from disease outbreak to laboratory confirmation,as well as that from disease outbreak to the first time of receiving NAIs treatment;and understood the diagnostic capability and timely treatment ability to cope with AIV H7N9 in Guangdong Province.NA inhibition assays were performed on 40 strains of AIV H7N9 collected in Guangdong Province from 2014 to 2016,and sensitivity of human infection with AIV H7N9 spreading in Guangdong Province to three NAIs(namely,oseltamivir,peramivir and zanamivir)was analyzed,with an aim to provide experimental evidence for reasonable medication for cases infected with AIV H7N9 in clinic.Whole genome sequencing on 40 strains of human infection with AIV H7N9 collected in Guangdong Province from 2014 to 2016 was carried out using Ion Torrent high-throughput sequencing;meanwhile,the related pathogenic sites were analyzed,and virus variations during spreading process were learned and tracked,which could provide scientific and effective foundation for effective prevention and treatment of AIV H7N9 in the future.Research objective1.Analyzing the epidemiological and clinical data of 183 cases of human infection with AIV H7N9 collected in Guangdong Province from 2013 to 2015,so as to learn the diagnostic and treatment capabilities in Guangdong Province.2.Analyzing the sensitivity of human infection with AIV H7N9 spreading in Guangdong Province to three NAIs(namely,oseltamivir,peramivir and zanamivir)by detecting virus phenotype.3.Analyzing the key points of drug resistance of Neuraminidase inhibitors and related pathogenic sites of human infection with AIV H7N9 spreading in Guangdong Province.Learning virus variations during spreading process could provide scientific and effective foundation for effective prevention and treatment of AIV H7N9 in the future.Research methods1.Data sources:Epidemiological and clinical data of 183 cases of human infection with AIV H7N9 collected in Guangdong Province from 2013 to 2015 were obtained from the National Avian Influenza Surveillance Network Database.2.Virus sources:40 virus strains for test were collected,isolated and restored from various prefectures in Guangdong Province by Center for Disease Control and Prevention of Guangdong Province from 2014 to 2016.3.Research methods:Time duration from disease outbreak to laboratory confirmation,as well as that from disease outbreak to the first time of receiving NAIs treatment was calculated and analyzed using box-and-whisker plot analysis and Mann-Whitney U test.Experimental strains were extracted in'accordance with stratified random sampling;and half maximal inhibitory concentration(IC50)of experimental strains on three NAIs(carboxylated oseltamivir,peramivir and zanamivir)was detected by NA inhibition assays.Differences in IC50 of three drugs to AIV H7N9 were analyzed and compared with one-way variance;and pairwise comparisons of IC50 among three drugs were conducted using Least Significant Difference(L-S-D).Whole genome sequencing on 40 strains of human infection with AIV H7N9 collected in Guangdong Province from 2014 to 2016 was carried out using Ion Torrent high-throughput sequencing,MEGA7.0 software was used for sequence alignment,and key points of drug resistance of Neuraminidase inhibitors and related pathogenic sites were found and analyzed.Amino acid sequence analysis of the potential glycosylation sites was performed using glycosylation predictor.Research contents1.Epidemiological and clinical data of 183 cases of human infection with AIV H7N9 in Guangdong Province from 2013 to 2015 were collected from the National Avian Influenza Surveillance Network Database;the time duration from disease outbreak to laboratory confirmation,as well as that from disease outbreak to the first time of receiving NAIs treatment was analyzed;and the diagnostic capability and timely treatment ability to cope with AIV H7N9 in Guangdong Province were learned.2.NA inhibition assays were carried out on the selected AIV H7N9 strains in Guangdong Province,and sensitivity of human infection with AIV H7N9 spreading in Guangdong Province to three NAIs(namely,carboxylated oseltamivir,peramivir and zanamivir)was analyzed.3.High-throughput sequencing was conducted on the selected AIV H7N9 strains in Guangdong Province,and related pathogenic sites of human infection with AIV H7N9 spreading in Guangdong Province were analyzed.Research results1.Analyzing the epidemiological and clinical data of 183 cases of human infection with AIV H7N9 in Guangdong Province collected from the National Avian Influenza Surveillance Network Database.The results suggest that the time duration from disease outbreak to laboratory confirmation in 2015(median:8.0days,interquartile ranges:4.25-9.0 days)was remarkably reduced.In addition,145 cases(79.23%)were treated by at least one NAI.It was discovered when analyzing the time duration from disease outbreak to the first time of receiving NAIs treatment that,time duration in 2015(median:5.0 days,interquartile ranges:3.0-7.0 days)was notably reduced(p = 0.03 by Mann-Whitney U test).2.None of the 40 strains resisted to the three drugs(carboxylated oseltamivir,peramivir and zanamivir),suggesting that strains were all sensitive to three NAIs.IC50(x±SD)nmol/L of applying peramivir,carboxylated oseltamivir and zanamivir was(0.56±0.23)nmol/L,(1.31±0.27)nmol/L and(4.38±0.26)nmol/L,respectively.Differences in IC50 of three drugs to AIV H7N9 were of statistical significance(P<0.001).Further pairwise comparison of IC50 among the three drugs suggested that difference in IC50 between peramivir and carboxylated oseltamivir,that between peramivir and zanami,vir,as well as that between carboxylated oseltamivir and zanamivir was of statistical significance(P<0.001).3.Glycoprotein HA receptor binding sites(H3 numbering)G186v and Q226L on surface of 40 strains developed amino acid mutations.It indicated that human infection with AIV H7N9 spreading in Guangdong Province has generally acquired the molecular basis to bind with human upper respiratory tract epithelial cell SA?-2,6Gal receptor.None of the 40 strains of in this research developed any kind of mutation combinations of 110Y/160A/226L/228S,158D/224K/226L and 196R/226L/228S in HA protein(H3 numbering).It demonstrated that AIV H7N9 spread in the manner of droplet,thus it was less likely to spread from person to person.4.26 out of the 40 strains(65.0%)developed mutation of PB2 protein on E627K site,1(2.5%)strain developed mutation of PB2 protein on D701N site,while 10(25.0%)developed mutation of PA protein on L336M site.All the 40(100.0%)strains encoded the full length of PB1-F2 proteins,suggesting that AIV H7N9 in Guangdong Province possessed strong adaptability to human body.5.All the 40 strains(100.0%)of human infection with AIV H7N9 developed HA protein(H3 numbering)mutation on D225G site,QISNT sequence deletion on 69-73 sites in NA protein stem area,M1 protein mutations on N30D and T215A sites,PB2 protein mutation on L89V site and NS1 protein mutation on P42S site.26 strains(65.0%)developed PB2 protein mutation on E627K site,and 10(25.0%)developed PA protein mutation on L336M site.The above variations suggested that human infection with AIV H7N9 spreading in Guangdong Province had enhanced virulence,which might be closely related to the high pathogenicity of AIV H7N9 to human.6.Mutation on S3 IN site discovered in M2 protein in all 40 strains(100.0%),indicating that AIV H7N9 spreading in Guangdong Province was basically insensitive to M2 ion channel inhibitors(amantadine drugs).The results revealed that none of the NA proteins of all H7N9 strains developed mutations on E119G,R152K,H274Y and R294K sites,indicating that these viruses did not develop resistance to NAIs.Research conclusions1.Compared with 2013-2014,the time duration from outbreak to laboratory confirmation in 2015 is shortened,while that from disease outbreak to the first time of receiving NAIs treatment is also reduced.This result manifest the gradual improvements in diagnostic and timely treatment capabilities to cope with AIV H7N9 in Guangdong Province.2.Human infection with AIV H7N9 in Guangdong Province in the past three years maintains high sensitivity to NAIs.And IC50 results reveal different sensitivities to different NAIs.Peramivir is associated with low toxicity,convenient administration route(oral or intravenous administration)and being unlikely to develop drug resistance.In conclusion,Peramivir is an anti-influenza virus drug with application value.3.Mutations on HA receptor binding sites(H3 numbering)G186V and Q226L extensively exist in the experimental strains,suggesting that human infection with AIV H7N9 spreading in Guangdong Province has generally acquired the molecular basis to bind with human upper respiratory tract epithelial cell SAa-2,6Gal receptor.4.PB2 protein mutation on E627K site and PA protein mutation on L336M site in experimental strains,together with the phenomenon that virus encodes the full length of PB1-F2 proteins,indicated strong adaptability of human infection with AIV H7N9 spreading in Guangdong Province to human body.5.HA protein mutation on D225G site,QISNT sequence deletion on 69-73 sites of neck of NA protein,M1 protein mutations on N30D and T215A sites,PB2 protein mutations on E627K and L89V sites,NS1 protein mutation on P42S site,and PA protein mutation on L336M site suggested enhanced virulence of human infection with AIV H7N9 spreading in Guangdong Province.It may be closely related to the high pathogenicity of AIV H7N9 to human.6.M2 protein mutation on S3 IN site can be seen in all experimental strains in this research,demonstrating that AIV H7N9 spreading in Guangdong Province is basically insensitive to M2 ion channel inhibitors(amantadine drugs).The results revealed that none of the NA protein of all experimental strains develops NA protein mutations,indicating that these viruses do not develop resistance to NAIs.NAIs should be kept as the preferred drugs during the early stage of human infection with AIV H7N9. |
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