Detection Of High Pathogenicity Avian Influenza H5N6 Virus By Multiplex Real-time RT-PCR Assay And Moleular Characteristic Of The Influenza Virus H5N6 In Human Infection

Objective:Establish A detection of type A influenza virus(Flu A)and highly pathogenic avian influenza(HPAI)H5N6 virus by using One-step multiple fluorescence quantitative RT-PCR technique and to investigate the gene sequences of hemagglutinin(HA)and neuraminidase(NA)of the novel highly pathogenic avian influenza H5N6 virus in 2014-2015,and analyze the viral mutation and molecular biology characteristics.Methods:1.Download a number of gene sequences from domestic and foreign human and poultry FluA and H5N6 from the NCBI(http://www.ncbi.nlm.nih.gov/)genebank.The homology of gene was determined by Mega6.0 software.Primer Express 3.0 software was used to design highly specific primers and TaqMan in its conserved region.And then BLAST(http://www.ncbi.nlm.nih.gov/tools/primer-blast/)sequence alignment verifies the specificity of primers and probes.2.We synthesized the DNA fragments of each gene connected on the plasmid vector Simple Vector PmdTM19-T and then transform E.DH5a competent bacteria and culture,After identification and gene sequencing,plasmid DNA was extracted by the NanoDrop ND-2000 for detecting plasmid DNA concentration and detecting the DNA copy number as the sensitivity of the standard quantitative mother liquor.According to the requirement,standard quantitative liquor was diluted from the highest concentration(108copies/mL)to the lowest concentration(102copies/mL)to in a row 10 times dilution.And stored at-80?.we analyze the sensitivity and repeatability by using this method to detect different concentrations of target gene standard,continuous detection 3 times.3.In the reaction system of the method,we added a dozen respiratory pathogenic microorganisms and inactivated strains of avian influenza H5N1,H5N6,H7N9 viruses was detected by the extractive template.4.The clinical pharyngeal swab samples of 135 influenza patients were collected from the First Affiliated Hospital of Zhejiang University Medical College and placed in 3.0mL virus transport culture medium in November 2014 to January 2015,The nucleic acid was extracted according to the viral RNA nucleic acid extraction kit,The samples were detected by this method,and compared with the results detected by Shanghai River biology Co.,Ltd.5.According to the software manual operation,analyze sequence alignment,homology analysis and evolution by using MEGA6.0.Phylogenetic tree analysis using WHO recommended method,in which HA genes with neighbor-joining,NA gene with the maximum likelihood,bootstrap is 1000.6.Gene sequence mutation analysis was performed using MEGA6.0 software.,the N-glycosylation site predict by using Netnglyc server(http://www.cbs.dtu.dk/services/NetNGlyc/)examined Asn-Xaa-Ser/Thr(where Xaa represents any amino acid other than proline).Results:1.sensitivity and repeatability analysis:The plasmid was prepared by serial dilution(10 times)with the plasmid standard(initial copy number 1.0×107 copies/mL)of the target gene(Flu A,H5,N6 and RP),then the standards was detected.The results showed that the detection sensitivity of each target gene was 1.0×102.Different concentrations of nucleic acid detection was detected three times,within their respective batches Ct value detected in standard deviation between 0.18 to 0.46,the coefficient of variation(CV)<5.00%,showed good reproducibility within its batch.2.Specificity analysis:A dozen respiratory pathogenic microorganisms stored by our office and three kinds of avian influenza strains provided by the State Key Laboratory of Infectious Diseases Diagnosis were detected by the kits of Shanghai River Bio-Tech Co.,Ltd and the multiplex real-time RT-PCR assay was established by us respectively,The results shows that the test results are identical,no cross-reaction was observed for these viruses and common respiratory pathogens,and the specificity of the multiplex assay for each gene was 100%.3.coincidence rate analysis:The results of 135 cases of swab samples collected in patients with influenza-like in fever clinics showed:Ct value of the positive results of the gene of interest between 19.35?32.58;23 samples with FluA infection were tested,135 RP gene were detected,and the patients with HPAI-H5N6 virus infection were not found by the multiplex assay for detecting 135 clinical specimens,which were completely accorded with the results detected by the regents provided by Shanghai River Bio-Tech Co.,Ltd.The coincidence rate was 100%.4.Comparison of homology and phylogenetic tree analysis of the H5N6-HAgene:The homology of HA amino acid sequences of the three patient HPAI-H5N6 virus comparing with each other was 96.5%?98.7%respectively.The phylogenetic trees showed that the three strains of patients were located in the two different subclades;The GZ/39715/14 and YN/0127/15 are in the same branch as the A/chicken/Zhejiang/727155/2014(H5N6),A/chicken/Shenzhen/2396/2013(H5N6),A/chicken/Jiangxi/NCDZT1126/2014(H5N6),A/duck/Laos/XBY004/2014(H5N6)detected avian infected strains in the Zhejiang,Guangdong,Jiangxi,Laos.Then SC/26221/14,A/duck/Sichuan/NCJPL7/2014(H5N6),A/duck/Jiangxi/13484/2014(H5N6)are in the same branch in China avian infected strains in the Sichuan,Jiangxi;the HA gene of influenza virus H5N6 in human infection from clade 2.3.4.4 H5 virus.5.Comparison of homology and phylogenetic tree analysis of the H5N6-NAgene:The homology of NA amino acid sequences of the three patient HPAI-H5N6 virus comparing with each other was 90.1%?98.2%respectively.The phylogenetic trees showed that the three strains of patients were located in the two different subclades;The GZ/39715/14 and YN/0127/15 are in the same branch as the A/chicken/Shenzhen/2396/2013(H5N6),A/duck/Zhejiang/727158/2014(H5N6),A/chicken/Jiangxi/NCDZT1126/2014(H5N6),A/duck/Laos/XBY004/2014(H5N6)detected avian infected strains in the Guangdong,Zhejiang,Jiangxi,Laos.Then SC/26221/14,A/duck/Jiangxi/13484/2014(H5N6),A/goose/Zhejiang/1120132/2014(H5N6),A/chicken/Shenzhen/1845/2013(H5N6)et al are in the same branch in China avian infected strains;The NA gene of influenza virus H5N6 in human infection from H6N6 virus.6.Analysis of HA amino acid sequence mutation site:Through the analysis of MEGA6.0 software,there was a conserved amino acid sequence QRG at positions 222-224.The mutation of HA gene contains S137A,T160A etc,Some strains present S137A and T160A mutations,which lead to the deletion of 158N-glycosylation,The cleavage site of the two strains of HA was PLRERRRKR/GLF and the other cleavage site was PLREKRRKR/GLF.7.Analysis of N-glycosylation sites:Six N-glycosylation sites were found in three strains of HPAI-H5N6 infected in HA strains,YN/0127/15 more than one Asn124.8.Guangdong strain HA protein is more likely to become B cell surface antigen epitope segment were:26-30;99-103;110-113;136-142;169-173;181-184;197-200;235-239.Sichuan strain HA protein is more likely to become B cell surface antigen epitope segment were:26-30;99-103;110-113;137-142;181-185;197-200;235-239.The results of two epitope such as:26-30;99-103;110-113;137-142;181-184;197-200;235-239 can be used as HPAI-H5N6 possible linear B cell epitopes.Conclusions1.Multiplex fluorescence quantitative RT-PCR technique is the use of TaqMan PCR technology add a specific dual-labeled fluorescent probes base on the original primer.The results are determined by the change of the amount of fluorescence in the detection system.we use this principle to establish a multiplex fluorescent RT-PCR method to detect Flu A and/or HPAI-H5N6 virus infection.2.Through the multi-angle evaluation of the method,the method is stable,fast,high sensitivity and specificity,reproducibility is good,The multiplex real-time RT-PCR assay was established and used to early diagnose for the patients with Flu A and/or HPAI-H5N6 virus infection.3.Clinical specimens verification:the simultaneous detection of patients by using our reagents separately with River reagents,we draw both compliance rate reached 100%.4.Through genetic evolution analysis:A novel highly pathogenic avian influenza H5N6 virus in human infection was reassorted with an HA from clade 2.3.4.4 H5 viruses and a NA from H6N6 viruses.5.The mutation of H5N6-HA gene and N-glycosylation may help to strength human infection.6.The results of two epitope such as:26-30;99-103;110-113;137-142;The above epitope can be used as HPAI-H5N6 possible linear B cell epitopes.We can use this as the basis of H5N6 influenza vaccine and for the next step to prepare for vaccine research.We should focus on observation of the virus in variation of these areas when the H5N6 is popular.