| The influenza virus is a major respiratory pathogen. Every year the influenza outbreaks occur at different scales.Hemagglutinin(HA) and neuraminidase(NA) are the two most important envelope proteins of influenza viruses, frequently encountering external antibody selection pressure. Mutation occuring in the essential protein such as HA or NA can raise possibility of new virus strain.This article divided into two parts.Part 1 Objective: To analyze the HA and NA genetic characteristics of influenza viruses in one case of mixed infection with Pandemic H1N1 and Seasonal H3N2. Method: The viruses nucleic acid from nasopharyngeal and it’s 100 times dilution from cell culture were tested by real-time PCR for identifying influenza subtypes. The viruses isolation of cell culture was also tested by Haemagglutination and Haemagglutination inhibition tests. The HA gene and NA gene of virus isolation in cell culture were amplified by reverse transcription-polymerase chain reaction(RT-PCR) and sequenced. The sequences identity were compared with WHO(2013-2014)vaccine virus and representative virus. Phylogenic analysis was performed by using bioinformatics software. Results: The nasopharyngeal swab and cell culture specimens were positive for Seasonal H3N2(CT 25.837 VS 15.675), and positive for Pandemic H1N1(CT 20.267 VS 25.169). The titer of HI was >1280 for Seasonal H3N2 and 80 for Pandemic H1N1. Hemagglutinin(HA) and neraminidase(NA) segements of both subtype were sequenced and compared with known reference strains A/Victoria/361/2011(H3N2) vaccine strain and A/Texas/50/2012(H3N2)(WHO recommended strain) used for A/H3N2 subtype, A/California/07/2009(H1N1) vaccine strain used for H1N1pdm09 subtype. Compared with the A/Victoria/361/2011(H3N2) and A/Texas/50/2012(H3N2), amino acid sequence indentities of HA1 segment from A/H3N2 subtype were 96.7% and 97.1% respectively. Compared with A/Texas/50/2012(H3N2) amino acid sequence indentity of NA segment was 99.6%. Compared with A/California/07/2009(H1N1) vaccine strain, amino acid sequence indentity of HA segment from H1N1pdm09 was 97.9%, while that of NA segment was 97.8%. By the comparison, HA and NA amino acid sequences showed some mutation sites, several mutation sites located in antigentic domain. None of the neuraminidase inhibitors(such as oseltamivir and zanamivir) resistant mutation from A/H3N2 and H1N1pdm09 were found.Conclusion: In the co-circulated period, the mixed infection of Pandemic H1N1 and Seasonal H3N2 influenza viruses could be tested by real-time PCR. Through our effort, we want to provide more information for special influenza virus further analysis.Part 2 Objective: This study analyzed the genetic evolution of the neuraminidase(NAs) of influenza A subtype N9 viruses and attempted to find the origin of novel avian A/H7N9 influenza virus. Method :The data of influenza A subtype N9 viruses NA sequences from NCBI were used to construct phylogenetic tree by the software of Clustal X 2.0 and MEGA 6.0. The overall rate of evolutionary and selective pressure of modern Eurasian cluster at the nucleotide level was estimated by BEAST 2.1.2 and Datamonkey interface. The entropy plot of NA proteins was analyzed by software of Bioedit. Results: Results showed that the novel avian A/H7N9 influenza viruses were located in the modern Eurasian cluster. In this cluster, the mean rate of nucleotide for NA was 3.8354×10-3 and mean synonymous(d S) and non- synonymous(d N) substitutions per site(d N/d S) ratio was 0.140413. Also in this cluster, high level amino acid mutation entropy was located at sites of 16, 19, 40, 53, 81,84, 112, 256, 335, 359, and 401. Conclusions: With this genetic evolution analysis it is found the origin of the of novel avian A/H7N9 influenza viruses neuraminidase may be the results of overall genetic evolution of influenza A subtype N9 viruses NAs. The influenza A virus(A/duck/Siberia/700/1996(H11N9)) isolated in 1996 was taken as the common ancestor of the more recent influenza A subtype N9 viruses NAs. The origin of novel avian A/H7N9 influenza viruses NAs was not the results of selective stress. |
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