Identification of H1N1 influenza A virus by sequencing the partial regions of HA and NA gene specifically amplified with nested RT-PCR

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Author:
CHEN Wei-lie(Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou 510060, China)
WEI Shao-jing(Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou 510060, China)
ZHANG Fu-chun(Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou 510060, China)
WANG Jian(Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou 510060, China)
TANG Yang-bo(Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou 510060, China)
Journal Title:
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
Issue:
Volume 16, Issue 05, 2010
DOI:
10.3760/cma.j.issn.1674-1927.2010.05.017
Key Word:
Polymerase chain reaction;Sequence analysis,DNA;Influenza A virus,H1N1 subtype;HA gene;NA gene

Abstract: Objective To establish an approach for identification of H1N1 influenza A (A H1N1 )virus by sequencing the partial regions of HA and NA gene amplified with nested RT-PCR. Methods The nested RT-PCR method with a total of 7 specific primers designed as two primer sets was used to amplify the partial regions of A H1N1 virus HA and NA genes. The PCR products were then sequenced. Phylogenetic analysis for PCR- obtained sequences and sequences of influenza A virus (mainly HA and NA) were performed to further validate the results. Sequence alignment of the proteins was also studied and analyzed for their characteristics. Results Nested RT-PCR of A H1N1 virus HA and NA genes from 4 patients infected with H1N1 influenza A virus respectively yielded 442 bp and 543 bp DNA segments. Phylogenetic tree analysis for all nucleotide sequences showed that the sequences of HA and NA from 4 patients clustered specifically with the sequences of HA and NA from the novel influenza A (H1N1) virus that emerged in 2009 and separated from seasonal influenza virus H1, H2, H3, N1 and N2, human avian influenza virus H5 and N1. Protein sequences alignment analysis showed that the amino acid sequences of proteolytic activation cleavage site on HA protein from 4 patients were PSIQSR ↓ GLF and did not appear to be characteristic of highly pathogenicity. The amino acid residue 275 on NA protein was His, suggesting absence of drugresistance mutation to H275Y. Conclusions The nested RT-PCR technique described in this study can specifically amplify the partial regions of A H1N1 virus HA and NA gene. The sequences obtained can be used for further identification of the virus, and also for the virulence and drug resistance analysis.

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