Full length sequencing of all nine subtypes of the neuraminidase gene of influenza A viruses using subtype specific primer sets.

An RT-PCR based method was developed using subtype specific overlapping primers to obtain full length amplification of neuraminidase (NA) gene from all subtypes (N1-N9) of influenza A viruses. This method was validated using reference strains of avian influenza viruses (AIV) (N1-N9), human influenza viruses (N1 and N2), and swine influenza viruses (N1-N3). Amplification of the NA gene was obtained with all viruses tested. Additionally, 200 field isolates of AIV from wild birds were tested by this method and the NA gene was amplified in all isolates. The NA subtype of all 200 isolates was determined by further sequencing of the amplified NA genes and all sequences were submitted to GenBank. The method described in this paper can be used to determine subtype of influenza isolates as well as their evolution and mutations if any, in the NA gene.

[1]  Yiping Fan,et al.  Response to Comment on "Large-Scale Sequence Analysis of Avian Influenza Isolates" , 2006, Science.

[2]  G. Pauli,et al.  Application of a Fluorogenic PCR Assay for Typing and Subtyping of Influenza Viruses in Respiratory Samples , 2000, Journal of Clinical Microbiology.

[3]  Shun-lin Hu,et al.  A reverse transcription-PCR for subtyping of the neuraminidase of avian influenza viruses. , 2009, Journal of virological methods.

[4]  M. Zambon,et al.  Multiplex PCR for Typing and Subtyping Influenza and Respiratory Syncytial Viruses , 1998, Journal of Clinical Microbiology.

[5]  K. Lohman,et al.  Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes , 2002, Journal of Clinical Microbiology.

[6]  R. Webster,et al.  Influenzavirus neuraminidase and neuraminidase-inhibition test procedures. , 1973, Bulletin of the World Health Organization.

[7]  T. Mettenleiter,et al.  Simultaneous one-tube full-length amplification of the NA, NP, M, and NS genes of influenza A viruses for reverse genetics. , 2009, Journal of virological methods.

[8]  Albert D. M. E. Osterhaus,et al.  Characterization of a Novel Influenza A Virus Hemagglutinin Subtype (H16) Obtained from Black-Headed Gulls , 2005, Journal of Virology.

[9]  S. Zou.  A practical approach to genetic screening for influenza virus variants , 1997, Journal of clinical microbiology.

[10]  Y. Guan,et al.  Universal primer set for the full-length amplification of all influenza A viruses , 2001, Archives of Virology.

[11]  I. Capua,et al.  Animal and Human Health Implications of Avian Influenza Infections , 2007, Bioscience reports.

[12]  M. Beer,et al.  Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses. , 2009, Veterinary microbiology.

[13]  Z. Grossman,et al.  Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel , 2005, Journal of Clinical Microbiology.

[14]  R. E. Gough,et al.  A standard haemagglutination inhibition test for Newcastle disease. (1). A comparison of macro and micro methods , 1974, Veterinary Record.

[15]  J. Pasick.  Advances in the molecular based techniques for the diagnosis and characterization of avian influenza virus infections. , 2008, Transboundary and emerging diseases.

[16]  M. Brunck,et al.  A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus , 2008, Virology Journal.

[17]  M. S. Lee,et al.  Identification and subtyping of avian influenza viruses by reverse transcription-PCR. , 2001, Journal of virological methods.

[18]  D. Stallknecht,et al.  Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method. , 2009, Journal of virological methods.

[19]  V. Hinshaw,et al.  Micro neuraminidase-inhibition assay for classification of influenza A virus neuraminidases. , 1983, Avian diseases.