Skip to main content
Log in

Genetic and antigenic analysis of H5N1 viruses for selection of HA-donor virus for vaccine strains

  • Original Article
  • Published:
Indian Journal of Virology Aims and scope Submit manuscript


Genetic and antigenic analysis of H5N1 viruses, isolated in India during a period from year 2006 to 2010, was carried out for selection of the potential H5-HA (haemagglutinin) gene donor virus for developing a reverse genetics based DIVA marker H5 vaccine for poultry in India. Out of the 47 H5N1 viruses (clade 2.2), 14 representative viruses were selected on the basis of amino acid sequence analysis of HA1 gene for further antigenic characterization. Using antigenic cartography, an antigenic map was constructed based on the data of cross-HI (haemagglutinin inhibition) titration of 14 sera versus 14 viruses to visualize the relatedness among the antigens and antigenic coverage of the sera. Sera against five H5N1 viruses (A/crow/Assam/142119/2008, A/chicken/West Bengal/100879/2008, A/chicken/West Bengal/155505/2009, A/chicken/West Bengal/80995/2008 and A/chicken/West Bengal/81760/2008) exhibited maximum (100 %) antigenic coverage, hence, were selected as the potential HA donor viruses. However, the virus strain A/chicken/West Bengal/80995/2008 matched completely with the consensus amino acid sequence of the 47 viruses, therefore, was considered the best HA donor candidate out of the five showing 100 % antigenic coverage. The present study demonstrates a stepwise methodology for logical selection of vaccine strain or HA gene donor strain for developing H5 vaccines using genetic and antigenic data.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others


  1. Abbas T, Wilking H, Staubach C, Ziller M, Conraths FJ. Priority areas for surveillance and prevention of avian influenza during the water-bird migration season in Pakistan. Geospat Health. 2011;6(1):107–16.

    PubMed  Google Scholar 

  2. Bublot M, Le Gros FX, Nieddu D, Pritchard N, Mickle TR, Swayne DE. Efficacy of two H5N9-inactivated vaccines against challenge with a recent H5N1highly pathogenic avian influenza isolate from a chicken in Thailand. Avian Dis. 2007;51:332–7.

    Article  PubMed  Google Scholar 

  3. Dubey SC, Nagarajan S, Tosh C, Bhatia S, Krishna. Avian influenza: a long-known disease and its current threat. Indian J Anim Sci. 2009;79:113–40.

    Google Scholar 

  4. Fouchier RA, Smith DJ. Use of antigenic cartography in vaccine seed strain selection. Avian Dis. 2010;54:220–3.

    Article  PubMed  Google Scholar 

  5. Horimoto T, Kawaoka Y. Pandemic threat posed by avian influenza A viruses. Clin Microbiol Rev. 2001;14:129–49.

    Article  PubMed  CAS  Google Scholar 

  6. Jadhao SJ, Lee CW, Sylte M, Suarez DL. Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza viruses in chickens. Vaccine. 2009;27:6247–60.

    Article  PubMed  CAS  Google Scholar 

  7. Lamb RA, Krug RM. Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology. 4th ed. Philadelphia: Lippincott William and Wilkins; 2001. p. 1487–532.

    Google Scholar 

  8. Liao YC, Ko CY, Tsai MH, Lee MS, Hsiung CA. ATIVS: analytical tool for influenza virus surveillance. Nucl Acids Res (Web Server issue) 2009; W643–6.

  9. Murugkar HV, Nagarajan S, Tosh C, Bhatia S, Venkatesh G, Jain R, Kumar S, Khandia R, Pandey M, Behera P, Tripathy S, Kulkarni DD, Dubey SC. H5N1 virus outbreaks in poultry in India. Vet Rec. 2008;162:255.

    Article  PubMed  CAS  Google Scholar 

  10. Naeem K, Siddique N. Use of strategic vaccination for the control of avian influenza in Pakistan. Dev Biol. 2006;124:145–50.

    CAS  Google Scholar 

  11. Nagarajan S, Murugkar HV, Tosh C, Behera P, Jain R, Tripathi S, Khandia R, Gupta V, Kulkarni DD, Dubey SC. Avian influenza virus (H5N1) in chickens in India. Vet Rec. 2009;164:128.

    Article  PubMed  CAS  Google Scholar 

  12. Pattnaik B, Pateriya AK, Khandia R, Tosh C, Nagarajan S, Gounalan S, Murugkar HV, Shankar BP, Shrivastava N, Behera P, Bhagat S, Peiris JSM, Pradhan HK. Phylogenetic analysis revealed genetic similarity of the H5N1 avian influenza viruses isolated from HPAI outbreaks in chickens in Maharashtra, India with those isolated from swan in Italy and Iran in 2006. Curr Sci. 2006;91:77–81.

    CAS  Google Scholar 

  13. Pedersen JC. Hemagglutination-inhibition test for avian influenza virus subtype identification and the detection and quantitation of serum antibodies to the avian influenza virus. Methods Mol Biol. 2008;436:53–66.

    PubMed  CAS  Google Scholar 

  14. Russell CA, Jones TC, Barr IG, Cox NJ, Garten RJ, Gregory V, Gust ID, Hampson AW, Hay AJ, Hurt AC, de Jong JC, Kelso A, Klimov AI, Kageyama T, Komadina N, Lapedes AS, Lin YP, Mosterin A, Obuchi M, Odagiri T, Osterhaus AD, Rimmelzwaan GF, Shaw MW, Skepner E, Stohr K, Tashiro M, Fouchier RA, Smith DJ. Influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses. Vaccine. 2008;26:D31–4.

    Article  PubMed  CAS  Google Scholar 

  15. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406–25.

    PubMed  CAS  Google Scholar 

  16. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. Mapping the antigenic and genetic evolution of influenza virus. Science. 2004;305:371–6.

    Article  PubMed  CAS  Google Scholar 

  17. Swayne DE, Beck JR, Garcia M, Stone HD. Influence of virus strain and antigen mass on efficacy of H5 avian influenza inactivated vaccines. Avian Pathol. 1999;28:245–55.

    Article  Google Scholar 

  18. Swayne DE, Lee CW, Spackman E. Inactivated North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus. Avian Pathol. 2006;35:141–6.

    Article  PubMed  CAS  Google Scholar 

  19. Swayne DE, Garcia M, Beck JR, Kinney N, Suarez DL. Protection against diverse highly pathogenic avian influenza viruses in chickens immunized with a recombinant fowl pox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine. 2000;18:1088–95.

    Article  PubMed  CAS  Google Scholar 

  20. Swayne DE, Pavade G, Hamilton K, Vallat B, Miyagishima K. Assessment of national strategies for control of high-pathogenicity avian influenza and low pathogenicity notifiable avian influenza in poultry, with emphasis on vaccines and vaccination. Rev Sci Tech OffIntEpiz. 2011;30(3):839–70.

    CAS  Google Scholar 

  21. Tosh C, Murugkar HV, Nagarajan S, Bhatia S, Pateriya AK, Behera P, Jain R, Kumar S, Khandia R, Vanamayya PR, Dubey SC, Ahlawat SPS. Outbreak of avian influenza virus H5N1 in India. Vet Rec. 2007;161:279.

    Article  PubMed  CAS  Google Scholar 

  22. Tosh C, Nagarajan S, Murugkar HV, Jain R, Behera P, Katare M, Kulkarni DD, Dubey SC. Phylogenetic evidence of multiple introduction of H5N1 virus in Malda district of West Bengal, India in 2008. Vet Microbiol. 2008;148:2–4.

    Google Scholar 

  23. Wu WL, Chen Y, Wang P, Song W, Lau SY, Rayner JM, Smith GJ, Webster RG, Peiris JSM, Lin T, Xia N, Guan Y, Chen H. Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007. J Virol. 2008;82(4):1798–807.

    Article  PubMed  CAS  Google Scholar 

  24. Yassine HM, Lee CW, Suarez DL, Saif YM. Genetic and antigenic relatedness of H3 subtype influenza A viruses isolated from avian and mammalian species. Vaccine. 2008;26:966–77.

    Article  PubMed  CAS  Google Scholar 

Download references


We are thankful to the Deputy Director General, Education Division, Indian Council of Agricultural Research for providing the necessary funds under National Fellow project and to the Director, Indian Veterinary Research Institute, Indian Council of Agricultural Research for providing necessary infrastructure and other facilities to undertake the work at HSADL, Bhopal.

Author information

Authors and Affiliations


Corresponding author

Correspondence to S. Bhatia.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bhatia, S., Kunal, A., Khandia, R. et al. Genetic and antigenic analysis of H5N1 viruses for selection of HA-donor virus for vaccine strains. Indian J. Virol. 24, 357–364 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: