Duration and Frequency of Shedding of Influenza Virus H9N2 Subtype by Infected Birds based on an Experimental Study

Document Type: Original Article


1 Assistant Professor of Avian Medicine, Department of Clinical Science, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

2 MPH, Leishmaniosis Research Center, Kerman University of Medical Sciences, Kerman, Iran

3 Graduate Student of Veterinary Medicine


Background & Aims: The H9N2 avian influenza subtype is endemic in many parts of Iran and has the ability to transmit from bird to human. In the present study, the risk of this subtype for humans was assessed by determination of the viral titer and shedding time in bird.
Method: The experiment was done on fifteen male budgerigars at 4 months of age. The birds were inoculated intranasally with allantoic fluid containing 106 EID50/ml of H9N2 virus. At days 1,3,5 and 7 post inoculation, viral presence and titer in the pharynx and cloacal were determined using TaqMan-real time-PCR.
Results:The study showed that following infection of companion birds, viral shedding from pharyngeal and cloacal secretions can start one day after infection continued up to 7 days. In the early days, the viral titer in pharyngeal secretions is very high and reduces over time, while in the dropping of affected bird, it is low in the early days and increases, over time. Maximum viral shedding in the pharyngeal and fecal exertion was seen on days 3 and 5, respectively.
Conclusion: The results of the present study showed that the H9N2 subtype sheds from pharyngeal and cloacal secretions and releases in the environment. Due to the zoonotic nature of the influenza disease and the increasing tendency of people to keep birds, informing the public about the disease, duration of shedding, risk for human and strategies of disease prevention is necessary.


  1. Liu J, Xiao H, Wu Y, Liu D, Qi X, Shi Y, et al. H7N9: a low pathogenic avian influenza A virus infecting humans. Curr Opin Virol 2014; 5(3): 91-7.
  2. Fusaro A, Monne I, Salviato A, Valastro V, Schivo A, Amarin NM, et al. Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications. J Virol 2011; 85(16): 8413-21.
  3. Butt AM, Siddique S, Idrees M, Tong Y. Avian influenza A (H9N2): computational molecular analysis and phylogenetic characterization of viral surface proteins isolated between 1997 and 2009 from the human population. Virol J 2010; 7(1): 319.
  4. Xiong X, Martin SR, Haire LF, Wharton SA, Daniels RS, Bennett MS, et al. Receptor binding by an H7N9 influenza virus from humans. Nature 2013; 499(7459): 496-9.
  5. Osterholm MT, Kelley NS, Sommer A, Belongia EA. Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. The Lancet infectious diseases 2012; 12(1): 36-44.
  6. Brien S, Kwong JC, Buckeridge DL. The determinants of 2009 pandemic A/H1N1 influenza vaccination: a systematic review. Vaccine 2012; 30(7): 1255-64.
  7. Rasmussen SA, Kissin DM, Yeung LF, Mac Farlane K, Chu SY, Turcios-Ruis RM, et al. Preparing for influenza after 2009 H1N1: special considerations for pregnant women and newborns. Am j Obstetr Gynecol 2011; 204(6): S13-S20.
  8. Moghadaszadeh M, Golchin M, Tavakkoli H, Ghambarpour R. Cloning, expression and purification of M2e-HA2 from Influenza A virus in Escherichia coli. Online Journal of Veterinary Research 2015; 19(2): 124-9.
  9. Price GE, Lo C-Y, Misplon JA, Epstein SL. Mucosal immunization with a candidate universal influenza vaccine reduces virus transmission in a mouse model. J Virol 2014; 88(11): 6019-30.
  10. Tavakkoli H, Asasi K, Mohammadi A. Effectiveness of two H9N2 low pathogenic avian influenza conventional inactivated oil emulsion vaccine on H9N2 viral replication and shedding in broiler chickens Iranian Journal of Veterinary Research 2011; 12(3): 214-21.
  11. Fallah Mehrabadi MH, Bahonar MR, Zeynolabedini Tehrani F, Mardani M, Sadrzadeh A, Ghafouri SA, et al. Seroepidemiology of Avian Influenza (H9N2) in Rural Domestic Poultry of Iran: A Cross-Sectional Study. Iranian Journal of Epidemiology 2015; 10(4): 1-9.
  12. Tavakkoli H. Replication of H9N2 avian influenza virus in lungs and kidneys after inoculation in broilers. Online Journal of Veterinary Research 2012; 16(6): 321-6.
  13. Tavakkol H, Kheirandish R. Assessment of the shedding pattern of a chicken origin H9N2 avian influenza subtype in ostrich (Struthio camelus) using TaqMan real time PCR. Online Journal of Veterinary Research 2012; 16(4): 266-73.
  14. Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. The American Journal of Higiene 1938; 27(3): 493-7.
  15. Ward C, Dempsey MH, Ring CJ, Kempson RE, Zhang L, Gor D, et al. Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. J Clin Virol 2004; 29(3): 179-88.
  16. Ebrahimi M, Grigorian S, Shoushtari A, Abedini F, Moeini H. Sequence analysis and phylogenetic profiling of the nonstructural (NS) genes of H9N2 influenza A viruses isolated in Iran during 1998-2007. Archives of Razi Institute 2014; 69(2): 127-35.
  17. Bashashati M, Vasfi Marandi M, Sabouri F. Genetic diversity of early (1998) and recent (2010) avian influenza H9N2 virus strains isolated from poultry in Iran. Arch Virol 2013; 158(10): 2089-100.
  18. Kianizadeh M, Pourbakhsh S.A, Toroghi R, Momayez R. Pathogenicity and haemagglutinin gene sequence analysis of Iranian avian influenza H9N2 viruses isolated during (1998–2001). Iranian Journal of Veterinary Research 2006; 7(3): 37-41.
  19. Soltanialvar M, Shoushtari H, Bozorgmehrifard M, Charkhkar S, Akbarnejad F. Sequence and phylogenetic analysis of neuraminidase genes of H9N2 avian influenza viruses isolated from commercial broiler chicken in Iran (2008 and 2009). Trop Anim Health Prod 2012; 44(3): 419-25.
  20. Haghighat-Jahromi M, Asasi K, Nili H, Dadras H, Shooshtari AH. Coinfection of avian influenza virus (H9N2 subtype) with infectious bronchitis live vaccine. Arch Virol 2008; 153(4): 651-5.
  21. Tavakkoli H, Salehi M. Low pathogenic avian influenza in zebra finch (Taeniopygia guttata): clinical signs, replication and excretion time. Human & Veterinary Medicine 2015; 7(3): 221-4.
  22. Tavakkoli H, Mosallanejad S, Noori S. Heterologous H9N2 avian influenza viral shedding pattern in Alectoris chukar. Online Journal of Veterinary Research 2013; 17(10): 566-70.
  23. Ebrahimi SM, Ziapour S, Tebianian M, Dabaghian M, Mohammadi M. Study of infection with an Iranian field-isolated H9N2 avian influenza virus in vaccinated and unvaccinated Japanese quail. Avian Dis 2011; 55(2): 195-200.
  24. Hadipour MH. H9N2 avian influenza virus antibody titers in human population in fars province, Iran. Revista Brasileira de Ciência Avícola 2010; 12(3): 160-4.
  25. Hadipour MM. Seroprevalence of H9N2 avian influenza virus in human population in Boushehr Province, Iran. Asian Journal of Animal and Veterinary Advances 2011; 6(2): 196-200.
  26. Alizadeh E, Kheiri MT, Bashar R, Tabatabaeian M, Hosseini SM. Avian Influenza (H9N2) among poultry workers in Iran. Iranian Journal of Microbiology 2009; 1(3): 3-6.
  27. Anvar E, Hosseini SM, Tavasoti Kheiri M, Mazaheri V, Fazaei K, Shabani M, et al. Serological survey of avian influenza (H9N2) among different occupational groups in Tehran and Qazvin provinces in IR Iran. Jundishapur Journal of Microbiology 2013; 6(4):
  28. Wang Q, Ju L, Liu P, Zhou J, Lu X, Li L, et al. Serological and virological surveillance of avian influenza A virus H9N2 subtype in humans and poultry in Shanghai, China, between 2008 and 2010. Zoonoses and public health 2015; 62(2): 131-40.
  29. Edge R, Heath J, Rowlingson B, Keegan T, Isba R. Seasonal influenza in medical students: an outbreak simulation model based on a social network approach. The Lancet 2014; 384(S29): 29-30.
  30. Gomaa MR, et al. Avian influenza A (H5N1) and A (H9N2) seroprevalence and risk factors for infection among Egyptians: a prospective, controlled seroepidemiological study. Journal of Infectious Diseases 2014; 211(9): 1399-407.
  31. Huang Y, Li X, Zhang H, Chen B, Giang Y, Yang L, et al. Human infection with an avian influenza A (H9N2) virus in the middle region of China. Journal of Medical Virology 2015; 87(10): 132-9.
  32. Khan SU, Anderson BD, Heil GL, Liang S, Gray GC. A Systematic Review and Meta-Analysis of the Seroprevalence of Influenza A (H9N2) Virus Infection Among Humans. J Infect Dis 2015; 212(4): 109-15.
  33. Trombetta C, Piccirella S, Perini D, Kistner O, Montomoli E. Emerging Influenza Strains in the Last Two Decades: A Threat of a New Pandemic? Vaccines 2015; 3(1): 172-85.
  34. Heithoff DM, Mahan MJ. Pandemic influenza virus: Tracking a Three-Headed Monster. Virulence 2015; 4(2): 112-119.