Volume 25, Number 12—December 2019

Research Letter

Laboratory-Confirmed Avian Influenza A(H9N2) Virus Infection, India, 2019


A 17-month-old boy in India with severe acute respiratory infection was laboratory confirmed to have avian influenza A(H9N2) virus infection. Complete genome analysis of the strain indicated a mixed lineage of G1 and H732. The strain also was found to be susceptible to adamantanes and neuraminidase inhibitors.

Low-pathogenicity avian influenza A(H9N2) viruses have a wide host range, and outbreaks in poultry have been recorded since the 1990s in China (1). In India, avian specimens indicated no serologic evidence of H5N1 and H9N2 during 1958–1981 (2); however, 5%–6% persons with direct exposure to poultry had H9N2 antibodies (3). Human cases of influenza H9N2 virus infection have been observed in Hong Kong, China, Bangladesh, and Pakistan (47).

An institutional review board approved an ongoing community-based surveillance in 93 villages of Korku tribes in Melghat District, Maharashtra State, India to determine incidence of respiratory syncytial virus (RSV)–associated deaths among children >2 years of age. A total of 2,085 nasopharyngeal swabs from children with severe or fatal pneumonia were transported to India’s National Institute of Virology to test for influenza, RSV, and other respiratory viruses. A nasopharyngeal swab from a 17-month-old boy received on February 12, 2019, tested positive by PCR for influenza A(H9N2) virus.

The child, a resident of Melghat, had fever, cough, breathlessness, and difficulty feeding for 2 days since illness onset on January 31, 2019. His high intermittent grade fever had no diurnal variation and no association with rash or mucocutaneous lesions. Examination revealed a conscious, restless child with a respiratory rate of 48 breaths/min and lower chest wall in-drawing with intermittent absence of breathing for >20 seconds. He was fully immunized for his age, with bacillus Calmette–Gu?rin, diphtheria, hepatitis B, poliovirus, and measles vaccines. Both length and weight for age were less than −3 SD. History of travel with his parents to a local religious gathering 1 week before symptom onset was elicited. The father had similar symptoms on return from the gathering but could not undergo serologic testing because of his migrant work. No history of poultry exposure was elicited. The child received an antibacterial drug and antipyretics and recovered uneventfully.

We tested the clinical sample using duplex real-time PCR for influenza A/B, H3N2, and 2009 pandemic H1N1 viruses; RSV A/B; human metapneumovirus; parainfluenza viruses types 1–4, rhinovirus; and adenovirus. The sample was strongly positive for influenza A virus (cycle threshold value 20) but negative for seasonal influenza viruses and all respiratory viruses. Real-time PCR analysis for avian influenza viruses H5N1, H7N9, H10N8, and H9N2 revealed positivity for H9N2 virus (cycle threshold value for H9 was 25). We confirmed this result by sequencing the matrix (M) and hemagglutinin (HA) genes of the isolate, designated A/India/TCM2581/2019/(H9N2); the M gene (260 bp) had 97.27% nucleotide identity with A/chicken/India/99321/2009(H9N2), and the HA gene (225,478 bp) had 96.93% nucleotide identity with A/chicken/India/12CL3074/2015(H9N2).

Figure. Phylogenetic tree of hemagglutinin gene (A) and neuraminidase gene (B) gene of influenza virus A/India/TCM 2581/2019(H9N2) from India (black circle) and reference strains. The numbers above the branches are the bootstrap...

We then generated whole-genome sequences by using the Miniseq NGS Platform (Illumina, https://www.illumina.comExternal Link) and a de novo assembly program (CLC Genomics Software 10.1.1 [8]). We used MEGA7 (https://megasoftware.netExternal Link) with a Tamura-Nei nucleotide substitution model including 1,000 replicates bootstrap support (9) for evolutionary analysis of 8 genes of A/India/TCM2581/2019/(H9N2) (submitted to GenBank under accession nos. MK673893–900). The HA, neuraminidase, and nucleoprotein gene phylogeny of A/India/TCM 2581/2019/(H9N2) grouped with the dominant G1 lineage (h94.1.1) and clustered with poultry strains from India and human strains from Bangladesh (Figure). The M, nonstructural, polymerase basic 1, polymerase basic 2, and polymerase acidic genes were related to an H7N3 isolate from Pakistan (10) (Appendix Figure 1). We confirmed that the A/India/TCM2581/2019(H9N2) strain had low pathogenicity, showing a KSKR/GLF amino acids motif at the cleavage site of HA (335−341 [H9 numbering]). We observed 6 potential glycosylation sites (11, 87, 123, 280, 287, and 472 [H9 numbering]) and loss of 2 sites (208 and 218 [H9 numbering]) in the HA gene of A/India/TCM2581/2019(H9N2) with respect to G1 viruses.

The virus was susceptible to adamantanes with S31 and to neuraminidase inhibitor with R292 and E119 (N2 numbering) (11). A/India/TCM2581/2019(H9N2) had Q226L and I155T in HA gene, which promote the human receptor binding. Compared with G1 vaccine strain A/Hong Kong/1073/99, the study strain had multiple mammalian-specific mutations that already exist in poultry-adapted H9N2. The study strain had amino acid changes R207K, H436Y, and M677T in the polymerase basic 1 gene; A515T in the polymerase acidic 1 gene; N30D, T215A, and T139A (all H3 numbering) in the matrix 1 gene; and P42S in the nonstructural 1 gene, all of which are known to be associated with mammalian host specificity and increased virulence in ferrets and mice (12). Known markers for virulence and transmission (E627K and D701N) in the polymerase basic 2 gene in the study strain were absent (Appendix Table 1).

Bayesian evolutionary analyses using BEAST version 1.8.1 (13) of the HA gene of H9N2 poultry strains from India indicated 3 clusters of multiple introductions at the estimated node age of 2000–2001 (Appendix Figure 2). Human strain A/India/TCM2581/2019(H9N2) and the other poultry viruses from India evolved with 5.163 ? 10–3 substitutions/site/year.

In conclusion, multiple introductions of H9N2 viruses in poultry have been observed in India. The identification of a human case of H9N2 virus infection highlights the importance of systemic surveillance in humans and animals to monitor this threat to human health.

Dr. Potdar is senior scientist heading the Influenza Group at the National Institute of Virology, Pune, India. Her primary research interest is molecular characterization and antiviral susceptibility of influenza viruses.



We thank Devendra Mourya for his valuable suggestions and support during the study. We also thank Niteen Wairagkar for critical review of the project. We acknowledge the health workers from Mahan Trust Melghat for their help in collecting clinical information and sample collection. We also acknowledge Sumit Bhardwaj and the staff of the Influenza Group and Biosafety Level 4 Laboratory at the National Institute of Virology for scientific and technical support for influenza testing and next-generation sequencing. Finally, we acknowledge the state Integrated Disease Surveillance Programme in Maharashtra for carrying out the preliminary house-to-house investigation.

Financial support was provided by the Bill and Melinda Gates foundation (grant no. OPP1128488) to E.A.F.S.


  1. Malik Peiris JS. Avian influenza viruses in humans. Rev Sci Tech. 2009;28:161–73. DOIExternal LinkPubMedExternal Link
  2. Pawar SD, Jamgaonkar AV, Umarani UB, Kode SS. Seroepidemiology of avian influenza H5N1, H9N2 & Newcastle disease viruses during 1954 to 1981 in India. Indian J Med Res. 2016;144:472–6. DOIExternal LinkPubMedExternal Link
  3. Pawar SD, Tandale BV, Raut CG, Parkhi SS, Barde TD, Gurav YK, et al. Avian influenza H9N2 seroprevalence among poultry workers in Pune, India, 2010. PLoS One. 2012;7:e36374. DOIExternal LinkPubMedExternal Link
  4. Pan Y, Cui S, Sun Y, Zhang X, Ma C, Shi W, et al. Human infection with H9N2 avian influenza in northern China. Clin Microbiol Infect. 2018;24:321–3. DOIExternal LinkPubMedExternal Link
  5. Butt KM, Smith GJ, Chen H, Zhang LJ, Leung YH, Xu KM, et al. Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J Clin Microbiol. 2005;43:5760–7. DOIExternal LinkPubMedExternal Link
  6. Shanmuganatham K, Feeroz MM, Jones-Engel L, Smith GJ, Fourment M, Walker D, et al. Antigenic and molecular characterization of avian influenza A(H9N2) viruses, Bangladesh. Emerg Infect Dis. 2013;19:19. DOIExternal LinkPubMedExternal Link
  7. Ali M, Yaqub T, Mukhtar N, Imran M, Ghafoor A, Shahid MF, et al. Avian influenza A(H9N2) virus in poultry worker, Pakistan, 2015. Emerg Infect Dis. 2019;25:136–9. DOIExternal LinkPubMedExternal Link
  8. Mourya DT, Yadav PD, Nyayanit DA, Majumdar TD, Jain S, Sarkale P, et al. Characterization of a strain of quaranfil virus isolated from soft ticks in India. Is quaranfil virus an unrecognized cause of disease in human and animals?". Heliyon. 2019;5:e01368. DOIExternal LinkPubMedExternal Link
  9. Iqbal M, Yaqub T, Reddy K, McCauley JW. Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses. PLoS One. 2009;4:e5788. DOIExternal LinkPubMedExternal Link
  10. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33:1870–4. DOIExternal LinkPubMedExternal Link
  11. Govorkova EA, Baranovich T, Seiler P, Armstrong J, Burnham A, Guan Y, et al. Antiviral resistance among highly pathogenic influenza A (H5N1) viruses isolated worldwide in 2002-2012 shows need for continued monitoring. Antiviral Res. 2013;98:297–304. DOIExternal LinkPubMedExternal Link
  12. Li X, Shi J, Guo J, Deng G, Zhang Q, Wang J, et al. Genetics, receptor binding property, and transmissibility in mammals of naturally isolated H9N2 Avian Influenza viruses. PLoS Pathog. 2014;10:e1004508. DOIExternal LinkPubMedExternal Link
  13. Potdar VA, Hinge DD, Dakhave MR, Manchanda A, Jadhav N, Kulkarni PB, et al. Molecular detection and characterization of Influenza ‘C’ viruses from western India. Infect Genet Evol. 2017;54:466–77. DOIExternal LinkPubMedExternal Link

Top Figure


Suggested citation for this article: Potdar V, Hinge D, Satav A, Sim?es EF, Yadav PD, Chadha MS. Laboratory-confirmed avian influenza A(H9N2) virus infection, India, 2019. Emerg Infect Dis. 2019 Dec [date cited]. https://doi.org/10.3201/eid2512.190636

DOI: 10.3201/eid2512.190636

Original Publication Date: 11/6/2019