Emergence of influenza A(H1N1)pdm09 genogroup 6B a

Eurosurveillance, Volume 21, Issue 5, 04 February 2016

Rapid communication

Emergence of influenza A(H1N1)pdm09 genogroup 6B and drug resistant virus, India, January to May 2015



To investigate the aetiology of the 2015 A(H1N1)pdm09 influenza outbreak in India, 1,083 nasopharyngeal swabs from suspect patients were screened for influenza A(H1N1)pdm09 in the state of Madhya Pradesh. Of 412 positive specimens, six were further characterised by phylogenetic analysis of haemagglutinin (HA) sequences revealing that they belonged to genogroup 6B. A new mutation (E164G) was observed in HA2 of two sequences. Neuraminidase genes in two of 12 isolates from fatal cases on prior oseltamivir treatment harboured the H275Y mutation.

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An epidemic of influenza A(H1N1)pdm09, affecting over 39,000 persons and causing more than 2,500 deaths occurred in India in 2015 [1]. We show that genotype 6B strains forming two sub-lineages circulated during the outbreak. Comparison of the sequences of six outbreak strains recovered in this work, to other published genotype 6B sequences, also reveals a unique combination of previously-reported mutations in the haemagglutinin (HA) gene. Two of the six sequences additionally display a E164G mutation in HA2, which has not been reported to date, moreover a N129D mutation in HA1 is observed for two sequences derived from patients with severe disease. Among strains analysed from 12 fatal cases on prior oseltamivir treatment, two harbour the H275Y mutation in the neuraminidase (NA) gene, which confers resistance to this antiviral.


Description of the study


Sampling and testing for influenza A(H1N1)pdm09

A total of 1,083 acute phase nasopharyngeal swab specimens from patients suspected of influenza (as prior defined [2]), were referred by 13 district health authorities of Madhya Pradesh, India between 29 January and 7 May 2015. Upon specimen collection, the travel history, treatment status, and symptoms of the patients were recorded in addition to age, sex and place of residence. The samples were handled in a designated biosafety level (BSL) 3 laboratory and viral RNA was extracted using QIAamp viral RNA mini kit (Qiagen). The RNA samples were screened by World Health Organization (WHO)–Centers for Disease Control and Prevention (CDC) approved quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) for influenza A(H1N1)pdm09 [3].


Molecular analyses of the strains

Six clinical samples testing positive for influenza A(H1N1)pdm09 by qRT-PCR were selected based on patients’ disease severity category A (n=2; A/India/DRDE_GWL897/2015 and A/India/DRDE GWL721/2015), B (n=2; A/India/DRDE_GWL703/2015, A/India/DRDE_GWL989/2015), and C (n=2; A/India/DRDE GWL719/2015 and A/India/DRDE_GWL812/2015) as previously described [2], and used for direct nucleotide (nt) sequencing of the haemagglutinin (HA) gene. A phylogenetic analysis was performed by comparing with nt sequence of 45 globally diverse influenza A(H1N1)pdm09 viruses retrieved from GenBank (as further shown in the phylogenetic tree) and the Global Initiative on Sharing Avian Influenza Data (GISAID) (Table 1). The phylogenetic tree in this analysis was constructed with maximum likelihood and bootstrap analysis of 1,000 replicates using Mega 5.03 software [4]. Further the amino acid substitutions were marked at the major branches for better clarity.

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