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Identification of Human Case of Avian Influenza A(H5N1) Infection, India

A 11-year-old boy with acute myeloid leukemia was brought for treatment of severe acute respiratory infection in the National Capital Region, New Delhi, India. Avian influenza A(H5N1) infection was laboratory confirmed. Complete genome analysis indicated hemagglutinin gene clade 2.3.2.1a. We found the strain to be susceptible to amantadine and neuraminidase inhibitors.


The Study
An 11-year old boy who had acute myeloid leukemia diagnosed in June 2021 in the department of pediatrics at the All India Institute of Medical Sciences (AIIMS) in New Delhi was brought in for treatment of fever, cough, coryza, and breathing difficulty in the first week of July 2021. The patient was a resident of Gurugram, National Capital Region (Delhi), India. His clinical work-up showed febrile neutropenia with pneumonia and shock, which progressed to acute respiratory distress syndrome, so he was mechanically ventilated. He then developed multiorgan dysfunction, which ultimately resulted in his death on July 12, 2021. An in-depth interview with family members indicated that the patient often frequented a family-owned poultry business and may have been exposed to birds with undetected infection, although no infected domestic or wild avian sources or any environmental contamination had been reported in or around the residence of the child.
Staff in the AIIMS department of pediatrics referred nasopharyngeal (NP) swab specimens collected on July 7 and bronchoalveolar lavage (BAL) fluid collected on July  We then generated whole-genome sequences from the original clinical NP samples and BAL-and MDCKgrown for passages 1 and 2 isolates using the Miniseq NGS Platform (Illumina, https://www.illumina.com) and a de-novo assembly program using QIAGEN CLC Genomics software 10.1.1 (https://www.quiagen. com). We constructed phylogenetic trees for 8 genes of A/India/NIV-SARI-4571/2021 (H5N1) virus using the neighbor-joining model with a Tamura-Nei nucleotide substitution performing 1,000 replicates of bootstrap support implemented in MEGA 7 (https:// megasoftware.net) software. We submitted sequences to GenBank (accession nos. OL311384-91).
The sequences generated for the original clinical sample and the passaged virus were identical, suggesting no passage-induced mutations. We performed BLAST analysis of all 8 genes of A/India/NIV-SA-RI-4571/2021. The HA gene showed 97% identity with A/duck/Bangladesh/32003/2017 (H5N1). The polymerase basic (PB) 1 and nonstructural genes showed 97% nucleotide similarity to the avian influenza A/duck/Mongolia/729/2019 (H4N6) virus, suggesting probable reassortment.
Using the WHO/CDC H5N1 inventory (11), we reviewed potential markers for the newly identified A/India/NIV-SARI-4571/2021 strain. The HA protein (H5) possessed a multiple basic amino acid cleavage site motif (PQRERRRKR*G), resulting in a highly pathogenic strain of clade 2.3.2.1 viruses. The sequence of the 220-loop receptor-binding site between amino acids Q222 and G224 remains conserved for the avian α 2-3 receptor.
We did not observe the NA and matrix 2 gene mutations responsible for neuraminidase inhibitors and amantadine resistance in the study strain. The virus remains purely avian-adapted, and we observed no markers for adaptation in mammals or pathogenicity for humans (Appendix Figure 2). We also did not observe PB2 hallmark mutations E627K and D701N, responsible for host adaptation and virulence in mammals in the study strain (12). The PDZ ligand domain (ESEV) at the C terminus remained conserved. We observed further compensatory mutations during the adaptation of H5N1 in mice, L89V, G309D, T339K, R477G, and I495V of PB2 (13) in the study strain. However, further studies are required to understand their role.
No evidence of H5 antibodies was detected in 18 close contacts of the deceased child. Available information and initial field investigations revealed that no additional cases were detected, indicating low human-to-human transmission. However, unreported high-pathogenicity avian influenza virus continues to exist in traded poultry in India, constituting a substantial risk for further human exposure (https:// www.who.int/emergencies/disease-outbreaknews/item/human-infection-with-avian-influenzaa(h5n1)-%EF%BD%B0-india). Although widespread avian influenza outbreaks have been documented globally, only a limited number have shown transmission of avian influenza viruses to humans (14). The severe immunocompromised status of the child in this study may have made him vulnerable, and direct exposure to infected poultry might have been the source of infection. Influenza B virus, simultaneously detected with influenza A in this case-patient, is known to persist in such cases and might have been identified because of a prior infection (15).

Conclusions
In December 2020 and early 2021, outbreaks of avian influenza H5N1 and H5N8 were reported in poultry in 15 states in India; the National Capital Region, Maharashtra, Punjab, and Kerala, in particular, were severely affected. The whole-genome sequencing of A/India/NIV-SARI-4571/2021 (H5N1) virus in our study provides valuable insight into the absence of hallmark mutations responsible for adaptation and virulence in mammals. The strain remained sensitive to amantadine and neuraminidase inhibitors. However, identification of a human H5N1 case in India highlights the need for systemic surveillance at the human-animal interface level.