Genomic surveillance of Influenza, SARS-CoV-2, and RSV in patients from Islamabad and Rawalpindi, Pakistan: a 2023-24 perspective.

Respiratory viruses, including influenza, SARS-CoV-2, and RSV, pose significant public health challenges in low-and-middle-income countries like Pakistan. This study, conducted from January-2023 to January-2024, investigated SARS-CoV-2, Influenza virus and RSV infections in individuals with respiratory symptoms and explored the genomic diversity of these viruses in Pakistan. This study analyzed 320 oropharyngeal/nasopharyngeal swabs, of which 57.1% (n = 183) tested positive for respiratory viruses using RT-PCR, including Influenza virus (59%; n = 108), SARS-CoV-2 (30%; n = 54), and RSV (11%; n = 21). Whole-genome sequencing was performed on 100 samples with Ct-value < 30, yielding 85 complete genomes: 48.24% (n = 41) were Influenza A (H3N2: 87.80%, n = 36; H1N1pdm09: 12.20%, n = 5), 45.88% (n = 39) were SARS-CoV-2 Omicron variants, and 5.88% (n = 5) were RSV-B. H3N2 sequences clustered mainly in clade 3 C.2a1b.2a.2a.3a.1 (94.4%, n = 34/36) with 99.32%–99.56% nucleotide identity to 2023 strains from Russia, USA, UK, and Pakistan, and 1.05%–1.87% divergence from the vaccine strain A/Thailand/8/2022. Key NA gene mutations included E50K, T3A in the signal peptide, and R150H, that have implications on antiviral resistance. H1N1 sequences, confined to clade 6B.1 A.5a.2a, showed high similarity (99.41%–100%) with 2022–2023 USA and Pakistan strains but increased divergence (1.44%–1.66%) from vaccine strain A/Wisconsin/588/2019, with significant mutations (K54Q, D94N, E224A) at antigenic sites., that can potentially enhance viral fitness. The SARS-CoV-2 Omicron subvariants included GW.5, XBB.1.22, and FL, closely resembling sequences from England, Singapore, and Canada. All RSV-B sequences belonged to the GB5.0.5a G_clade, with 97.8% to 99.5% similarity to strains from England, Australia, Bangladesh, Senegal, and the USA. Notably, RSV-B exhibited a deletion (L250 to I268) in the G protein and key mutations in the F protein (S190N, S211N, and L173del), which could affect therapeutic and vaccine efficacy. These findings highlight the need for integrated surveillance of these viruses to inform public health responses.