Genomic Diversity and Evolution of SARS-CoV-2 Lineages in Pakistan.

The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.