Since its emergence in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide including Pakistan. During the pandemic, whole genome sequencing has played an important role in understanding the evolution and genomic diversity of SARS-CoV-2. Although an unprecedented number of SARS-CoV-2 full genomes have been submitted in GISAID and NCBI, data from Pakistan is scarce. We report the sequencing, genomic characterization, and phylogenetic analysis of five SARS-CoV-2 strains isolated from patients in Pakistan. The oropharyngeal swabs of patients that were confirmed positive for SARS-CoV-2 through real-time RT-PCR at National Institute of Health, Pakistan, were selected for whole-genome sequencing. Sequencing was performed using NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEW ENGLAND BioLabs Inc., MA, US) and Illumina iSeq 100 instrument (Illumina, San Diego, US). Based on whole-genome analysis, three Pakistani SARS-CoV-2 strains clustered into the 20A (GH) clade along with the strains from Oman, Slovakia, United States, and Pakistani strain EPI_ISL_513925. The two 19B (S)-clade strains were closely related to viruses from India and Oman. Overall, twenty-nine amino acid mutations were detected in the current study genome sequences, including fifteen missense and four novel mutations. Notably, we have found a D614G (aspartic acid to glycine) mutation in spike protein of the sequences from the GH clade. The G614 variant carrying the characteristic D614G mutation has been shown to be more infectious that lead to its rapid spread worldwide. This report highlights the detection of GH and S clade strains and G614 variant from Pakistan warranting large-scale whole-genome sequencing of strains prevalent in different regions to understand virus evolution and to explore their genetic diversity.

SARS‐CoV‐2 variants of concern (VOCs) have emerged worldwide and gained significant importance due to their high transmissibility and global spread, thus meriting close monitoring. In Pakistan, limited information is available on circulation of these variants as the alpha variant has been reported the main circulating lineage. The current study was designed to detect and explore the genomic diversity of SARS‐CoV‐2 lineages circulating during the third wave of the pandemic in the indigenous population. From May 01 to June 09, 2021, a total of 16 689 samples were tested using TaqPath™ COVID‐19 kit for the presence of SARS‐CoV‐2. Overall, 2562 samples (15.4%) were COVID‐19 positive. Out of these positive samples, 2124 (12.7%) did not show the spike gene amplification (spike gene target failure ([SGTF]), whereas 438 (2.6%) showed spike gene amplification (non‐SGTF). A subset (n = 58/438) of non‐SGTF samples were randomly selected for whole‐genome sequencing. Among VOCs, 45% (n = 26/58) were delta, 46% (n = 27/58) were beta, and one was gamma variant. The delta variant cases were reported mainly from Islamabad (n = 15; 58%) followed by Rawalpindi and Azad Kashmir (n = 1; 4% each). Beta variant cases originated mainly from Karachi (n = 8; 30%) and Islamabad (n = 11; 41%) and the gamma variant case was reported in a traveler from Italy. The delta, beta, and gamma variants possessed lineage‐specific spike mutations. Notably, two rare mutations (E484Q and L5F) were found in the delta variant. Furthermore, in the beta variant, two significant rare non‐synonymous spike mutations (A879S and K444R) were also reported. High prevalence of beta and delta variants in local population may increase the number of cases in the near future and provides an early warning to national health authorities to take timely decisions and devise suitable interventions to contain a possible fourth wave.

Pakistan, a dengue-endemic country, has encountered several outbreaks during the past decade. The current study aimed to explore the serotype and genomic diversity of dengue virus responsible for the 2022 outbreak in Pakistan. From August to October 2022, NS-1 positive blood samples (n = 343) were collected from dengue patients, among which, (85%; n = 293) were positive based on RT-PCR. In terms of gender and age, dengue infection was more prevalent in male patients (63%; n = 184), with more adults (21–30 years; n = 94) being infected. The serotyping results revealed DENV-2 to be the most predominant serotype (62%; n = 183), followed by DENV-1 (37%; n = 109) and DENV-3 (0.32%; n = 1). Moreover, a total of 10 samples (DENV-2; n = 8, DENV-1; n = 2) were subjected to whole-genome sequencing. Among these, four were collected in early 2022, and six were collected between August and October 2022. Phylogenetic analysis of DENV-2 sequenced samples (n = 8) revealed a monophyletic clade of cosmopolitan genotype IVA, which is closely related to sequences from China and Singapore 2018, and DENV-1 samples (n = 2) show genotype III, which is closely related to Pakistan isolates from 2019. We also reported the first whole genome sequence of a coinfection case (DENV1-DENV2) in Pakistan detected through a meta-genome approach. Thus, dengue virus dynamics reported in the current study warrant large-scale genomic surveillance to better respond to future outbreaks.

Being an endemic country for Crimean Congo hemorrhagic fever (CCHF), this study aimed to explore the genetic diversity of CCHF virus (CCHFV) detected in Pakistan during 2019. Serum samples from patients with clinical signs of hemorrhagic fever attending tertiary care hospitals in Pakistan were tested for CCHFV RNA using real-time PCR at Department of Virology, National Institute of Health. The partial S-gene fragments were directly sequenced to determine the prevailing CCHFV genotypes and their molecular epidemiology in Pakistan. During January-December 2019, 280 samples from suspected CCHF patients were tested and 28 (10%) were found positive on real-time PCR. Positive cases were detected from 14 districts and across all four provinces of Pakistan with majority reported during August-September. The mean age of CCHFV positive patients was 37.25 years (range 5–65 years) with a high frequency in males (92.8%; n = 26) and a case fatality rate of 40.7% was observed. Phylogenetic analysis showed that S- segment of 2019 PAK CCHFV strains (n = 13) belonged to Asia-1 genotype and clustered with regional strains from Iran, Oman, and Afghanistan. We conclude that Asia-1 genotype of CCHF virus remains endemic in Pakistan. Our findings emphasize to establish a laboratory based surveillance program to monitor the disease burden and identify outbreak hotspots for effective control. Crimean-Congo hemorrhagic fever, a tick-borne viral disease is a zoonotic priority disease in Pakistan. During the current study, we found a mortality rate of 40.7% due CCHF which is high in comparison to studies from regional countries Iran, Afghanistan and Oman. The disease was commonly associated with people involved in animal husbandry and farming however other occupations were also affected. High proportion of cases was observed in the summer months, around the Eid-Al-Ahda festival. Genetic analysis revealed Pakistani CCHF viruses to be closely related with strains from bordering countries Iran and Afghanistan as well as with Oman indicating that animal trade and movement of people serve as a common source for the disease transmission.

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