Whole-genome sequencing of SARS-CoV-2 reveals the detection of G614 variant in Pakistan

Abstract

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.

Fig 1. Daily reported COVID-19 cases and deaths in Pakistan since March 2020 to October 16, 2020.

The data of confirmed positive cases and deaths were obtained from Government of Pakistan dashboard (https://covid.gov.pk/stats/pakistan).

Fig 2. Inference of SARS-CoV-2 phylogenetic relationships.

The phylogenetic tree was reconstructed using Maximum Likelihood method with bootstrap 1000 replicates (A). Globally subsampled SARS-CoV-2 genomes supplemented with additional complete genomes from countries surrounding Pakistan were included. Pakistani strains are highlighted with grey arrows. B) Expansion of clade containing sequences NIH-44905 and NIH-HAS001. C) Expansion of clade containing sequences NIH-45090, NIH-45579, and NIH-45143.

Fig 3. Comparison of SARS-CoV-2 isolates sequenced in the current study with the prototype strain and Pakistan isolates.

The nucleotide changes are shown in A and the amino acid variations are presented in B.