The SARS‐CoV‐2 coronavirus is responsible for the current COVID‐19 pandemic, with an ongoing toll of over 5 million infections and 333 thousand deaths worldwide within the first 5 months. Insight into the phylodynamics and mutation variants of this virus is vital to understanding the nature of its spread in different climate conditions. The incidence rate of COVID‐19 is increasing at an alarming pace within subtropical Southeast Asian nations with high temperatures and humidity. To understand this spread, we analyzed 444 genome sequences of SARS‐CoV‐2 available on the GISAID platform from 6 Southeast Asian countries. Multiple sequence alignments and maximum likelihood phylogenetic analyses were performed to analyze and characterize the non‐synonymous mutant variants circulating in this region. Global mutation distribution analysis showed that the majority of the mutations found in this region are also prevalent in Europe and North America, and the concurrent presence of these mutations at a high frequency in other countries indicate possible transmission routes. Unique spike protein and non‐structural protein mutations were observed circulating within confined area of a given country. We divided the circulating viral strains into 4 major groups and 3 sub‐groups on the basis of the most frequent non‐synonymous (NS) mutations. Strains with a unique set of 4 co‐evolving mutations were found to be circulating at a high frequency within India, specifically. Group 2 strains characterized by two co‐evolving NS mutants which alter in RdRp (P323L) and spike (S) protein (D614G), were found to be common in Europe and North America. These European and North American variants have rapidly emerged as dominant strains within Southeast Asia, increasing from a 0% prevalence in January to an 81% by May 2020. These variants may have an evolutionary advantage over their ancestral types and could present a large threat to Southeast Asia for the coming winter.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is continuously spreading worldwide at an unprecedented scale in 2020. Within the first six months of the COVID-19 pandemic, it has evolved into six clades according to GISAID where three (G, GH, and GR) are now globally prevalent (>75%). Here we report the prevalence of these dominant clades, both individually and in combination, with disease progression and death-case scenario that leads to infer fitness of the SARS-CoV-2 by compromising its virulence. Unlike G or GH clades, the GR clade strains represent a significant negative association with the death-case ratio (R= -0.558, p=0.019). Docking analysis revealed the molecular scenario behind more infectiousness of S protein D614G mutation and reasoned more favorable binding of G614 with the elastase-2. Viral RNA-dependent-RNA-polymerase (RdRp) mutation p.P323L facilitated significantly higher (p<0.0001) genome-wide mutations because more flexible RdRp (mutant)-NSP8 interaction may accelerate replication. Superior RNA stability and structural variation at NSP3:C241T might change the protein’s conformation with a speculated impact on 5'UTR, nucleocapsid, and replication complex interactions. Another silent 5'UTR:C241T mutation might affect translational efficiency and viral packaging. These G-featured coevolving mutations might together increase the viral load, quicker cell death, and potentially a stronger immune response within the host, hence can modulate intra-host genomic plasticity. In addition, viroporin ORF3a:p.Q57H mutation of GH-clade prevents ion permeability by constricting the channel pore more tightly due to additional ionic interaction with the cysteine (C81) of transmembrane-domain-2, which possibly reduces viral release and immune response. GR strains (four G clade mutations with N:p.RG203-204KR) would have maintained more stability with stronger RNA interaction, a flexible linker region, and the molecular effect of hypo-phosphorylation at SR-stretch. These empirical assumptions need further retrospective and prospective studies to understand detailed molecular and evolutionary events featuring the fitness and virulence of SARS-CoV-2.

Tracing the globally circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) phylogenetic clades by high-throughput sequencing is costly, timeconsuming, and labor-intensive. We here propose a rapid, simple, and cost-effective amplification refractory mutation system (ARMS)-based multiplex reverse-transcription polymerase chain reaction (PCR) assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. Our multiplex PCR is designed in a mutually exclusive way to identify V-S and G-GH-GR clade variants separately. The pentaplex assay included all five variants and the quadruplex comprised of the triplex variants alongside either V or S clade mutations that created two separate subsets. The procedure was optimized with 0.2-0.6 µM primer concentration, 56-60°C annealing temperature, and 3-5 ng/µl complementary DNA to validate on 24 COVID-19-positive samples. Targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of primers. This multiplex ARMS-PCR assay is a fast, low-cost alternative and convenient to discriminate the circulating phylogenetic clades of SARS-CoV-2.

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The mortality of coronavirus disease 2019 (COVID‐19) disease is very high among the elderly or individuals having comorbidities such as obesity, cardiovascular diseases, lung infections, hypertension, and/or diabetes. Our study characterizes the metagenomic features in severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2)‐infected patients with or without type 2 diabetes, to identify the microbial interactions associated with its fatal consequences.This study compared the baseline nasopharyngeal microbiome of SARS‐CoV‐2‐infected diabetic and nondiabetic patients with controls adjusted for age and gender. The metagenomics based on next‐generation sequencing was performed using Ion GeneStudio S5 Series and the data were analyzed by the Vegan‐package in R. All three groups possessed significant bacterial diversity and dissimilarity indexes (p < 0.05). Spearman's correlation coefficient network analysis illustrated 183 significant positive correlations and 13 negative correlations of pathogenic bacteria (r = 0.6–1.0, p < 0.05), and 109 positive correlations between normal flora and probiotic bacteria (r > 0.6, p < 0.05). The SARS‐CoV‐2 diabetic group exhibited a significant increase in pathogens and secondary infection‐causing bacteria (p < 0.05) with a simultaneous decrease of normal flora (p < 0.05). The dysbiosis of the bacterial community might be linked with severe consequences of COVID‐19‐infected diabetic patients, although a few probiotic strains inhibited numerous pathogens in the same pathological niches. This study suggested that the promotion of normal flora and probiotics through dietary supplementation and excessive inflammation reduction by preventing secondary infections might lead to a better outcome for those comorbid patients.