Genomic analysis of SARS-CoV-2 variants of concern identified from the ChAdOx1 nCoV-19 immunized patients from Southwest part of Bangladesh (preprint)

IntroductionBangladesh introduced ChAdOx1 nCoV-19 since February, 2021 and in six months, only a small population (3.5%) received their first dose of vaccination like other low-income countries. The remaining populations are struggling with increased rate of infection due to beta and delta variants. Although this uncontrolled COVID-19 pandemic did not leave even the immunized group because of immune escaping capacity of those new variants. MethodsA total of 4718 nasopharygeal samples were collected from 1st March until 15th April, 2021, of which, 834 (18%) were SARS-CoV-2 positive. Randomly generated 135 positive cases were selected for telephone interview and 108 were available and provided consent. The prevalence of SARS-CoV-2 variants and disease severity among both immunized and unimmunized group was measured. A total of 63 spike protein sequence and 14 whole genome sequences were performed from both groups and phylogenetic reconstruction and mutation analysis were compared. ResultsA total of 40 respondents (37%, N=108) received single-dose and 2 (2%) received both doses of ChAdOx1 nCoV-19 vaccine which significantly reduce dry cough, loss of appetite and difficulties in breathing compared to none. There was no significant difference in hospitalization, duration of hospitalization or reduction of other symptoms like running nose, muscle pain, shortness of breathing or generalized weakness between immunized and unimmunized group. Spike protein sequence assumed 21 (87.5%) B.1.351, one B.1.526 and two 20B variants in immunized group compared to 27 (69%) B.1.351, 5 (13%) B.1.1.7, 4 (10%) 20B, 2 B.1.526 and one B.1.427 variant in unimmunized group. Those variants were further confirmed by 14 whole genome sequence analysis. Complete genome analysis included seven B.1.351 Beta V2, three B.1.1.7 Alpha V1, one B.1.526 Eta and rest three 20B variant. ConclusionSingle dose of ChAdOx1 couldnt prevent the new infection or disease severity by the COVID-19 variants of concern, B.1.351, in Bangladesh.

Metagenomic diagnosis and pathogenic network profile of SARS-CoV-2 in patients co-morbidly affected by type 2 diabetes (preprint)

Background: The mortality of COVID-19 disease is very high among males or elderly or individuals having comorbidities with obesity, cardiovascular diseases, lung infections, hypertension, and/or diabetes. Our study characterizes the metagenomic feature of SARS-CoV-2 infected patients with or without type 2 diabetes to identify the microbial interactions associated with its fatal consequences. Method: This study compared the baseline nasopharyngeal microbiome of SARS-CoV-2 infected diabetic and non-diabetic patients with controls adjusted with age and gender. The mNGS were performed using Ion GeneStudio S5 Series and the data were analyzed by the Vegan-package in R. Results: All three groups possessed significant bacterial diversity and dissimilarity indexes (p<0.05). Spearmans 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 among normal-flora and probiotic bacteria (r>0.6, p<0.05). The SARS-CoV-2 diabetic group exhibited a significant increase of pathogens (p<0.05) and opportunistic pathogens (p<0.05) with a simultaneous decrease of normal-flora (p<0.05). The molecular docking analysis of Salivaricin, KLD4 (alpha), and enterocin produced by several enriched probiotic strains presented strong binding affinity with Shiga toxin, outer membrane proteins (ompA, omp33) or hemolysin. Conclusion: The dysbiosis of the bacterial community might be linked with severe consequences of COVID-19 infected diabetic patients, although few probiotic strains inhibited numerous pathogens in the same pathological niches. This study suggested that the promotion of normal-flora and probiotics through dietary changes and reduction of excessive pro-inflammatory states by preventing pathogenic environment might lead to a better outcome for those co-morbid patients.

Evolving Infection Paradox of SARS-CoV-2: Fitness Costs Virulence? (preprint)

Background: SARS-CoV-2 is continuously spreading worldwide at an unprecedented scale and evolved into seven clades according to GISAID where four (G, GH, GR and GV) are globally prevalent in 2020. These major predominant clades of SARS-CoV-2 are continuously increasing COVID-19 cases worldwide; however, after an early rise in 2020, the death-case ratio has been decreasing to a plateau. G clade viruses contain four co-occurring mutations in their genome (C241T+C3037T+C14408T: RdRp.P323L+A23403G:spike.D614G). GR, GH, and GV strains are defined by the presence of these four mutations in addition to the clade-featured mutation in GGG28881-28883AAC:N. RG203-204KR, G25563T:ORF3a.Q57H, and C22227T:spike.A222V+C28932T-N.A220V+G29645T, respectively. The research works are broadly focused on the spike protein mutations that have direct roles in receptor binding, antigenicity, thus viral transmission and replication fitness. However, mutations in other proteins might also have effects on viral pathogenicity and transmissibility. How the clade-featured mutations are linked with viral evolution in this pandemic through gearing their fitness and virulence is the main question of this study. Methodology: We thus proposed a hypothetical model, combining a statistical and structural bioinformatics approach, endeavors to explain this infection paradox by describing the epistatic effects of the clade-featured co-occurring mutations on viral fitness and virulence. Results and Discussion: The G and GR/GV clade strains represent a significant positive and negative association, respectively, with the death-case ratio (incidence rate ratio or IRR = 1.03, p <0.001 and IRR= 0.99/0.97, p < 0.001), whereas GH clade strains showed no association with the Docking analysis showed the higher infectiousness of a spike mutant through more favorable binding of G614 with the elastase-2. RdRp mutation p.P323L significantly increased genome-wide mutations (p<0.0001) since more expandable RdRp (mutant)-NSP8 interaction may accelerate replication. Superior RNA stability and structural variation at NSP3:C241T might impact upon protein or RNA interactions. Another silent 5'UTR:C241T mutation might affect translational efficiency and viral packaging. These G-featured co-occurring mutations might increase the viral load, alter immune responses in host and hence can modulate intra-host genomic plasticity. An additional viroporin ORF3a:p.Q57H mutation, forming GH-clade, prevents ion permeability by cysteine (C81)-histidine (H57) inter-transmembrane-domain interaction mediated tighter constriction of the channel pore and possibly reduces viral release and immune response. GR strains, four G clade mutations and N:p.RG203-204KR, would have stabilized RNA interaction by more flexible and hypo-phosphorylated SR-rich region. GV strains seemingly gained the evolutionary advantage of superspreading event through confounder factors; nevertheless, N:p.A220V might affect RNA binding. Conclusion: These hypotheses need further retrospective and prospective studies to understand detailed molecular and evolutionary events featuring the fitness and virulence of SARS-CoV-2.

A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades (preprint)

Tracing the globally circulating SARS-CoV-2 mutants is essential for the outbreak alerts and far-reaching epidemiological surveillance. The available technique to identify the phylogenetic clades through high-throughput sequencing is costly, time-consuming, and labor-intensive that hinders the viral genotyping in low-income countries. Here, we propose a rapid, simple and cost-effective amplification-refractory mutation system (ARMS)-based multiplex reverse-transcriptase PCR assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. This approach is applied on 24 COVID-19 positive samples as confirmed by CDC approved real-time PCR assay for SARS-CoV-2. 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 in the primer concentration (0.2-0.6 {micro}M) and annealing temperature (56-60{degrees}C) of PCR using 3-5 ng/{micro}l cDNA template synthesized upon random- and oligo(dT)-primer based reverse transcription. The different primer concentration for the triplex and quadruplex adjusted to different strengths ensured an even amplification with a maximum resolution of all targeted amplicons. The targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of our designed primers. This multiplex ARMS-PCR assay is sample, cost-effective, and convenient that can successfully discriminate the circulating phylogenetic clades of SARS-CoV-2.