Genomic characterization of the dominating Beta, V2 variant carrying vaccinated (Oxford-AstraZeneca) and nonvaccinated COVID-19 patient samples in Bangladesh: A metagenomics and whole-genome approach.

Bangladesh is experiencing a second wave of COVID-19 since March 2021, despite the nationwide vaccination drive with ChAdOx1 (Oxford-AstraZeneca) vaccine from early February 2021. Here, we characterized 19 nasopharyngeal swab (NPS) samples from COVID-19 suspect patients using genomic and metagenomic approaches. Screening for SARS-CoV-2 by reverse transcriptase polymerase chain reaction and metagenomic sequencing revealed 17 samples of COVID-19 positive (vaccinated = 10, nonvaccinated = 7) and 2 samples of COVID-19 negative. We did not find any significant correlation between associated factors including vaccination status, age or sex of the patients, diversity or abundance of the coinfected organisms/pathogens, and the abundance of SARS-CoV-2. Though the first wave of the pandemic was dominated by clade 20B, Beta, V2 (South African variant) dominated the second wave (January 2021 to May 2021), while the third wave (May 2021 to September 2021) was responsible for Delta variants of the epidemic in Bangladesh including both vaccinated and unvaccinated infections. Noteworthily, the receptor binding domain (RBD) region of S protein of all the isolates harbored similar substitutions including K417N, E484K, and N501Y that signify the Beta, while D614G, D215G, D80A, A67V, L18F, and A701V substitutions were commonly found in the non-RBD region of Spike proteins. ORF7b and ORF3a genes underwent a positive selection (dN/dS ratio 1.77 and 1.24, respectively), while the overall S protein of the Bangladeshi SARS-CoV-2 isolates underwent negative selection pressure (dN/dS = 0.621). Furthermore, we found different bacterial coinfections like Streptococcus agalactiae, Neisseria meningitidis, Elizabethkingia anophelis, Stenotrophomonas maltophilia, Klebsiella pneumoniae, and Pseudomonas plecoglossicida, expressing a number of antibiotic resistance genes such as tetA and tetM. Overall, this approach provides valuable insights on the SARS-CoV-2 genomes and microbiome composition from both vaccinated and nonvaccinated patients in Bangladesh.

Temporal landscape of mutational frequencies in SARS-CoV-2 genomes of Bangladesh: possible implications from the ongoing outbreak in Bangladesh.

Along with intrinsic evolution, adaptation to selective pressure in new environments might have resulted in the circulatory SARS-CoV-2 strains in response to the geoenvironmental conditions of a country and the demographic profile of its population. With this target, the current study traced the evolutionary route and mutational frequency of 198 Bangladesh-originated SARS-CoV-2 genomic sequences available in the GISAID platform over a period of 13 weeks as of 14 July 2020. The analyses were performed using MEGA X, Swiss Model Repository, Virus Pathogen Resource and Jalview visualization. Our analysis identified that majority of the circulating strains strikingly differ from both the reference genome and the first sequenced genome from Bangladesh. Mutations in nonspecific proteins (NSP2-3, NSP-12(RdRp), NSP-13(Helicase)), S-Spike, ORF3a, and N-Nucleocapsid protein were common in the circulating strains with varying degrees and the most unique mutations (UM) were found in NSP3 (UM-18). But no or limited changes were observed in NSP9, NSP11, Envelope protein (E) and accessory factors (NSP7a, ORF 6, ORF7b) suggesting the possible conserved functions of those proteins in SARS-CoV-2 propagation. However, along with D614G mutation, more than 20 different mutations in the Spike protein were detected basically in the S2 domain. Besides, mutations in SR-rich region of N protein and P323L in RDRP were also present. However, the mutation accumulation showed a significant association (p = 0.003) with sex and age of the COVID-19-positive cases. So, identification of these mutational accumulation patterns may greatly facilitate vaccine development deciphering the age and the sex-dependent differential susceptibility to COVID-19.

Evolutionary dynamics of SARS-CoV-2 nucleocapsid protein and its consequences.

The emerged novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health crisis that warrants an accurate and detailed characterization of the rapidly evolving viral genome for understanding its epidemiology, pathogenesis, and containment. Here, we explored 61,485 sequences of the nucleocapsid (N) protein, a potent diagnostic and prophylactic target, for identifying the mutations to review their roles in real-time polymerase chain reaction based diagnosis and observe consequent impacts. Compared to the Wuhan reference strain, a total of 1034 unique nucleotide mutations were identified in the mutant strains (49.15%, n = 30,221) globally. Of these mutations, 367 occupy primer binding sites including the 3'-end mismatch to the primer-pair of 11 well-characterized primer sets. Noteworthily, CDC (USA) recommended the N2 primer set contained a lower mismatch than the other primer sets. Moreover, 684 amino acid (aa) substitutions were located across 317 (75.66% of total aa) unique positions including 82, 21, and 83 of those in the RNA binding N-terminal domain (NTD), SR-rich region, and C-terminal dimerization domain, respectively. Moreover, 11 in-frame deletions, mostly (n = 10) within the highly flexible linker region, were revealed, and the rest was within the NTD region. Furthermore, we predicted the possible consequence of high-frequency mutations (≥20) and deletions on the tertiary structure of the N protein. Remarkably, we observed that a high frequency (67.94% of mutated sequences) co-occuring mutations (R203K and G204R) destabilized and decreased overall structural flexibility. The N protein of SARS-CoV-2 comprises an average of 1.2 mutations per strain compared to 4.4 and 0.4 in Middle East respiratory syndrome-related coronavirus and SARS-CoV, respectively. Despite being proposed as the alternative target to spike protein for vaccine and therapeutics, the ongoing evolution of the N protein may challenge these endeavors, thus needing further immunoinformatics analyses. Therefore, continuous monitoring is required for tracing the ongoing evolution of the SARS-CoV-2 N protein in prophylactic and diagnostic interventions.

Microbial co-infections in COVID-19: Associated microbiota and underlying mechanisms of pathogenesis.

The novel coronavirus infectious disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has traumatized the whole world with the ongoing devastating pandemic. A plethora of microbial domains including viruses (other than SARS-CoV-2), bacteria, archaea and fungi have evolved together, and interact in complex molecular pathogenesis along with SARS-CoV-2. However, the involvement of other microbial co-pathogens and underlying molecular mechanisms leading to extortionate ailment in critically ill COVID-19 patients has yet not been extensively reviewed. Although, the incidence of co-infections could be up to 94.2% in laboratory-confirmed COVID-19 cases, the fate of co-infections among SARS-CoV-2 infected hosts often depends on the balance between the host's protective immunity and immunopathology. Predominantly identified co-pathogens of SARS-CoV-2 are bacteria such as Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Acinetobacter baumannii, Legionella pneumophila and Clamydia pneumoniae followed by viruses including influenza, coronavirus, rhinovirus/enterovirus, parainfluenza, metapneumovirus, influenza B virus, and human immunodeficiency virus. The cross-talk between co-pathogens (especially lung microbiomes), SARS-CoV-2 and host is an important factor that ultimately increases the difficulty of diagnosis, treatment, and prognosis of COVID-19. Simultaneously, co-infecting microbiotas may use new strategies to escape host defense mechanisms by altering both innate and adaptive immune responses to further aggravate SARS-CoV-2 pathogenesis. Better understanding of co-infections in COVID-19 is critical for the effective patient management, treatment and containment of SARS-CoV-2. This review therefore necessitates the comprehensive investigation of commonly reported microbial co-pathogens amid COVID-19, their transmission pattern along with the possible mechanism of co-infections and outcomes. Thus, identifying the possible co-pathogens and their underlying molecular mechanisms during SARS-CoV-2 pathogenesis may shed light in developing diagnostics, appropriate curative and preventive interventions for suspected SARS-CoV-2 respiratory infections in the current pandemic.

Mutational insights into the envelope protein of SARS-CoV-2.

The ongoing mutations in the structural proteins of SARS-CoV-2 are the major impediment for prevention and control of the COVID-19 disease. Presently we focused on evolution of the envelope (E) protein, one of the most enigmatic and less studied protein among the four structural proteins (S, E, M and N) associated with multitude of immunopathological functions of SARS-CoV-2. In the present study, we comprehensively analyzed 81,818 high quality E protein sequences of SARS-CoV-2 globally available in the GISAID database as of 20 August 2020. Compared to Wuhan reference strain, our mutational analysis explored only 1.2 % (982/81818) mutant strains undergoing a total of 115 unique amino acid (aa) substitutions in the E protein, highlighting the fact that most (98.8 %) of the E protein of SARS-CoV-2 strains are highly conserved. Moreover, we found 58.77 % (134 of 228) nucleotides (nt) positions of SARS-CoV-2 E gene encountering a total of 176 unique nt-level mutations globally, which may affect the efficacy of real time RT-PCR-based molecular detection of COVID-19. Importantly, higher aa variations observed in the C-terminal domain (CTD) of the E protein, particularly at Ser55-Phe56, Arg69 and the C-terminal end (DLLV: 72-75) may alter the binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 and thus could play a key role in COVID-19 pathogenesis. Furthermore, this study revealed the V25A mutation in the transmembrane domain which is a key factor for the homopentameric conformation of E protein. Our analysis also observed a triple cysteine motif harboring mutation (L39M, A41S, A41V, C43F, C43R, C43S, C44Y, N45R) which may hinder the binding of E protein with spike glycoprotein. These results therefore suggest the continuous monitoring of the structural proteins including the envelope protein of SARS-CoV-2 since the number of genome sequences from across the world are continuously increasing.