Phylogenetic and whole genome analysis of first seven SARS-CoV-2 isolates in Bangladesh
ABSTRACT: Aim: Whole genome and peptide mutation analysis can specify effective vaccine and therapeutics against severe acute respiratory coronavirus-2 (SARS-CoV-2). Materials & methods: Whole genome similarity for Bangladeshi SARS-CoV-2 was determined using ClustalW and BLASTn. Phylogenetic analysis was conducted using neighbor-joining method. Results: 100% of isolates in Bangladesh were in the G clade. We found 99.98–100% sequence similarity among Bangladeshi isolates and isolates of England, Greece, USA, Saudi Arabia and India. Deletion of bases at 5? untranslated region and 3? untranslated region was detected. Substitution 261 (E?D) at NSP13 and 1109 (F?L) at spike (S) protein were detected. Substitution 377 (D?G) at nucleocapsid with common substitution 614 (D?G) at S were also detected. Conclusion: This study will provide baseline data for development of an effective vaccine or therapeutics against SARS-CoV-2. Graphical abstract
Project description:Graphical abstract Highlights • Phylogenetic analysis revealed that SARS-CoV-2 isolates sequenced from Dhaka and Chittagong were the lineage of Europe and India, respectively.• 42 mutations were identified and about half of them were synonymous. Identified missense mutations had less effect on viral pathogenesis.• 3 large deletions were identified which cause loss of one or more proteins resulting in leading the virus less pathogenic.• Effect of mutations on the interaction between spike proteins and human ACE2 was investigated by molecular docking approaches.• A binding domain of spike protein was found to interact with human ACE2 which was conserved in all isolates. As the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), rages across the world, killing hundreds of thousands and infecting millions, researchers are racing against time to elucidate the viral genome. Some Bangladeshi institutes are also in this race, sequenced a few isolates of the virus collected from Bangladesh. Here, we present a genomic analysis of these isolates. The analysis revealed that SARS-CoV-2 isolates sequenced from Dhaka and Chittagong were the lineage of Europe and India, respectively. Our analysis identified a total of 42 mutations, including three large deletions, half of which were synonymous. Most of the missense mutations in Bangladeshi isolates found to have weak effects on the pathogenesis. Some mutations may lead the virus to be less pathogenic than the other countries. Molecular docking analysis to evaluate the effect of the mutations on the interaction between the viral spike proteins and the human ACE2 receptor, though no significant difference was observed. This study provides some preliminary insights into the origin of Bangladeshi SARS-CoV-2 isolates, mutation spectrum and its possible pathomechanism, which may give an essential clue for designing therapeutics and management of COVID-19 in Bangladesh.
Project description:The novel human coronavirus (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) pandemic worldwide. Control of COVID-19 pandemic is vital for public health and is the prerequisite to maintain social stability. However, the origin and transmission route of SARS-CoV-2 is unclear, bringing huge difficult to virus control. Monitoring viral variation and screening functional mutation sites are crucial for prevention and control of infectious diseases. In this study, we developed a user-friendly software, named BioAider, for quick sequence annotation and mutation analysis on large-scale genome-sequencing data. Herein, we detected 14 substitution hotspots within 3,240 SARS-CoV-2 genome sequences, including 3 groups of potentially linked substitution. NSP13-Y541C was crucial substitution which might affect the unwinding activity of the viral helicase. In particular, we discovered a SR-rich region of SARS-CoV-2 distinct from SARS-CoV, indicating more complex replication mechanism and unique N-M interaction of SARS-CoV-2. Interestingly, the quantity of SSRX repeat fragments in SARS-CoV-2 provided further evidence of its animal origin. Overall, we developed an efficient tool for rapid identification of viral genome mutations which could facilitate viral genomic studies. Using this tool, we have found critical clues for the transmission route of SARS-CoV-2 which would provide theoretical support for the epidemic control of pathogenic coronaviruses.
Project description:The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.
Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.
Project description:The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' ? 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.
Project description:SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.
Project description:SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.
Project description:After 56 days without coronavirus disease 2019 (COVID-19) cases, reemergent cases were reported in Beijing, China on June 11, 2020. Here, we report the genetic characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequenced from the clinical specimens of 4 human cases and 2 environmental samples. The nucleotide similarity among six SARS-CoV-2 genomes ranged from 99.98% to 99.99%. Compared with the reference strain of SARS-CoV-2 (GenBank No. NC_045512), all six genome sequences shared the same substitutions at nt241 (C ? T), nt3037 (C ? T), nt14408 (C ? T), nt23403 (A ? G), nt28881 (G ? A), nt28882 (G ? A), and nt28883 (G ? C), which are the characteristic nucleotide substitutions of L-lineage European branch I. This was also proved by the maximum likelihood phylogenetic tree based on the full-length genome of SARS-CoV-2. They also have a unique shared nucleotide substitution, nt6026 (C ? T), which is the characteristic nucleotide substitution of SARS-CoV-2 in Beijing's Xinfadi outbreak. It is noteworthy that there is an amino acid D614G mutation caused by nt23403 substitution in all six genomes, which may enhance the virus's infectivity in humans and help it become the leading strain of the virus to spread around the world today. It is necessary to continuously monitor the genetic variation of SARS-CoV-2, focusing on the influence of key mutation sites of SARS-CoV-2 on viral transmission, clinical manifestations, severity, and course of disease.
Project description:The severe acute respiratory syndrome (SARS) epidemic was characterized by increased pathogenicity in the elderly due to an early exacerbated innate host response. SARS-CoV is a zoonotic pathogen that entered the human population through an intermediate host like the palm civet. To prevent future introductions of zoonotic SARS-CoV strains and subsequent transmission into the human population, heterologous disease models are needed to test the efficacy of vaccines and therapeutics against both late human and zoonotic isolates. Here we show that both human and zoonotic SARS-CoV strains can infect cynomolgus macaques and resulted in radiological as well as histopathological changes similar to those seen in mild human cases. Viral replication was higher in animals infected with a late human phase isolate compared to a zoonotic isolate. Host responses to the three SARS-CoV strains were similar and only apparent early during infection with the majority of genes associated with interferon signalling pathways.This study characterizes critical disease models in the evaluation and licensure of therapeutic strategies against SARS-CoV for human use 4 strains, time course, lungs
Project description:Remdesivir (RDV) exhibits potent antiviral activity against SARS-CoV-2 and is currently the only drug approved for the treatment of COVID-19. However, little is currently known about the potential for pre-existing resistance to RDV and the possibility of SARS-CoV-2 genetic diversification that might impact RDV efficacy as the virus continue to spread globally. In this study, >90,000 SARS-CoV-2 sequences from globally circulating clinical isolates, including sequences from recently emerged United Kingdom and South Africa variants, and >300 from mink isolates were analyzed for genetic diversity in the RNA replication complex (nsp7, nsp8, nsp10, nsp12, nsp13, and nsp14) with a focus on the RNA-dependent RNA polymerase (nsp12), the molecular target of RDV. Overall, low genetic variation was observed with only 12 amino acid substitutions present in the entire RNA replication complex in ?0.5% of analyzed sequences with the highest overall frequency (82.2%) observed for nsp12 P323L that consistently increased over time. Low sequence variation in the RNA replication complex was also observed among the mink isolates. Importantly, the coronavirus Nsp12 mutations previously selected in vitro in the presence of RDV were identified in only 2 isolates (0.002%) within all the analyzed sequences. In addition, among the sequence variants observed in ?0.5% clinical isolates, including P323L, none were located near the established polymerase active site or sites critical for the RDV mechanism of inhibition. In summary, the low diversity and high genetic stability of the RNA replication complex observed over time and in the recently emerged SARS-CoV-2 variants suggests a minimal global risk of pre-existing SARS-CoV-2 resistance to RDV.