Seroprevalence of IgG Antibody against SARS-CoV-2 Nucleocapsid protein and Associated Risk Factors (preprint)

Estimation of antibody development against SARS-CoV-2 is essential means for understanding the immune response against the virus. We reported IgG antibody development status against Nucleocapsid protein of the virus and compared with lifestyle (health and food habits), co-existing diseases, vaccination and COVID-19 infection status. ELISA (Enzyme Linked Immunosorbent Assay) was performed to assess IgG antibodies targeted against the Nucleocapsid protein of SARS-CoV-2 in participants (n=500). In this seroprevalence study, serological data were estimated for a period of 10 months in the participants who were aged 10 years and above. Sociodemographic and risk factors related data were collected through a written questionnaire and chi-square test was performed to determine the association with seropositivity. The overall seroprevalence of anti-SARS-CoV-2 antibodies among the study subjects was 47.8%. Estimates were highest among the participants of 21-40 years old (55.1%), and lowest in older aged (>60 years) participants (39.5%). Among the Sinopharm vaccinated individuals 81.8% had developed anti-Nucleocapsid antibody. Physical exercise and existence of comorbidities like hypertension and diabetes were the distinguishing factors between seropositive and seronegative individuals. Seropositivity rate largely varied among symptomatic (67%) and asymptomatic (33.1%) COVID-19 infected participants. The findings suggest that residents of Dhaka city had a higher prevalence of anti-nucleocapsid antibody in the second year of the pandemic. This indicates the improvement of immunological status among the population. Finally, the study emphasizes on maintaining active and healthy lifestyle to improve immunity. However, the absence of IgG antibodies in many cases of COVID-19 infected individuals suggests that antibodies wane with time.

Whole Genome CRISPR Screening Strategy for Identification of Host Genes Contributing to Envelope Mediated Entry of SARS-CoV-2 Spike and VSV-G (preprint)

Background Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using the live viruses and cellular lethality for selection, these screens identify large numbers of genes without any specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole genome screen and counter screen strategy based on a pseudoviral platform that allowed identification of genes specific to SARS-CoV-2 spike and vesicular stomatitis virus glycoprotein VSV-G mediated entry.Methods To focus the screen onto the entry step, we used non-lytic fluorescent reporters in combination with a comparative counter screen strategy to distinguish host genes affecting the pseudoviral reporter from those unique to envelope-mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein in the pseudovirus was used to bypassed retro-transcription and integration by directly activating a floxed GFP reporter upon entry to reduce the number of gene hits and increase specificity for viral entry.Results Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and LDLR, respectively and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry associated genes.Conclusion Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication. This approach provides a pathway for increasing the specificity of CRISPR whole genome screens for identifying host genes contributing to specific steps in viral infection.

The genetic variants analysis of circulating SARS-CoV-2 in Bangladesh. (preprint)

Genomic mutation of the virus may impact the viral adaptation to the local environment, their transmission, disease manifestation, and the effectiveness of existing treatment and vaccination. The objectives of this study were to characterize genomic variations, non-synonymous amino acid substitutions, especially in target proteins, mutation events per samples, mutation rate, and overall scenario of coronaviruses across the country. To investigate the genetic diversity, a total of 184 genomes of virus strains sampled from different divisions of Bangladesh with sampling dates between the 10th of May 2020 and the 27th of June 2020 were analyzed. To date, a total of 634 mutations located along the entire genome resulting in non-synonymous 274 amino acid substitutions in 22 different proteins were detected with nucleotide mutation rate estimated to be 23.715 substitutions per year. The highest non-synonymous amino acid substitutions were observed at 48 different positions of the papain-like protease (nsp3). Although no mutations were found in nsp7, nsp9, nsp10, and nsp11, yet orf1ab accounts for 56% of total mutations. Among the structural proteins, the highest non-synonymous amino acid substitution (at 36 positions) observed in spike proteins, in which 9 unique locations were detected relative to the global strains, including 516E>Q in the boundary of the ACE2 binding region. The most dominated variant G614 (95%) based in spike protein is circulating across the country with co-evolving other variants including L323 (94%) in RNA dependent RNA polymerase (RdRp), K203 (82%) and R204 (82%) in nucleocapsid, and F120 (78%) in NSP2. These variants are mostly seen as linked mutations and are part of a haplotype observed in Europe. Data suggest effective containment of clade G strains (4.8%) with sub-clusters GR 82.4%, and GH clade 6.4%. HighlightsO_LIWe have sequenced 137 and analyzed 184 whole-genomes sequences of SARS-CoV-2 strains from different divisions of Bangladesh. C_LIO_LIA total of 634 mutation sites across the SARS-CoV-2 genome and 274 non-synonymous amino acid substitutions were detected. C_LIO_LIThe mutation rate of SARS-CoV-2 estimated to be 23.715 nucleotide substitutions per year. C_LIO_LINine unique variants were detected based on non-anonymous amino acid substitutions in spike protein relative to the global SARS-CoV-2 strains. C_LI