Substitution spectra of SARS-CoV-2 genome from Pakistan reveals insights into the evolution of variants across the pandemic

Changing morbidity and mortality due to COVID-19 across the pandemic has been linked with factors such as the emergence of SARS-CoV-2 variants and vaccination. Mutations in the Spike glycoprotein enhanced viral transmission and virulence. We investigated whether SARS-CoV-2 mutation rates and entropy were associated COVID-19 in Pakistan, before and after the introduction of vaccinations. We analyzed 1,705 SARS-CoV-2 genomes using the Augur phylogenetic pipeline. Substitution rates and entropy across the genome, and in the Spike glycoprotein were compared between 2020, 2021 and 2022 (as periods A, B and C). Mortality was greatest in B whilst cases were highest during C. In period A, G clades were predominant, and substitution rate was 5.25 × 10-4 per site per year. In B, Delta variants dominated, and substitution rates increased to 9.74 × 10-4. In C, Omicron variants led to substitution rates of 5.02 × 10-4. Genome-wide entropy was the highest during B particularly, at Spike E484K and K417N. During C, genome-wide mutations increased whilst entropy was reduced. Enhanced SARS-CoV-2 genome substitution rates were associated with a period when more virulent SARS-CoV-2 variants were prevalent. Reduced substitution rates and stabilization of genome entropy was subsequently evident when vaccinations were introduced. Whole genome entropy analysis can help predict virus evolution to guide public health interventions

Higher entropy observed in SARS-CoV-2 genomes from the first COVID-19 wave in Pakistan

Background: We investigated the genome diversity of SARS-CoV-2 associated with the early COVID-19 period to investigate evolution of the virus in Pakistan.Materials and methods: We studied ninety SARS-CoV-2 strains isolated between March and October 2020. Whole genome sequences from our laboratory and available genomes were used to investigate phylogeny, genetic variantion and mutation rates of SARS-CoV-2 strains in Pakistan. Site specific entropy analysis compared mutation rates between strains isolated before and after June 2020.Results: In March, strains belonging to L, S, V and GH clades were observed but by October, only L and GH strains were present. The highest diversity of clades was present in Sindh and Islamabad Capital Territory and the least in Punjab province. Initial introductions of SARS-CoV-2 GH (B.1.255, B.1) and S (A) clades were associated with overseas travelers. Additionally, GH (B.1.255, B.1, B.1.160, B.1.36), L (B, B.6, B.4), V (B.4) and S (A) clades were transmitted locally. SARS-CoV-2 genomes clustered with global strains except for ten which matched Pakistani isolates. RNA substitution rates were estimated at 5.86 x10-4. The most frequent mutations were 5\u27 UTR 241C \u3e T, Spike glycoprotein D614G, RNA dependent RNA polymerase (RdRp) P4715L and Orf3a Q57H. Strains up until June 2020 exhibited an overall higher mean and site-specific entropy as compared with sequences after June. Relative entropy was higher across GH as compared with GR and L clades. More sites were under selection pressure in GH strains but this was not significant for any particular site.Conclusions: The higher entropy and diversity observed in early pandemic as compared with later strains suggests increasing stability of the genomes in subsequent COVID-19 waves. This would likely lead to the selection of site-specific changes that are advantageous to the virus, as has been currently observed through the pandemic

Latent M. tuberculosis infection is associated with increased inflammatory cytokine and decreased suppressor of cytokine signalling (SOCS)-3 in the diabetic host

Tuberculosis (TB) outcomes are worsened by type II diabetes mellitus (DM). Protective immunity against Mycobacterium tuberculosis (MTB) is driven by cytokines. Latent TB (LTBi) is common but its effect on the diabetic host is not well understood. We investigated mycobacterial antigen-stimulated responses in peripheral blood mononuclear cell (PBMC) isolated from healthy endemic controls (EC), those with LTBi, DM groups with and without LTBi, as compared with TB patients. Cytokines were measured using a Luminex-based assay. Gene expression was determined by RT-PCR. In DM-LTBi cases, PPD-stimulated proinflammatory cytokines; IFN-γ, IL-6, IL-2, TNF-α and GM-CSF and anti-inflammatory cytokines, IL-5 and IL-13 were raised as compared with EC. DM-LTBi PPD-stimulated IFN-γ, IL-6 and TNF-α mRNA titres were found raised in DM-LTBi, whilst suppressor of cytokine signalling (SOCS)-3 expression was lowered. Within DM cases, stratification based on HbA1c levels revealed raised IFN-γ but lowered IL-6 gene expression in those with controlled levels as compared with uncontrolled glycaemic levels. Further, SOCS1 expression levels were found higher in DM cases with controlled glycaemia when compared with EC. Overall, we show that diabetics with LTBi manifest raised levels of inflammatory and anti-inflammatory cytokines concomitant with reduced SOCS3 mRNA expression. Reduced glycaemic control results in further inflammatory dysregulation impacting conversing impacting IFN-γ and IL-6 activation. These results suggest that dysregulated immune activation in diabetes is exacerbated by LTBi, lack of glycaemic control may further compromise immunity against MTB infection

Substitution spectra of SARS-CoV-2 genome from Pakistan reveals insights into the evolution of variants across the pandemic

Abstract Changing morbidity and mortality due to COVID-19 across the pandemic has been linked with factors such as the emergence of SARS-CoV-2 variants and vaccination. Mutations in the Spike glycoprotein enhanced viral transmission and virulence. We investigated whether SARS-CoV-2 mutation rates and entropy were associated COVID-19 in Pakistan, before and after the introduction of vaccinations. We analyzed 1,705 SARS-CoV-2 genomes using the Augur phylogenetic pipeline. Substitution rates and entropy across the genome, and in the Spike glycoprotein were compared between 2020, 2021 and 2022 (as periods A, B and C). Mortality was greatest in B whilst cases were highest during C. In period A, G clades were predominant, and substitution rate was 5.25 × 10–4 per site per year. In B, Delta variants dominated, and substitution rates increased to 9.74 × 10–4. In C, Omicron variants led to substitution rates of 5.02 × 10–4. Genome-wide entropy was the highest during B particularly, at Spike E484K and K417N. During C, genome-wide mutations increased whilst entropy was reduced. Enhanced SARS-CoV-2 genome substitution rates were associated with a period when more virulent SARS-CoV-2 variants were prevalent. Reduced substitution rates and stabilization of genome entropy was subsequently evident when vaccinations were introduced. Whole genome entropy analysis can help predict virus evolution to guide public health interventions

Sequential viral introductions and spread of BA.1 across Pakistan provinces during the Omicron wave

Background: COVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at different times. We focused on Omicron variants to understand the genomic diversity and phylogenetic relatedness of SARS-CoV-2 strains in various regions of Pakistan.Methods: We studied 276,525 COVID-19 cases and 1,031 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees.Results: The highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 93% of all genomes, with BA.2 (32.6%) and BA.5 (38.4%) predominating. The first Omicron wave was associated with the sequential identification of BA.1 in Sindh, then Islamabad Capital Territory, Punjab, Khyber Pakhtunkhwa (KP), Azad Jammu Kashmir (AJK), Gilgit-Baltistan (GB) and Balochistan. Phylogenetic analysis revealed Sindh to be the source of BA.1 and BA.2 introductions into Punjab and Balochistan during early 2022. BA.4 was first introduced in AJK and BA.5 in Punjab. Most recent common ancestor (MRCA) analysis revealed relatedness between the earliest BA.1 genome from Sindh with Balochistan, AJK, Punjab and ICT, and that of first BA.1 from Punjab with strains from KPK and GB.Conclusions: Phylogenetic analysis provides insights into the introduction and transmission dynamics of the Omicron variant in Pakistan, identifying Sindh as a hotspot for viral dissemination. Such data linked with public health efforts can help limit surges of new infections

Upregulated type I interferon responses in asymptomatic COVID-19 infection are associated with improved clinical outcome

Understanding key host protective mechanisms against SARS-CoV-2 infection can help improve treatment modalities for COVID-19. We used a blood transcriptome approach to study biomarkers associated with differing severity of COVID-19, comparing severe and mild Symptomatic disease with Asymptomatic COVID-19 and uninfected Controls. There was suppression of antigen presentation but upregulation of inflammatory and viral mRNA translation associated pathways in Symptomatic as compared with Asymptomatic cases. In severe COVID-19, CD177 a neutrophil marker, was upregulated while interferon stimulated genes (ISGs) were downregulated. Asymptomatic COVID-19 cases displayed upregulation of ISGs and humoral response genes with downregulation of ICAM3 and TLR8. Compared across the COVID-19 disease spectrum, we found type I interferon (IFN) responses to be significantly upregulated (IFNAR2, IRF2BP1, IRF4, MAVS, SAMHD1, TRIM1), or downregulated (SOCS3, IRF2BP2, IRF2BPL) in Asymptomatic as compared with mild and severe COVID-19, with the dysregulation of an increasing number of ISGs associated with progressive disease. These data suggest that initial early responses against SARS-CoV-2 may be effectively controlled by ISGs. Therefore, we hypothesize that treatment with type I interferons in the early stage of COVID-19 may limit disease progression by limiting SARS-CoV-2 in the host

Sequential viral introductions and spread of BA.1 across Pakistan provinces during the Omicron wave

Abstract Background COVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at different times. We focused on Omicron variants to understand the genomic diversity and phylogenetic relatedness of SARS-CoV-2 strains in various regions of Pakistan. Methods We studied 276,525 COVID-19 cases and 1,031 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees. Results The highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 93% of all genomes, with BA.2 (32.6%) and BA.5 (38.4%) predominating. The first Omicron wave was associated with the sequential identification of BA.1 in Sindh, then Islamabad Capital Territory, Punjab, Khyber Pakhtunkhwa (KP), Azad Jammu Kashmir (AJK), Gilgit-Baltistan (GB) and Balochistan. Phylogenetic analysis revealed Sindh to be the source of BA.1 and BA.2 introductions into Punjab and Balochistan during early 2022. BA.4 was first introduced in AJK and BA.5 in Punjab. Most recent common ancestor (MRCA) analysis revealed relatedness between the earliest BA.1 genome from Sindh with Balochistan, AJK, Punjab and ICT, and that of first BA.1 from Punjab with strains from KPK and GB. Conclusions Phylogenetic analysis provides insights into the introduction and transmission dynamics of the Omicron variant in Pakistan, identifying Sindh as a hotspot for viral dissemination. Such data linked with public health efforts can help limit surges of new infections

BBIBP-CorV (Sinopharm) vaccination- induced immunity is affected by age, gender and prior COVID-19 and activates responses to spike and other antigens

Long-term solutions against SARS-CoV-2 infections require understanding of immune protection induced by different vaccine COVID-19 formulations. We investigated humoral and cellular immunity induced by Sinopharm (BBIBP-CorV) in a region of high SARS-CoV-2 seroprevalence.Levels of IgG antibodies to SARS-CoV-2 spike protein and its receptor-binding domain (RBD) were determined 24-weeks. Cellular immunity was investigated using a commercially available IFN-γ release assay to SARS-CoV-2 spike (Ag1 and 2) and extended genome antigens (Ag3).Increasing IgG seropositivity to Spike protein and RBD was observed post-vaccination. Seropositivity was reduced in those over 50 years and raised in females and those with prior COVID-19. After 20 weeks post-vaccination, only one third of participants had positive T cell responses to SARS-CoV-2 antigens. Prior COVID-19 impacted IFNγ responses, with reactivity enhanced in those infected earlier. The frequency of IFNγ responses was highest to extended genome antigen set.Overall, BBIBP-CorV- induced antibody responses were impacted by age, gender and prior COVID-19. Cellular immunity was present in a limited number of individuals after 20 weeks but was enhanced by prior infection. This suggests the need for booster vaccinations in older individuals. BBIBP-CorV-induced cellular activation is broader than to spike, requiring further study to understand how to monitor vaccine effectiveness

Investigating the impact of prior COVID-19 on IgG antibody and interferon γ responses after BBIBP-CorV vaccination in a disease endemic population: A prospective observational study

Background and aims: COVID-19 vaccinations have reduced morbidity and mortality from the disease. Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) have been associated with immune protection. Seroprevalence studies revealed high immunoglobulin G (IgG) antibody levels to SARS-CoV-2 in the Pakistani population before vaccinations. We investigated the effect of BBIBP-CorV vaccination on circulating IgG antibodies and interferon (IFN)-γ from T cells measured in a cohort of healthy individuals, with respect to age, gender, and history of COVID-19. Methods: The study was conducted between April and October 2021. BBIBP-CorV vaccinated participants were followed up to 24 weeks. Antibodies to SARS-CoV-2 Spike protein and its receptor-binding domain (RBD) were measured. IFNγ secreted by whole blood stimulation of Spike protein and extended genome antigens was determined. Results: Study participants with a history of prior COVID-19 displayed a higher magnitude of IgG antibodies to Spike and RBD. IgG seropositivity was greater in those with prior COVID-19, aged 50 years or younger and in females. At 24 weeks after vaccination, 37.4% of participants showed IFN-γ responses to SARS-CoV-2 antigens. T cell IFN-γ release was higher in those with prior COVID-19 and those aged 50 years or less. Highest IFN-γ release was observed to extended genome antigens in individuals both with and without prior COVID-19. Conclusion: We found that IgG seropositivity to both Spike and RBD was affected by prior COVID-19, age and gender. Importantly, seropositive responses persisted up to 24 weeks after vaccination. Persistence of vaccine induced IgG antibodies may be linked to the high seroprevalence observed earlier in unvaccinated individuals. Increased T cell reactivity to Spike and extended genome antigens reflects cellular activation induced by BBIBP-CorV. COVID-19 vaccination may have longer lasting immune responses in populations with a higher seroprevalence. These data inform on vaccination booster policies for high-risk group