Nanopore Sequencing SARS-CoV-2 genome in Qatar

Background: The current pandemic, COVID-19, is cause by an RNA coronavirus that was recently identified as SARS-CoV-2. RNA viruses tend to have a high mutation rate; the rate is around a million times greater than that of their hosts. The mutagenic potential of the virus depends on many factors, including the fidelity of nucleic acid-replicating viral enzymes, such as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). The rate of mutation drives viral evolution and genome variability, consequently allowing viruses to escape the immunity of the host and develop resistance to drugs. Therefore, the characterization of SARS-CoV-2 variants might lead to implement better therapeutics treatments, vaccines design and identify new diagnostics approaches. Aim: The aim of this study was to establish a fast sequencing method to identify SARS-CoV-2 mutations in Qatar. This will help to assess if there are new viral variants that are spreading in country. Methods: RNA was isolated from samples collected from Qatar COVID-19 positive patients. The Artic Network V3 primer scheme and Oxford Nanopore ligation sequencing kit were used to prepare the sequencing libraries. Libraries were loaded on to R9.4.1 flow cells and ran on a GridION. Bioinformatics analysis was done following the Artic Network SARA-CoV-2 bioinformatics tools. Results: Genome coverage of sequenced samples was >80% and the depth was average at 200x. The coverage was highly dependable on sample viral load; samples of CT value lower than 30 resulted in better sequence coverage. The sequenced genomes were deposited in GISAID and were mainly clustering with genomes deposited from the UK. Sequences were compared to Illumina and sanger sequences and they showed compatible results. Conclusion: The use of ONT to sequence SARA-CoV-2 is a quick, affordable, and reliable technique to determine viral mutation. Using this technique, the first sequences from Qatar were deposited in to GISAID. Up to date, 700 genomes have been sequences from Qatari samples

Inter-Versus Intra-Host Sequence Diversity of pH1N1 and Associated Clinical Outcomes

The diversity of RNA viruses dictates their evolution in a particular host, community or environment. Here, we reported within- and between-host pH1N1virus diversity at consensus and sub-consensus levels over a three-year period (2015–2017) and its implications on disease severity. A total of 90 nasal samples positive for the pH1N1 virus were deep-sequenced and analyzed to detect low-frequency variants (LFVs) and haplotypes. Parallel evolution of LFVs was seen in the hemagglutinin (HA) gene across three scales: among patients (33%), across years (22%), and at global scale. Remarkably, investigating the emergence of LFVs at the consensus level demonstrated that within-host virus evolution recapitulates evolutionary dynamics seen at the global scale. Analysis of virus diversity at the HA haplotype level revealed the clustering of low-frequency haplotypes from early 2015 with dominant strains of 2016, indicating rapid haplotype evolution. Haplotype sharing was also noticed in all years, strongly suggesting haplotype transmission among patients infected during a specific influenza season. Finally, more than half of patients with severe symptoms harbored a larger number of haplotypes, mostly in patients under the age of five. Therefore, patient age, haplotype diversity, and the presence of certain LFVs should be considered when interpreting illness severity. In addition to its importance in understanding virus evolution, sub-consensus virus diversity together with whole genome sequencing is essential to explain variabilities in clinical outcomes that cannot be explained by either analysis alone

Within-Host Diversity of SARS-CoV-2 in COVID-19 Patients With Variable Disease Severities.

The ongoing pandemic of SARS-COV-2 has already infected more than eight million people worldwide. The majority of COVID-19 patients either are asymptomatic or have mild symptoms. Yet, about 15% of the cases experience severe complications and require intensive care. Factors determining disease severity are not yet fully characterized. Here, we investigated the within-host virus diversity in COVID-19 patients with different clinical manifestations. We compared SARS-COV-2 genetic diversity in 19 mild and 27 severe cases. Viral RNA was extracted from nasopharyngeal samples and sequenced using the Illumina MiSeq platform. This was followed by deep-sequencing analyses of SARS-CoV-2 genomes at both consensus and sub-consensus sequence levels. Consensus sequences of all viruses were very similar, showing more than 99.8% sequence identity regardless of the disease severity. However, the sub-consensus analysis revealed significant differences in within-host diversity between mild and severe cases. Patients with severe symptoms exhibited a significantly (-value 0.001) higher number of variants in coding and non-coding regions compared to mild cases. Analysis also revealed higher prevalence of some variants among severe cases. Most importantly, severe cases exhibited significantly higher within-host diversity (mean = 13) compared to mild cases (mean = 6). Further, higher within-host diversity was observed in patients above the age of 60 compared to the younger age group. These observations provided evidence that within-host diversity might play a role in the development of severe disease outcomes in COVID-19 patients; however, further investigations are required to elucidate this association.This work was supported by Qatar University under internal grant (QUCG-BRC-20/21-1) and Qatar National Research Fund grant under grant (RRC-2-039)

Epidemiology of respiratory infections among adults in Qatar (2012-2017).

Limited data is available about the etiology of influenza like illnesses (ILIs) in Qatar. This study aimed at providing preliminary estimates of influenza and other respiratory infections circulating among adults in Qatar. We retrospectively collected data of about 44,000 patients who visited Hamad General Hospital clinics, sentinel sites, and all primary healthcare centers in Qatar between 2012 and 2017. All samples were tested for influenza viruses, whereas about 38,000 samples were tested for influenza and a panel of respiratory viruses using Fast Track Diagnostics (FTD) RT-PCR kit. Among all ILIs cases, 20,278 (46.5%) tested positive for at least one respiratory pathogen. Influenza virus was predominating (22.6%), followed by human rhinoviruses (HRVs) (9.5%), and human coronaviruses (HCoVs) (5%). A detection rate of 2-3% was recorded for mycoplasma pneumonia, adenoviruses, human parainfluenza viruses (HPIVs), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV). ILIs cases were reported throughout the year, however, influenza, RSV, and HMPV exhibited strong seasonal peaks in the winter, while HRVs circulated more during fall and spring. Elderly (>50 years) had the lowest rates of influenza A (13.9%) and B (4.2%), while presenting the highest rates of RSV (3.4%) and HMPV (3.3%). While males had higher rates of HRVs (11.9%), enteroviruses (1.1%) and MERS CoV (0.2%), females had higher proportions of influenza (26.3%), HPIVs (3.2%) and RSV (3.6%) infections. This report provides a comprehensive insight about the epidemiology of ILIs among adults in the Qatar, as a representative of Gulf States. These results would help in improvement and optimization of diagnostic procedures, as well as control and prevention of the respiratory infections.This study was supported by funds from Hamad Medical Corporation (grant # 16335/16) and Qatar University (grant # QUCG-BRC-2018/2019-1)

Profiling of intestinal microbiota in patients infected with respiratory influenza a and b viruses

Little is known about the association between respiratory viral infections and their impact on intestinal microbiota. Here, we compared the effect of influenza types, A and B, and influenza shedding in patients’ stools on the gut microbiota diversity and composition. Deep sequencing analysis was performed for the V4 region of the 16S rRNA gene. Fecal samples were collected from 38 adults with active respiratory influenza infection and 11 age-matched healthy controls. Influenza infection resulted in variations in intestinal bacterial community composition rather than in overall diversity. Overall, infected patients experienced an increased abundance of Bacteroidetes and a corresponding decrease in Firmicutes. Differential abundance testing illustrated that differences in gut microbiota composition were influenza type-dependent, identifying ten differentially abun-dant operational taxonomic units (OTUs) between influenza A-and influenza B-infected patients. Notably, virus shedding in fecal samples of some patients had significantly reduced gut bacterial diversity (p = 0.023). Further taxonomic analysis revealed that the abundance of Bacteroides fragilis was significantly higher among shedders compared to non-shedders (p = 0.037). These results pro-vide fundamental evidence of the direct effect of influenza infection on gut microbiota diversity, as reported in patients shedding the virus

Burden and disease pathogenesis of influenza and other respiratory viruses in diabetic patients

Over the past two decades, diabetes mellitus (DM) has been receiving increasing attention among autoimmune diseases. The prevalence of type 1 and type 2 diabetes has increased rapidly and has become one of the leading causes of death worldwide. Therefore, a better understanding of the genetic and environmental risk factors that trigger the onset of DM would help develop more efficient therapeutics and preventive measures. The role and mechanism of respiratory viruses in inducing autoimmunity have been frequently reported. On the other hand, the association of DM with respiratory infections might result in severe complications or even death. Since influenza is the most common respiratory infection, DM patients experience disease severity and increased hospitalization during influenza season. Vaccinating diabetic patients against influenza would significantly reduce hospitalization due to disease severity. However, recent studies also report the role of viral vaccines in inducing autoimmunity, specifically diabetes. This review reports causes of diabetes, including genetic and viral factors, with a special focus on respiratory viruses. We further brief the burden of influenza-associated complications and the effectiveness of the influenza vaccine in DM patients

Molecular epidemiology of influenza, RSV, and other respiratory infections among children in Qatar: A six years report (2012–2017)

Background: Studies on the etiology of respiratory infections among children in Qatar and surrounding countries are limited. Objectives: To describe the prevalence and seasonality of RSV, influenza, and other respiratory pathogens among children in Qatar. Methods: We retrospectively collected and analyzed data of 33,404 children (<15 years) presented with influenza-like illness from 2012 to 2017. Results: At least one respiratory pathogen was detected in 26,138 (78%) of patients. Together, human rhinoviruses (HRV), respiratory syncytial virus (RSV), and influenza viruses comprised nearly two-thirds of all cases, affecting 24%, 19.7%, and 18.5%, respectively. A prevalence of 5-10% was recorded for adenovirus, parainfluenza viruses (PIVs), human bocavirus (HboV), and human coronaviruses (HCoVs). Human metapneumovirus (HMPV), enteroviruses, M. pneumonia, and parechovirus had prevalences below 5%. While RSV, influenza, and HMPV exhibited strong seasonal activity in the winter, HRV was active during low RSV and influenza circulation. The burden of RSV exceeds that of influenza among young age groups, whereas influenza correlated positively with age. Further, HRV, adenovirus, influenza, and RSV infection rates varied significantly between male and females. Conclusion: This comprehensive multi-year study provides insights into the etiology of ILI among children in Qatar, which represents the Gulf region. Our results reinforce the significance of active surveillance of respiratory pathogens to improve infection prevention and control strategies, particularly among children

Comparative analysis of within-host diversity among vaccinated COVID-19 patients infected with different SARS-CoV-2 variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly evolving RNA virus that mutates within hosts and exists as viral quasispecies. Here, we evaluated the within-host diversity among vaccinated and unvaccinated individuals (n = 379) infected with different SARS-CoV-2 Variants of Concern. The majority of samples harbored less than 14 intra-host single-nucleotide variants (iSNVs). A deep analysis revealed a significantly higher intra-host diversity in Omicron samples than in other variants (p value < 0.05). Vaccination status and type had a limited impact on intra-host diversity except for Beta-B.1.315 and Delta-B.1.617.2 vaccinees, who exhibited higher diversity than unvaccinated individuals (p values: <0.0001 and <0.0021, respectively). Three immune-escape mutations were identified: S255F in Delta and R346K and T376A in Omicron-B.1.1.529. The latter 2 mutations were fixed in BA.1 and BA.2 genomes, respectively. Overall, the relatively higher intra-host diversity among vaccinated individuals and the detection of immune-escape mutations, despite being rare, suggest a potential vaccine-induced immune pressure in vaccinated individuals.The authors are grateful for the leadership and assistance provided by the Ministry of Public Health in Qatar, the virology laboratory staff at Hamad Medical Corporation, and Qatar Biobank (QBB) team. This project was funded by Qatar National Research Fund (QNRF; Project number UREP28-164-3-048) and Qatar University (Project number QUCG-BRC-22/23-547). The article processing charges were paid from grant no. QUCG-BRC-2022/23-578

Pfizer-BioNTech mRNA BNT162b2 Covid-19 vaccine protection against variants of concern after one versus two doses.

Key messages and recommendations Thispopulation-based study documentsBNT162b2vaccine protection week-by-week after the first dose. 75% of protection against infection and disease is reached 15-21 daysafter the first dose. Protection increased most rapidly against hospitalization and death and slowest against B.1.351infection.While protection of one dose beyond 21 days could not be assessed, findings support delaying the second vaccinedosein situations of limited vaccine suppliesand high incidences

Immune Imprinting and Protection against Repeat Reinfection with SARS-CoV-2

More than 2 years into the coronavirus disease 2019 (Covid-19) pandemic, the global population carries heterogeneous immune histories derived from various exposures to infection, viral variants, and vaccination.1 Evidence at the level of binding and neutralizing antibodies and B-cell and T-cell immunity suggests that a history of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can have a negative effect on subsequent protective immunity.1 In particular, the immune response to B.1.1.529 (omicron) subvariants could be compromised by differential immune imprinting in persons who have had a previous infection with the original virus or the B.1.1.7 (alpha) variant.