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Clusters of nosocomial coronavirus disease 2019 (COVID-19) have been reported globally during the recent pandemic. Unfortunately, these clusters negatively affect inpatient morbidity, mortality, and hospital functioning. Using epidemiological data and whole-genome sequencing (WGS) of SARS-CoV-2, this study investigated the outbreak of COVID-19 at a university hospital. Eight inpatients and 13 healthcare workers tested positive for SARS-CoV-2 during a 1-month period. WGS of the virus in 11 patients revealed that the two variants of concern belonging to the Omicron sublineages, BA.2.3 and BA1.1.2, caused an outbreak when the proportion of the Omicron lineage in the community changed. When variants of concern undergo mutation, a response to the outbreak should be made with multiple variants in mind, even in the absence of epidemiological data showing close contact or other potential vectors of infection. Awareness of infection prevention and control should be raised to safeguard patient safety.

During the COVID-19 pandemic, fungal infections, especially pulmonary aspergillosis, mucormycosis, and invasive candidiasis, have emerged as a significant health concern. Beyond Candida albicans, the most common cause of invasive candidiasis, other rare ascomycetous yeast species have been described in tertiary care units, potentially posing a broader health threat. We have isolated, from September 2020 to June 2021, nine Diutina catenulata strains from urine samples of six patients. This was intriguing as this fungus had not been previously identified in our institution, nor after June 2021. Therefore, we decided to outline the clinical features of the patients with this rare pathogen, to describe phenotypic characteristics, including antifungal susceptibility profiles, of this yeast species and to identify the genetic makeup through whole-genome sequencing analysis to evaluate if this was a cluster of genetically similar D. catenulata isolates in our institution. The strains were identified through MALDI-TOF MS analyses and Sanger sequencing of two rDNA regions. All patients yielding D. catenulata from urine samples needed ventilator support and used urinary catheters during hospitalization for treatment of COVID-19. None of them had received COVID-19 vaccines. Morphological and biochemical profiles of the nine strains were largely consistent, although fluconazole susceptibility varied, ranging from 4 to 32 μg/mL. Phylogenomic analysis revealed minimal genetic variation among the isolates, with low intrapopulation variation, supported by the identification of only 84 SNPs across all strains. Therefore, we propose that the yeast strains isolated were part of a cluster of D. catenulata funguria in the context of COVID-19., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Respiratory syncytial virus (RSV) is the most predominant viral pathogen worldwide in children with lower respiratory tract infections. The Coronavirus disease 2019 (COVID-19) pandemic and resulting nonpharmaceutical interventions perturbed the transmission pattern of respiratory pathogens in South Africa. A seasonality shift and RSV resurgence was observed in 2020 and 2021, with several infected children observed. Conventional RSV-positive nasopharyngeal swabs were collected from various hospitals in the Free State province, Bloemfontein, South Africa, from children suffering from respiratory distress and severe acute respiratory infection between 2020 to 2021. Overlapping genome fragments were amplified and complete genomes were sequenced using the Illumina MiSeq platform. Maximum likelihood phylogenetic and evolutionary analysis were performed on both RSV-A/-B G-genes with published reference sequences from GISAID and GenBank. Our study strains belonged to the RSV-A GA2.3.2 and RSV-B GB5.0.5a clades. The upsurge of RSV was due to pre-existing strains that predominated in South Africa and circulating globally also driving these off-season RSV outbreaks during the COVID-19 pandemic. The variants responsible for the resurgence were phylogenetically related to pre-pandemic strains and could have contributed to the immune debt resulting from pandemic imposed restrictions. The deviation of the RSV season from the usual pattern affected by the COVID-19 pandemic highlights the need for ongoing genomic surveillance and the identification of genetic variants to prevent unforeseen outbreaks in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Vaccines and host genetic factors can influence the SARS-CoV-2 evolution and emergence of new variants. Even vaccinated cases get affected as virus continues to evolve, raising concerns about vaccine efficacy and the emergence of immune escape variants. Here, we have analyzed 2295 whole-genome sequences of SARS-CoV-2 collected from vaccinated and unvaccinated cases to evaluate the impact of vaccines on virus diversity within hosts. Our comparative analysis revealed a significant higher incidence of intra-host single nucleotides variants (iSNVs) in vaccinated cases compared to unvaccinated ones (p value<0.0001). Furthermore, we have found that specific mutational processes, including APOBEC (C > T) mediated and ADAR1 (A > G) mediated mutations, were found more prevalent in vaccinated cases. Vaccinated cases exhibited higher accumulation of nonsynonymous mutation than unvaccinated cases. Fixed iSNVs were predominantly located in the ORF1ab and spike genes, several key omicron defining immune escape variants S477N, Q493R, Q498R, Y505H, L452R, and N501Y were identified in the RBD domain of spike gene in vaccinated cases. Our findings suggest that vaccine plays an important role in the evolution of the virus genome. The virus genome acquires random mutations due to error-prone replication of the virus, host modification through APOBEC and ADAR1 mediated editing mechanism, and oxidative stress. These mutations become fixed in the viral population due to the selective pressure imposed by vaccination., Competing Interests: Declaration of competing interest The authors declared no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Background: SARS-CoV-2 variants of concern (VOCs) emerged and rapidly replaced the original strain worldwide. The increased transmissibility of these new variants led to increases in infections, hospitalizations, and mortality. However, there is a scarcity of retrospective investigations examining the severity of all the main VOCs in presence of key public health measures and within various social determinants of health (SDOHs)., Objective: This study aims to provide a retrospective assessment of the clinical severity of COVID-19 VOCs in the context of heterogenous SDOHs and vaccination rollout., Methods: We used a population-based retrospective cohort design with data from the British Columbia COVID-19 Cohort, a linked provincial surveillance platform. To assess the relative severity (hospitalizations, intensive care unit [ICU] admissions, and deaths) of Gamma, Delta, and Omicron infections during 2021 relative to Alpha, we used inverse probability treatment weighted Cox proportional hazard modeling. We also conducted a subanalysis among unvaccinated individuals, as assessed severity differed across VOCs and SDOHs., Results: We included 91,964 individuals infected with a SARS-CoV-2 VOC (Alpha: n=20,487, 22.28%; Gamma: n=15,223, 16.55%; Delta: n=49,161, 53.46%; and Omicron: n=7093, 7.71%). Delta was associated with the most severe disease in terms of hospitalization, ICU admissions, and deaths (hospitalization: adjusted hazard ratio [aHR] 2.00, 95% CI 1.92-2.08; ICU: aHR 2.05, 95% CI 1.91-2.20; death: aHR 3.70, 95% CI 3.23-4.25 relative to Alpha), followed generally by Gamma and then Omicron and Alpha. The relative severity by VOC remained similar in the unvaccinated individual subanalysis, although the proportion of individuals infected with Delta and Omicron who were hospitalized was 2 times higher in those unvaccinated than in those fully vaccinated. Regarding SDOHs, the proportion of hospitalized individuals was higher in areas with lower income across all VOCs, whereas among Alpha and Gamma infections, 2 VOCs that cocirculated, differential distributions of hospitalizations were found among racially minoritized groups., Conclusions: Our study provides robust severity estimates for all VOCs during the COVID-19 pandemic in British Columbia, Canada. Relative to Alpha, we found Delta to be the most severe, followed by Gamma and Omicron. This study highlights the importance of targeted testing and sequencing to ensure timely detection and accurate estimation of severity in emerging variants. It further sheds light on the importance of vaccination coverage and SDOHs in the context of pandemic preparedness to support the prioritization of allocation for resource-constrained or minoritized groups., (©Sean P Harrigan, Héctor A Velásquez García, Younathan Abdia, James Wilton, Natalie Prystajecky, John Tyson, Chris Fjell, Linda Hoang, Jeffrey C Kwong, Sharmistha Mishra, Linwei Wang, Beate Sander, Naveed Z Janjua, Hind Sbihi. Originally published in JMIR Public Health and Surveillance (https://publichealth.jmir.org), 27.08.2024.)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to challenge the global healthcare with emerging variants and higher infectivity as well as morbidities. This study investigated potential age-related variations through genomic characterization of the virus under common clinical settings. A cohort comprising 71 SARS-CoV-2 strains from both infected infants and accompanying adults, diagnosed via RT-PCR at a tertiary pediatric hospital and research center, underwent Illumina paired-end sequencing. The subsequent analysis involved standard genomic screening, phylogeny construction, and mutational analyses. The analyzed SARSCoV- 2 strains were compared with globally circulating variants. The overall distribution revealed 67.61 % Delta, 25.7 % Omicron, and 1 % either Kappa or Alpha variants. In 2021, Delta predominated at ∼ 94 %, with Alpha/Kappa accounting for around 5 %. However, in 2022, over 94 % of the samples were Omicron variants, signifying a substantial shift from Delta dominance. Delta variants constituted 69.5 % of infections in adults and 78.5 % in infants, while Omicron variants were responsible for 31 % of cases in infants and 18 % in adults. The Spike region harbored the majority of mutations, with T19R being the most prevalent mutation in the Delta lineage. Notably, the frequencies of this mutation varied between infants and adults. In Omicron samples, G142D emerged as the most prevalent mutation. Our dataset predominantly featured clade 21A and lineage B.1.617.2. This study underscores the differential clinical presentations and genomic characteristics of SARS-CoV-2 in pediatric patients and accompanying adults. Understanding the dynamic evolution of the SARS- CoV-2 in both pediatric and adults can help in strengthening prophylactic measures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Severe COVID-19 has been associated with coinfections with bacterial and fungal pathogens. Notably, patients with COVID-19 who develop Staphylococcus aureus bacteremia exhibit higher rates of mortality than those infected with either pathogen alone. To understand this clinical scenario, we collected and examined S. aureus blood and respiratory isolates from a hospital in New York City during the early phase of the pandemic from both SARS-CoV-2+ and SARS-CoV-2- patients. Whole genome sequencing of these S. aureus isolates revealed broad phylogenetic diversity in both patient groups, suggesting that SARS-CoV-2 coinfection was not associated with a particular S. aureus lineage. Phenotypic characterization of the contemporary collection of S. aureus isolates from SARS-CoV-2+ and SARS-CoV-2- patients revealed no notable differences in several virulence traits examined. However, we noted a trend toward overrepresentation of S. aureus bloodstream strains with low cytotoxicity in the SARS-CoV-2+ group. We observed that patients coinfected with SARS-CoV-2 and S. aureus were more likely to die during the acute phase of infection when the coinfecting S. aureus strain exhibited high or low cytotoxicity. To further investigate the relationship between SARS-CoV-2 and S. aureus infections, we developed a murine coinfection model. These studies revealed that infection with SARS-CoV-2 renders mice susceptible to subsequent superinfection with low cytotoxicity S. aureus . Thus, SARS-CoV-2 infection sensitizes the host to coinfections, including S. aureus isolates with low intrinsic virulence., Importance: The COVID-19 pandemic has had an enormous impact on healthcare across the globe. Patients who were severely infected with SARS-CoV-2, the virus causing COVID-19, sometimes became infected with other pathogens, which is termed coinfection. If the coinfecting pathogen is the bacterium Staphylococcus aureus , there is an increased risk of patient death. We collected S. aureus strains that coinfected patients with SARS-CoV-2 to study the disease outcome caused by the interaction of these two important pathogens. We found that both in patients and in mice, coinfection with an S. aureus strain lacking toxicity resulted in more severe disease during the early phase of infection, compared with infection with either pathogen alone. Thus, SARS-CoV-2 infection can directly increase the severity of S. aureus infection., Competing Interests: V.J.T. has consulted for Janssen Research & Development, LLC, and has received honoraria from Genentech and Medimmune. He is also an inventor on patents and patent applications filed by New York University, which are currently under commercial license to Janssen Biotech Inc. Janssen Biotech Inc. had provided research funding and other payments associated with a licensing agreement. B.S. has consulted for Regeneron and MicroGenDx. K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and AbbVie. R.T. completed a NYU-Regeneron postdoctoral training program in laboratory animal medicine.

The first case of COVID-19 was detected in Bangladesh on 8 March 2020. Since then, the Government of Bangladesh (GoB) has implemented various measures to limit the transmission of COVID-19, including widespread testing facilities across the nation through a laboratory network for COVID-19 molecular testing. This study aimed to analyze the dynamics of SARS-CoV-2 in Bangladesh by conducting COVID-19 testing and genomic surveillance of the virus variants throughout the pandemic. Nasopharyngeal swabs were collected from authorized GoB collection centers between April 2020 and June 2023. The viral RNA was extracted and subjected to real-time PCR analysis in icddr,b's Virology laboratory. A subset of positive samples underwent whole-genome sequencing to track the evolutionary footprint of SARS-CoV-2 variants. We tested 149,270 suspected COVID-19 cases from Dhaka (n = 81,782) and other districts (n = 67,488). Of these, 63% were male. The highest positivity rate, 27%, was found in the >60 years age group, followed by 26%, 51-60 years, 25% in 41-50 years, and the lowest, 9% in under five children. Notably, the sequencing of 2742 SARS-CoV-2 genomes displayed a pattern of globally circulating variants, Alpha, Beta, Delta, and Omicron, successively replacing each other over time and causing peaks of COVID-19 infection. Regarding the risk of SARS-CoV-2 infection, it was observed that the positivity rate increased with age compared to the under-5 age group in 2020 and 2021. However, these trends did not remain consistent in 2022, where older age groups, particularly those over 60, had a lower positivity rate compared to other age groups due to vaccination. The study findings generated data on the real-time circulation of different SARS-CoV-2 variants and the upsurge of COVID-19 cases in Bangladesh, which impacted identifying hotspots and restricting the virus from further transmission. Even though there is currently a low circulation of SARS-CoV-2 in Bangladesh, similar approaches of genomic surveillance remain essential for monitoring the emergence of new SARS-CoV-2 variants or other potential pathogens that could lead to future pandemics.

Application of whole genome sequencing (WGS) has allowed monitoring of the emergence of variants of concern (VOC) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) globally. Genomic investigation of emerging variants and surveillance of clinical progress has reduced the public health impact of infection during the COVID-19 pandemic. These steps required developing and implementing a proficiency testing program (PTP), as WGS has been incorporated into routine reference laboratory practice. In this study, we describe how the PTP evaluated the capacity and capability of one New Zealand and 14 Australian public health laboratories to perform WGS of SARS-CoV-2 in 2022. The participants' performances in characterising a specimen panel of known SARS-CoV-2 isolates in the PTP were assessed based on: (1) genome coverage, (2) Pango lineage, and (3) sequence quality, with the choice of assessment metrics refined based on a previously reported assessment conducted in 2021. The participants' performances in 2021 and 2022 were also compared after reassessing the 2021 results using the more stringent metrics adopted in 2022. We found that more participants would have failed the 2021 assessment for all survey samples and a significantly higher fail rate per sample in 2021 compared to 2022. This study highlights the importance of choosing appropriate performance metrics to reflect better the laboratories' capacity to perform SARS-CoV-2 WGS, as was done in the 2022 PTP. It also displays the need for a PTP for WGS of SARS-CoV-2 to be available to public health laboratories ongoing, with continuous refinements in the design and provision of the PTP to account for the dynamic nature of the COVID-19 pandemic as SARS-CoV-2 continues to evolve., (Copyright © 2024 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Background: DNA methylation integrates environmental signals with transcriptional programs. COVID-19 infection induces changes in the host methylome. While post-acute sequelae of COVID-19 (PASC) is a long-term complication of acute illness, its association with DNA methylation is unknown. No universal blood marker of PASC, superseding single organ dysfunctions, has yet been identified., Methods: In this single centre prospective cohort study, PASC, post-COVID without PASC, and healthy participants were enrolled to investigate their symptoms association with peripheral blood DNA methylation data generated with state-of-the-art whole genome sequencing. PASC-induced quality-of-life deterioration was scored with a validated instrument, SF-36. Analyses were conducted to identify potential functional roles of differentially methylated loci, and machine learning algorithms were used to resolve PASC severity., Findings: 103 patients with PASC (22.3% male, 77.7% female), 15 patients with previous COVID-19 infection but no PASC (40.0% male, 60.0% female), and 27 healthy volunteers (48.1% male, 51.9% female) were enrolled. Whole genome methylation sequencing revealed 39 differentially methylated regions (DMRs) specific to PASC, each harbouring an average of 15 consecutive positions, that differentiate patients with PASC from the two control groups. Motif analyses of PASC-regulated DMRs identify binding domains for transcription factors regulating circadian rhythm and others. Some DMRs annotated to protein coding genes were associated with changes of RNA expression. Machine learning support vector algorithm and random forest hierarchical clustering reveal 28 unique differentially methylated positions (DMPs) in the genome discriminating patients with better and worse quality of life., Interpretation: Blood DNA methylation levels identify PASC, stratify PASC severity, and suggest that DNA motifs are targeted by circadian rhythm-regulating pathways in PASC., Funding: This project has been funded by the following agencies: NIH-AI173035 (A. Jaitovich and R. Alisch); and NIH-AG066179 (R. Alisch)., Competing Interests: Declaration of interests AM, RA and AJ share pending patent application (20220364187): “Detecting, predicting severity of, and/or predicting treatment response to respiratory virus infection”., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The COVID-19 pandemic has significantly impacted education systems worldwide, with Brazil being one of the countries with the longest school closures. Over a million children and teenagers have been affected, leading to increased hunger and nutritional deficiencies. This study aimed to implement long-term surveillance of SARS-CoV-2 infections in public and private schools in Campo Grande, Brazil, after returning to in-person classes., Methods: The study involved testing and genomic surveillance at 23 public and private schools in Campo Grande, Mato Grosso do Sul, Brazil, from October 18, 2021 to November 21, 2022. The participants eligible for enrollment were students aged 6-17 years and staff members from school institutions. At the time of collection, participants were asked if they had symptoms in the last two weeks. Whole-genome sequencing of SARS-CoV-2 was conducted to identify circulating variants and to compare them with those detected in the municipality. The demographic data and clinical history of the participants were described, and a logistic regression model was used to understand how the RT-qPCR results could be related to different characteristics., Results: The study included 999 participants, most of whom were women. A total of 85 tests were positive, with an overall positivity rate of 3.2%. The dynamics of case frequency were consistent with those observed in the municipality during the study period. The most common symptoms reported were cough, rhinorrhea, headache, and sore throat. Symptoms were significantly associated with SARS-CoV-2 infection. Eleven lineages were identified in school community samples, with a frequency of occurrence per period similar to that found in the sequences available for the municipality. The most prevalent lineages within the sampling period were BA.2 (59.3%) and BA.5 (29.6%)., Conclusions: Our findings demonstrate that schools can play a crucial role in epidemiological surveillance, helping trigger rapid responses to pathogens such as SARS-CoV-2. Long-term surveillance can be used to track outbreaks and assess the role of children and adults in transmission. It can also contribute to pandemic preparedness, enabling a rapid response to emergencies, such as COVID-19., (© 2024. The Author(s).)

In December 2023, we observed a notable shift in the COVID-19 landscape, when JN.1 omicron emerged as the predominant SARS-CoV-2 variant with a 95% incidence. We characterized the clinical profile, and genetic changes in JN.1, an emerging SARS-CoV-2 variant of interest. Whole genome sequencing was performed on SARS-CoV-2 positive clinical specimens, followed by sequence analysis. Mutations within the spike protein sequences were analysed and compared with the previously reported lineages and sub-lineages, to identify the potential impact of the unique mutations on protein structure and possible alterations in the functionality. Several unique and dynamic mutations were identified herein. Molecular docking analysis showed changes in the binding affinity, and key interacting residues of wild-type and mutated structures with key host cell receptors of SARS-CoV-2 entry viz., ACE2, CD147, CD209L and AXL. Our data provides key insights on the emergence of newer variants and highlights the necessity for robust and sustained global genomic surveillance of SARS-CoV-2., (© 2024. The Author(s).)

Background: Respiratory syncytial virus (RSV), a highly transmissible virus, is the leading cause of lower respiratory tract infections. We examined molecular changes in the RSV genome before and after the coronavirus disease 2019 (COVID-19) pandemic in Korea, and investigated whether drug-resistant mutations were present., Methods: In this prospective, single-center study, RSV-positive respiratory samples were collected between September 2019 and December 2022. Long-read whole-genome sequencing (WGS) was performed, and the presence of known drug-resistant substitutions for palivizumab, nirsevimab, and suptavumab was investigated., Results: Overall, 288 respiratory samples were collected from 276 children. WGS data were available for 133 samples (71 and 62 samples from the pre- and post-pandemic periods, respectively). All RSV-A strains (n = 56) belonged to the GA2.3.5 (ON1) genotype, whereas all RSV-B strains (n = 77) belonged to the GB5.0.5a (BA) genotype. No significant differences in genotypes were observed between the pre- and post-pandemic periods. In addition, no notable mutations related to nirsevimab or palivizumab resistance were detected in the F gene. However, the L172Q and S173L substitutions, which are known to confer resistance to suptavumab, were present in all RSV-B samples., Conclusion: Despite the unprecedented interruption of RSV seasonality, there were no significant molecular changes in circulating RSV strains in Korea related to nirsevimab or palivizumab resistance before and after the COVID-19 pandemic. However, RSV-specific drug-resistance substitutions for suptavumab were identified., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2024 The Korean Academy of Medical Sciences.)

This study examines the epidemiological and genomic characteristics, along with the transmission dynamics, of SARS-CoV-2 within prison units I and II in Campo Grande, Mato Grosso do Sul, Brazil. Conducted between May and October 2022, it reveals how the virus spreads in the confined settings of prisons, emphasizing the roles of overcrowded cells, frequent transfers, and limited healthcare access. The research involved 1927 participants (83.93% of the total prison population) and utilized nasopharyngeal swabs and RT-qPCR testing for detection. Contact tracing monitored exposure within cells. Out of 2108 samples, 66 positive cases were identified (3.13%), mostly asymptomatic (77.27%), with the majority aged 21-29 and varying vaccination statuses. Next-generation sequencing generated 28 whole genome sequences, identifying the Omicron variant (subtypes BA.2 and BA.5) with 99% average coverage. Additionally, the study seeks to determine the relationship between immunization levels and the incidence of SARS-CoV-2 cases within this enclosed population. The findings underscore the necessity of comprehensive control strategies in prisons, including rigorous screening, isolation protocols, vaccination, epidemiological monitoring, and genomic surveillance to mitigate disease transmission and protect both the incarcerated population and the broader community.

Determining the genetic contribution of susceptibility to severe SARS-CoV-2 infection outcomes is important for public health measures and individualized treatment. Through intense research on this topic, several hundred genes have been implicated as possibly contributing to the severe infection phenotype(s); however, the findings are complex and appear to be population-dependent. We aimed to determine the contribution of human rare genetic variants associated with a severe outcome of SARS-CoV-2 infections and their burden in the Slovenian population. A panel of 517 genes associated with severe SARS-CoV-2 infection were obtained by combining an extensive review of the literature, target genes identified by the COVID-19 Host Genetic Initiative, and the curated Research COVID-19 associated genes from PanelApp, England Genomics. Whole genome sequencing was performed using PCR-free WGS on DNA from 60 patients hospitalized due to severe COVID-19 disease, and the identified rare genomic variants were analyzed and classified according to the ACMG criteria. Background prevalence in the general Slovenian population was determined by comparison with sequencing data from 8025 individuals included in the Slovenian genomic database (SGDB). Results show that several rare pathogenic/likely pathogenic genomic variants in genes CFTR , MASP2 , MEFV , TNFRSF13B , and RNASEL likely contribute to the severe infection outcomes in our patient cohort. These results represent an insight into the Slovenian genomic diversity associated with a severe COVID-19 outcome.

Experimental evidence suggests that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) remains viable within aerosols with a half-life of approximately 3 hours; however, it remains unclear how long airborne SARS-CoV-2 can transmit infection. Whole genome sequencing during an outbreak suggested in-room transmission of SARS-CoV-2 to two patients admitted nearly 2 and 5 hours, respectively, after discharge of an asymptomatic infected patient. These findings suggest that airborne SARS-CoV-2 may transmit infection for over 4 hours, even in a hospital setting., (Published by Elsevier Inc.)

Monitoring the presence of RNA from emerging pathogenic viruses, such as SARS-CoV-2, in wastewater (WW) samples requires suitable methods to ensure an effective response. Genome sequencing of WW is one of the crucial methods, but it requires high-quality RNA in sufficient quantities, especially for monitoring emerging variants. Consequently, methods for viral concentration and RNA extraction from WW samples have to be optimized before sequencing. The purpose of this study was to achieve high coverage (≥ 90 %) and sequencing depth (at least ≥200×) even for low initial RNA concentrations (< 10 5 genome copies (GC)/L) in WW. A further objective was to determine the range of SARS-CoV-2 RNA concentrations that allow high-quality sequencing, and the optimal sample volume for analysis. Ultrafiltration (UF) methods were used to concentrate viral particles from large influent samples (up to 500 mL). An RNA extraction protocol using silica beads, neutral phenol-chloroform treatment, and a PCR inhibitor removal kit was chosen for its effectiveness in extracting RNA and eliminating PCR inhibitors, as well as its adaptability for use with large influent samples. Recovery rates ranged from 24 % to 63 % (N = 17) for SARS-CoV-2 naturally present in WW samples. 200 mL WW samples can be enough for UF concentration, as they showed high quality sequencing analyses with between 5 × 10 4 GC/L and 6 × 10 3 GC/L. Below 6 × 10 3 GC/L, high-quality sequencing was also achieved for ∼40 % of the samples using 500 mL of WW. Sequencing analysis for variant detection was performed on 200 mL WW samples with coverage of >95 % and sequencing depth of >1000×. Analyses revealed the predominance of variant EG.5, known as Eris (66 %-100 %). The use of UF methods in combination with a suitable RNA extraction protocol appear promising for sequencing enveloped viruses in WW in a context of viral emergence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Isabelle Bertrand, Olivier Rohr, Nicolas Boudaud reports financial support was provided by ANRS. Isabelle Bertrand, Christophe Gantzer, Vincent Marechal, Nicolas Boudaud, Olivier Rohr reports a relationship with OBEPINE that includes: board membership. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Tracking new variants of SARS-CoV-2 is vital for managing COVID-19 spread and allocating resources. Domestic antigen testing has created surveillance gaps that make it hard to identify new viral variants. We conducted whole genome sequencing of wastewater viral genes from major and minor treatment facilities in Dehradun from March 2022 onwards. Based on our analysis, the samples that achieved higher sequencing depth and covered >90 % of the viral genome uncovered a major variant pattern resembling the XAP recombinant lineage that is reported for the first time in the City of Dehradun, Uttrakhand and is the first ever records in India as on date. This novel XAP recombinant lineage had 9, 2, 30, 1, 2, 5, 1, 1, 1 aminoacid changes (total 54 mutations) in Orf1a, Orf1b, S, E, M, N, Orf3a, Orf6 and Orf8 regions of the gene respectively that shares 49 mutations common to the ancestral lineages BA.1* and BA.2*, with 6 unique mutations. Subsequent comparison and analysis of the clinical sequence data from the region post-detection of this rare and unusual variant showed no causalities infected with the newly detected XAP lineage. These findings are indicative of future alarming situation with plausible threats of fresh spur of Omicron variant led infections in the urban community., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: In Senegal, molecular diagnosis was widely used for the detection and management of COVID-19 patients. However, genomic surveillance was very limited in the public sector. This study aimed to share the experience of a Senegalese public sector laboratory in response to the COVID-19 pandemic, and to describe the distribution of variants circulating in 2020 and 2021., Methodology: From July 2020 to December 2021, SARS-CoV-2 qRT-PCR was performed on nasopharyngeal samples from travelers and symptomatic patients at the Bacteriology and Virology Laboratory (LBV) of the Aristide le Dantec University Teaching Hospital. Samples with a cycle threshold (Ct) ≤ 30 were selected for whole-genome sequencing (WGS) using the Nanopore technology. In-house scripts were developed to study the spatial and temporal distribution of SARS-CoV-2 variants in Senegal, using our sequences and those retrieved from the GISAID database., Results: Of 8,207 patients or travelers screened for SARS-CoV-2, 970 (11.8%) were positive and 386 had a Ct ≤ 30. WGS was performed on 133 samples. Concomitantly with high-quality sequences deposited in the GISAID database covering nine cities in Senegal in 2020 and 2021 (n = 1,539), we observed a high circulation of the 20A (B.1, B.1.416 and B.1.620) and 20B (B.1.1.420) lineages in 2020, while most of the samples belonged to Delta variants (AY34 and AY.34.1, 22%) in 2021., Conclusions: Despite its late involvement, COVID-19 diagnosis was routinely performed in LBV, but genomic characterization remained challenging. The genomic diversity of SARS-CoV-2 strains in Senegal reflected that observed worldwide during the first waves of the pandemic., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Adjiratou Aissatou Ba, Romain Coppée, Assane Dieng, Jarra Manneh, Mengue Fall, Khadim Gueye, Pauline Yacine Sene, Samba Ndiour, Dianké Samaté, Pascaline Manga, Oumy Diop Diongue, Abdoulaye Camara, Abdou Padane, Sada Diallo; Sokhna Bousso Gueye; Makhtar Camara, Abdul Karim Sesay, Coumba Toure-Kane, Souleymane Mboup, Charlotte Charpentier, Halimatou Diop-Ndiaye.)

Background: Cell-free DNA (cfDNA) fragmentomic characteristics are promising analytes with abundant physiological signals for non-invasive disease diagnosis and monitoring. Previous studies on plasma cfDNA fragmentomics commonly employed a two-step centrifugation process for removing cell debris, involving a low-speed centrifugation followed by a high-speed centrifugation. However, the effects of centrifugation conditions on the analysis of cfDNA fragmentome remain uncertain., Methods: We collected blood samples from 10 healthy individuals and divided each sample into two aliquots for plasma preparation with one- and two-step centrifugation processes. We performed whole genome sequencing (WGS) of the plasma cfDNA in the two groups and comprehensively compared the cfDNA fragmentomic features. Additionally, we reanalyzed the fragmentomic features of cfDNA from 16 healthy individuals and 16 COVID-19 patients, processed through one- and two-step centrifugation in our previous study, to investigate the impact of centrifugation on disease signals., Results: Our results showed that there were no significant differences observed in the characteristics of nuclear cfDNA, including size, motif diversity score (MDS) of end motifs, and genome distribution, between plasma samples treated with one- and two-step centrifugation. The cfDNA size shortening in COVID-19 patients was observed in plasma samples with one- and two-step centrifugation methods. However, we observed a significantly higher relative abundance and longer size of cell-free mitochondrial DNA (mtDNA) in the one-step samples compared to the two-step samples. This difference in mtDNA caused by the one- and two-step centrifugation methods surpasses the pathological difference between COVID-19 patients and healthy individuals., Conclusions: Our findings indicate that one-step low-speed centrifugation is a simple and potentially suitable method for analyzing nuclear cfDNA fragmentation characteristics. These results offer valuable guidance for cfDNA research in various clinical scenarios., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion., (© 2024 Wiley Periodicals LLC.)

Background: Viral recombination that occurs by exchanging genetic materials between two viral genomes coinfecting the same host cells is associated with the emergence of new viruses with different virulence. Herein, we detected a patient coinfected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants and identified various recombinants in the SARS-CoV-2 full-length spike gene using long-read and Sanger sequencing., Methods: Samples from five patients in Japan with household transmission of coronavirus disease 2019 (COVID-19) were analyzed using molecular assays for detection and identification of SARS-CoV-2. Whole-genome sequencing was conducted using multiplex PCR with short-read sequencing., Results: Among the five SARS-CoV-2-positive patients, the mutation-specific assay identified the Delta variant in three, the Omicron variant in one, and an undetermined in one. The undermined patient was identified as Delta using whole-genome sequencing, but samples showed a mixed population of Delta and Omicron variants. This patient was analyzed for viral quasispecies by long-read and Sanger sequencing using a full-length spike gene amplicon. In addition to the Delta and Omicron sequences, the viral quasispecies analysis identified nine different genetic recombinant sequences with various breakpoints between Delta and Omicron sequences. The nine detected recombinant sequences in the spike gene showed over 99% identity with viruses that were detected during the Delta and Omicron cocirculation period from the United States and Europe., Conclusions: This study demonstrates that patients coinfected with different SARS-CoV-2 variants can generate various viral recombinants and that various recombinant viruses may be produced during the cocirculation of different variants., (© 2024 The Author(s). Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Reading the viral genome through whole genome sequencing (WGS) enables the detection of changes in the viral genome. The rapid changes in the SARS-CoV-2 viral genome may cause immune escape leading to an increase in the pathogenicity or infectivity. Monitoring mutations through genomic surveillance helps understand the amino acid changes resulting from the mutation. These amino acid changes, especially in the spike glycoprotein, may have implications on the pathogenicity of the virus by rendering it immune-escape. The region of Vidarbha in Maharashtra represents 31.6 % of the state's total area. It holds 21.3 % of the total population. In total, 7457 SARS-CoV-2 positive samples belonging to 16 Indian States were included in the study, out of which 3002 samples passed the sequencing quality control criteria. The metadata of 7457 SARS-CoV-2 positive samples included in the study was sourced from the Integrated Health Information Platform (IHIP). The metadata of 3002 sequenced samples, including the FASTA sequence, was submitted to the Global Initiative on Sharing Avian Influenza Data (GISAID) and the Indian biological data centre (IBDC). This study identified 104 different SARS-CoV-2 pango-lineages classified into 19 clades. We have also analysed the mutation profiles of the variants found in the study, which showed eight mutations of interest, including L18F, K417N, K417T, L452R, S477N, N501Y, P681H, P681R, and mutation of concern E484K in the spike glycoprotein region. The study was from November 2020 to December 2022, making this study the most comprehensive genomic surveillance of SARS-CoV-2 conducted for the region., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, in December 2019, it has spread rapidly, and many coronavirus disease (COVID-19) cases have occurred in Gwangju, South Korea. Viral mutations following the COVID-19 epidemic have increased interest in the characteristics of epidemics in this region, and pathogen genetic analysis is required for infection control and prevention., Methods: In this study, SARS-CoV-2 whole-genome analysis was performed on samples from patients with COVID-19 in Gwangju from 2020 to 2022 to identify the trends in COVID-19 prevalence and to analyze the phylogenetic relationships of dominant variants. B.41 and B.1.497 prevailed in 2020, the early stage of the COVID-19 outbreak; then, B.1.619.1 mainly occurred until June 2021. B.1.617.2, classified as sublineages AY.69 and AY.122, occurred continuously from July to December 2021. Since strict measures to strengthen national quarantine management had been implemented in South Korea until this time, the analysis of mutations was also able to infer the epidemiological relationship between infection transmission routes. Since the first identification of the Omicron variant in late December 2021, the spread of infection has been very rapid, and weekly whole-genome analysis of specimens has enabled us to monitor new Omicron sublineages occurring in Gwangju., Conclusions: Our study suggests that conducting regional surveillance in addition to nation-level genomic surveillance will enable more rapid and detailed variant surveillance, which will be helpful in the overall prevention and management of infectious diseases., (© 2024 The Author(s). Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

The social restriction measures implemented due to the COVID-19 pandemic have impacted the pattern of occurrences of respiratory viruses. According to surveillance results in the Gwangju region of South Korea, respiratory syncytial virus (RSV) did not occur during the 2020/2021 season. However, there was a delayed resurgence in the 2021/2022 season, peaking until January 2022. To analyze this, a total of 474 RSV positive samples were investigated before and after the COVID-19 pandemic. Among them, 73 samples were selected for whole-genome sequencing. The incidence rate of RSV in the 2021/2022 season after COVID-19 was found to be approximately three-fold higher compared to before the pandemic, with a significant increase observed in the age group from under 2 years old to under 5 years old. Phylogenetic analysis revealed that, for RSV-A, whereas four lineages were observed before COVID-19, only the A.D.3.1 lineage was observed during the 2021/2022 season post-pandemic. Additionally, during the 2022/2023 season, the A.D.1, A.D.3, and A.D.3.1 lineages co-circulated. For RSV-B, while the B.D.4.1.1 lineage existed before COVID-19, both the B.D.4.1.1 and B.D.E.1 lineages circulated after the pandemic. Although atypical RSV occurrences were not due to new lineages, there was an increase in the frequency of mutations in the F protein of RSV after COVID-19. These findings highlight the need to continue monitoring changes in RSV occurrence patterns in the aftermath of the COVID-19 pandemic to develop and manage strategies in response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects various mammalian species, with farmed minks experiencing the highest number of outbreaks. In Spain, we analyzed 67 whole genome sequences and eight spike sequences from 18 outbreaks, identifying four distinct lineages: B.1, B.1.177, B.1.1.7, and AY.98.1. The potential risk of transmission to humans raises crucial questions about mutation accumulation and its impact on viral fitness. Sequencing revealed numerous not-lineage-defining mutations, suggesting a cumulative mutation process during the outbreaks. We observed that the outbreaks were predominantly associated with different groups of mutations rather than specific lineages. This clustering pattern by the outbreaks could be attributed to the rapid accumulation of mutations, particularly in the ORF1a polyprotein and in the spike protein. Notably, the mutations G37E in NSP9, a potential host marker, and S486L in NSP13 were detected. Spike protein mutations may enhance SARS-CoV-2 adaptability by influencing trimer stability and binding to mink receptors. These findings provide valuable insights into mink coronavirus genetics, highlighting both host markers and viral transmission dynamics within communities.

Background: University spring break carries a two-pronged SARS-CoV-2 variant transmission risk. Circulating variants from universities can spread to spring break destinations, and variants from spring break destinations can spread to universities and surrounding communities. Therefore, it is critical to implement SARS-CoV-2 variant surveillance and testing strategies to limit community spread before and after spring break to mitigate virus transmission and facilitate universities safely returning to in-person teaching., Methods: We examined the SARS-CoV-2 positivity rate and changes in variant lineages before and after the university spring break for two consecutive years. 155 samples were sequenced across four time periods: pre- and post-spring break 2021 and pre- and post-spring break 2022; following whole genome sequencing, samples were assigned clades. The clades were then paired with positivity and testing data from over 50,000 samples., Results: In 2021, the number of variants in the observed population increased from four to nine over spring break, with variants of concern being responsible for most of the cases; Alpha percent composition increased from 22.2% to 56.4%. In 2022, the number of clades in the population increased only from two to three, all of which were Omicron or a sub-lineage of Omicron. However, phylogenetic analysis showed the emergence of distantly related sub-lineages. 2022 saw a greater increase in positivity than 2021, which coincided with a milder mitigation strategy. Analysis of social media data provided insight into student travel destinations and how those travel events may have impacted spread., Conclusions: We show the role that repetitive testing can play in transmission mitigation, reducing community spread, and maintaining in-person education. We identified that distantly related lineages were brought to the area after spring break travel regardless of the presence of a dominant variant of concern., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SW is currently employed by Illumina. BK, SP, JP, RD, LI, and AS are employed by Labcorp. The other authors declare no competing interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Napolitano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The JN.1 sub-variant is a new variant of the SARS-CoV-2 Omicron strain, derived from the BA.2.86 sub-variant. It was first detected in late 2023 and has quickly spread to many countries, becoming the most prevalent variant in some regions. JN.1 exhibits a unique mutation (L455S) in the spike protein compared to the BA.2.86 lineage, which may affect its transmissibility and immune evasion capabilities. JN.1 has been designated as a "variant of interest" by the World Health Organization due to its rapidly increasing spread and is being closely monitored for its impact on the COVID-19 pandemic. This study describes the emergence of SARS-CoV-2 JN.1 sub-variant in Tunisia, and reports its mutation profiles.Nasopharyngeal samples collected over a four-month period (October 2023 to January 2024) were subjected to RNA extraction and real-time RT-PCR confirmation of SARS-CoV-2 infection. The whole-genome sequencing was performed by an iSeq 100 sequencer and COVIDSeq kit reagents (Illumina, USA).Mutation analysis, using the NextClade platform and GISAID database, revealed the presence of JN.1 in 15 out of 80 positive cases (18.75%) during the study period.The emergence of JN.1 highlights the ongoing evolution of SARS-CoV-2 and the need for continued surveillance and research to better understand the characteristics and impact of emerging variants.

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses., Competing Interests: The authors declare no conflict of interest.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies., (© 2024 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Objective: This study aims to analyze the molecular characteristics of the novel coronavirus (SARS-CoV-2) Omicron variant BA.2.76 in Jining City, China., Methods: Whole-genome sequencing was performed on 87 cases of SARS-CoV-2 infection. Evolutionary trees were constructed using bioinformatics software to analyze sequence homology, variant sites, N-glycosylation sites, and phosphorylation sites., Results: All 87 SARS-CoV-2 whole-genome sequences were classified under the evolutionary branch of the Omicron variant BA.2.76. Their similarity to the reference strain Wuhan-Hu-1 ranged from 99.72 to 99.74%. In comparison to the reference strain Wuhan-Hu-1, the 87 sequences exhibited 77-84 nucleotide differences and 27 nucleotide deletions. A total of 69 amino acid variant sites, 9 amino acid deletions, and 1 stop codon mutation were identified across 18 proteins. Among them, the spike (S) protein exhibited the highest number of variant sites, and the ORF8 protein showed a Q27 stop mutation. Multiple proteins displayed variations in glycosylation and phosphorylation sites., Conclusion: SARS-CoV-2 continues to evolve, giving rise to new strains with enhanced transmission, stronger immune evasion capabilities, and reduced pathogenicity. The application of high-throughput sequencing technologies in the epidemic prevention and control of COVID-19 provides crucial insights into the evolutionary and variant characteristics of the virus at the genomic level, thereby holding significant implications for the prevention and control of the COVID-19 pandemic., (© 2024. The Author(s).)

In January 2021, the monitoring of circulating variants of SARS-CoV-2 was initiated in Germany under the Corona Surveillance Act, which was discontinued after July 2023. This initiative aimed to enhance pandemic containment, as specific amino acid changes, particularly in the spike protein, were associated with increased transmission and reduced vaccine efficacy. Our group conducted whole genome sequencing using the ARTIC protocol (currently V4) on Illumina's NextSeq 500 platform (and, starting in May 2023, on the MiSeq DX platform) for SARS-CoV-2 positive specimen from patients at Heidelberg University Hospital, associated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. In total, we sequenced 26,795 SARS-CoV-2-positive samples between January 2021 and July 2023. Valid sequences, meeting the requirements for upload to the German electronic sequencing data hub (DESH) operated by the Robert Koch Institute (RKI), were determined for 24,852 samples, and the lineage/clade could be identified for 25,912 samples. The year 2021 witnessed significant dynamics in the circulating variants in the Rhine-Neckar/Heidelberg region, including A.27.RN, followed by the emergence of B.1.1.7 (Alpha), subsequently displaced by B.1.617.2 (Delta), and the initial occurrences of B.1.1.529 (Omicron). By January 2022, B.1.1.529 had superseded B.1.617.2, dominating with over 90%. The years 2022 and 2023 were then characterized by the dominance of B.1.1.529 and its sublineages, particularly BA.5 and BA.2, and more recently, the emergence of recombinant variants like XBB.1.5. Since the global dominance of B.1.617.2, the identified variant distribution in our local study, apart from a time delay in the spread of new variants, can be considered largely representative of the global distribution. om a time delay in the spread of new variants, can be considered largely representative of the global distribution., Competing Interests: Declaration of competing interest None declared., (Copyright © 2023. Published by Elsevier B.V.)

Background: Early during the COVID-19 pandemic, it was important to better understand transmission dynamics of SARS-CoV-2, the virus that causes COVID-19. Household contacts of infected individuals are particularly at risk for infection, but delays in contact tracing, delays in testing contacts, and isolation and quarantine posed challenges to accurately capturing secondary household cases., Methods: In this study, 346 households in the Seattle region were provided with respiratory specimen collection kits and remotely monitored using web-based surveys for respiratory illness symptoms weekly between October 1, 2020, and June 20, 2021. Symptomatic participants collected respiratory specimens at symptom onset and mailed specimens to the central laboratory in Seattle. Specimens were tested for SARS-CoV-2 using RT-PCR with whole genome sequencing attempted when positive. SARS-CoV-2-infected individuals were notified, and their household contacts submitted specimens every 2 days for 14 days., Results: In total, 1371 participants collected 2029 specimens that were tested; 16 individuals (1.2%) within 6 households tested positive for SARS-CoV-2 during the study period. Full genome sequences were generated from 11 individuals within 4 households. Very little genetic variation was found among SARS-CoV-2 viruses sequenced from different individuals in the same household, supporting transmission within the household., Conclusions: This study indicates web-based surveillance of respiratory symptoms, combined with rapid and longitudinal specimen collection and remote contact tracing, provides a viable strategy to monitor households and detect household transmission of SARS-CoV-2., Trial Registration Identifier: NCT04141930, Date of registration 28/10/2019., (© 2024. The Author(s).)

Introduction: SARS-CoV-2 is known to infect respiratory tissue cells. However, less is known about infection of ocular tissue and potential infectivity of lacrimal fluid. With this study, we want to compare viral loads in eye and nasopharyngeal swabs and analyze these for infectious virus., Methods: Between May 2020 and April 2021 ocular and nasopharyngeal swabs were collected from 28 SARS-CoV-2 infected patients treated on the corona virus disease 2019 (COVID-19)-ward of the University Hospital of Innsbruck, Austria. Samples with PCR detectable SARS-CoV-2 were analyzed via whole genome sequencing and an attempt was made to isolate infectious virus., Results: At the time point of sample collection, 22 individuals were still PCR positive in nasopharyngeal samples and in 6 of these patients one or both ocular samples were additionally positive. CT-values in eyes were generally higher compared to corresponding nasopharyngeal samples and we observed a tendency for lower CT-values, i.e. increased viral load, in nasopharyngeal swabs of individuals with at least one infected eye, compared to those where ocular samples were PCR negative. Ocular and nasopharyngeal sequences from the same patient were assigned to the same variant, either the D614G or the Alpha variant. Infectious virus was successfully isolated from 9 nasopharyngeal swabs, however only from one of the seven PCR positive ocular samples., Conclusion: We could detect SARS-CoV-2 in eyes of some of the infected patients albeit at lower levels compared to nasopharyngeal swabs. However, our results also indicate that lacrimal fluid might be infectious in patients with high viral load., Competing Interests: DB declares to hold stocks of Pfizer and Oxford Nanopore Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kimpel, Rössler, Bante, Borena, von Laer, Zehetner, Rauchegger, Seiwald and Falkensammer.)

The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19), a disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a global pandemic in March 2020. Reverse transcription-polymerase chain reaction (RT-PCR) is the reference technique for molecular diagnosis of SARS-CoV-2 infection. The SARS-CoV-2 virus is constantly mutating, and more transmissible variants have emerged, making genomic surveillance a crucial tool for investigating virus transmission dynamics, detecting novel genetic variants, and assessing mutation impact. The S gene, which encodes the spike protein, is frequently mutated, and it plays an important role in transmissibility. Spike protein mutations affect infectivity and vaccine effectiveness. SARS-CoV-2 variants are tracked using whole genome sequencing (WGS) and S-gene analysis. WGS, Sanger sequencing, and many S-gene-targeted RT-PCR methods have been developed. WGS and Sanger sequencing are standard methods for detecting mutations and can be used to identify known and unknown mutations. Melting curve analysis, endpoint genotyping assay, and S-gene target failure are used in the RT-PCR-based method for the rapid detection of specific mutations in SARS-CoV-2 variants. Therefore, these assays are suitable for high-throughput screening. The combinatorial use of RT-PCR-based assays, Sanger sequencing, and WGS enables rapid and accurate tracking of SARS-CoV-2 variants. In this review, we described RT-PCR-based detection and surveillance techniques for SARS-CoV-2., (Copyright © 2024. Published by Elsevier Inc.)

The evolution of SARS-CoV-2 within immunocompromised hosts who fail to clear the virus over many months has been proposed as a route to the development of Variants of Concern (VoCs). We present a case of an immunocompromised male patient with a prolonged SARS-CoV-2 infection. During hospitalisation, 7 weeks after first diagnosis, his condition worsened to require continuous ventilation support. Resolution of symptoms was observed after convalescent plasma therapy. Whole genome sequencing of the virus showed Pango lineage B.1.221. Between the first sample and the second from bronchoalveolar lavage fluid 7 weeks later, we identified eight mutations, including minor variants, which could be used to estimate the chronology of mutations. This suggests an elevated mutation rate, in-host accumulation of mutations and further evidence for sources of VoCs. Prolonged SARS-CoV-2 infections in immunocompromised hosts increase the likelihood of hospital stays and morbidity, and also pose an increased risk to global public health., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

ABSTRACT Global and even national genome surveillance approaches do not provide the resolution necessary for rapid and accurate direct response by local public health authorities. Hence, a regional network of microbiological laboratories in collaboration with the health departments of all districts of the German federal state of Mecklenburg-Western Pomerania (M-V) was formed to investigate the regional molecular epidemiology of circulating SARS-CoV-2 lineages between 11/2020 and 03/2022. More than 4750 samples from all M-V counties were sequenced using Illumina and Nanopore technologies. Overall, 3493 (73.5%) sequences fulfilled quality criteria for time-resolved and/or spatially-resolved maximum likelihood phylogenic analyses and k-mean/ median clustering (KMC). We identified 116 different Pangolin virus lineages that can be assigned to 16 Nextstrain clades. The ten most frequently detected virus lineages belonged to B.1.1.7, AY.122, AY.43, BA.1, B.1.617.2, BA.1.1, AY.9.2, AY.4, P.1 and AY.126. Time-resolved phylogenetic analyses showed the occurrence of virus clades as determined worldwide, but with a substantial delay of one to two months. Further spatio-temporal phylogenetic analyses revealed a regional outbreak of a Gamma variant limited to western M-V counties. Finally, KMC elucidated a successive introduction of the various virus lineages into M-V, possibly triggered by vacation periods with increased (inter-) national travel activities. The COVID-19 pandemic in M-V was shaped by a combination of several SARS-CoV-2 introductions, lockdown measures, restrictive quarantine of patients and the lineage specific replication rate. Complementing global and national surveillance, regional surveillance adds value by providing a higher level of surveillance resolution tailored to local health authorities.

The emergence of the SARS-CoV-2 Beta (B.1.351) variant in November 2020 raised concerns of increased transmissibility and severity. We describe the epidemiology of 949 confirmed SARS-CoV-2 Beta variant cases in England, identified between December 2020 and June 2022. Most cases were detected in the first 3 months. A total of 10 deaths (1.1%; 10/949) were identified among all cases and of those with travel information, 38 (4.9%; 38/781) cases with hospital admissions within 14 days of a positive test being detected. 52.9% (413/781) cases were imported. This study reinforces the importance of monitoring of travel-associated cases to inform public health response and reduce transmissibility of new variants., (© 2023 Crown copyright. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.)

The ongoing coronavirus disease 2019 (COVID-19) pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the critical role of genomic surveillance in tracking rapidly spreading viruses and their evolving lineages. The emergence of the SARS-CoV-2 tiling array, a comprehensive tool capable of capturing the entire viral genome, has presented a promising avenue for variants. This study introduces the SARS-CoV-2 tiling array as a novel method for port inspection. Using next-generation sequencing as a benchmark, 35 positive samples underwent sequencing through both methodologies, including the Alpha variant (B.1.1.7), Delta variants (AY.120, AY.122, AY.23.1), and Omicron variants (BA.1, BA.2, BA.2.75, BA.4, BA.5, BE.1, BF.7, BN.1, BQ.1, XBB.1) within the sample set. The whole-genome tiling array demonstrated successful identification of various sublineages of SARS-CoV-2. The average sequencing coverage rates were 99.22% (96.82%-99.92%) for the whole-genome tiling array and 98.56% (92.81%-99.59%) for Illumina sequencing, respectively. The match rates of these two methods ranged from 92.81%-99.59%, with an average rate of 98.56%. Among the benefits of the whole-genome tiling array are its cost-effectiveness and equipment simplification, making it particularly suitable for identifying SARS-CoV-2 variants in the front-line inspection department. The aforementioned findings provide valuable insights into the surveillance of COVID-19 and present a pragmatic solution for improving quarantine measures at entry points., (© 2023 Wiley Periodicals LLC.)

The SARS-CoV-2 BA.2.86 Omicron subvariant was first detected in wastewater in Sweden in week 31 2023, using 21 highly specific markers from the 50 investigated. We report BA.2.86's introduction and subsequent spread to all 14 regions performing wastewater sampling, and on 70 confirmed COVID-19 cases, along with the emergence of sublineages JN.1 and JN.2. Further, we investigated two novel mutations defining the unique BA.2.86 branching in Sweden. Our integrated approach enabled variant tracking, offering evidence for well-informed public health interventions.

Introduction: The SARS-CoV-2 pandemic represented a formidable scientific and technological challenge to public health due to its rapid spread and evolution. To meet these challenges and to characterize the virus over time, the State of California established the California SARS-CoV-2 Whole Genome Sequencing (WGS) Initiative, or "California COVIDNet". This initiative constituted an unprecedented multi-sector collaborative effort to achieve large-scale genomic surveillance of SARS-CoV-2 across California to monitor the spread of variants within the state, to detect new and emerging variants, and to characterize outbreaks in congregate, workplace, and other settings., Methods: California COVIDNet consists of 50 laboratory partners that include public health laboratories, private clinical diagnostic laboratories, and academic sequencing facilities as well as expert advisors, scientists, consultants, and contractors. Data management, sample sourcing and processing, and computational infrastructure were major challenges that had to be resolved in the midst of the pandemic chaos in order to conduct SARS-CoV-2 genomic surveillance. Data management, storage, and analytics needs were addressed with both conventional database applications and newer cloud-based data solutions, which also fulfilled computational requirements., Results: Representative and randomly selected samples were sourced from state-sponsored community testing sites. Since March of 2021, California COVIDNet partners have contributed more than 450,000 SARS-CoV-2 genomes sequenced from remnant samples from both molecular and antigen tests. Combined with genomes from CDC-contracted WGS labs, there are currently nearly 800,000 genomes from all 61 local health jurisdictions (LHJs) in California in the COVIDNet sequence database. More than 5% of all reported positive tests in the state have been sequenced, with similar rates of sequencing across 5 major geographic regions in the state., Discussion: Implementation of California COVIDNet revealed challenges and limitations in the public health system. These were overcome by engaging in novel partnerships that established a successful genomic surveillance program which provided valuable data to inform the COVID-19 public health response in California. Significantly, California COVIDNet has provided a foundational data framework and computational infrastructure needed to respond to future public health crises., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wadford, Baumrind, Baylis, Bell, Bouchard, Crumpler, Foote, Gilliam, Glaser, Hacker, Ledin, Messenger, Morales, Smith, Sevinsky, Corbett-Detig, DeRisi, Jacobson and the COVIDNet Consortium.)

Background: Remdesivir (RDV) has been shown to reduce hospitalization and mortality in COVID-19 patients. Resistance mutations caused by RDV are rare and have been predominantly reported in patients who are on prolonged therapy and immunocompromised. We investigate the effects of RDV treatment on intra-host SARS-CoV-2 diversity and low-frequency mutations in moderately ill hospitalized COVID-19 patients and compare them to patients without RDV treatment., Methods: From March 2020 to April 2022, sequential collections of nasopharyngeal and mid-turbinate swabs were obtained from 14 patients with and 30 patients without RDV treatment. Demographic and clinical data on all patients were reviewed. A total of 109 samples were sequenced and mutation analyses were performed., Results: Previously reported drug resistant mutations in nsp12 were not identified during short courses of RDV therapy. In genes encoding and surrounding the replication complex (nsp6-nsp14), low-frequency minority variants were detected in 7/14 (50%) and 18/30 (60%) patients with and without RDV treatment, respectively. We did not detect significant differences in within-host diversity and positive selection between the RDV-treated and untreated groups., Conclusions: Minimal intra-host variability and stochastic low-frequency variants detected in moderately ill patients suggests little selective pressure in patients receiving short courses of RDV. The barrier to RDV resistance is high in patients with moderate disease. Patients undergoing short regimens of RDV therapy should continue to be monitored., (© 2023 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans has been monitored at an unprecedented level due to the public health crisis, yet the stochastic dynamics underlying such a process is dubious. Here, considering the number of acquired mutations as the displacement of the viral particle from the origin, we performed biostatistical analyses from numerous whole genome sequences on the basis of a time-dependent probabilistic mathematical model. We showed that a model with a constant variant-dependent evolution rate and nonlinear mutational variance with time (i.e., anomalous diffusion) explained the SARS-CoV-2 evolutionary motion in humans during the first 120 wk of the pandemic in the United Kingdom. In particular, we found subdiffusion patterns for the Primal, Alpha, and Omicron variants but a weak superdiffusion pattern for the Delta variant. Our findings indicate that non-Brownian evolutionary motions occur in nature, thereby providing insight for viral phylodynamics.

In early 2020, the emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population quickly developed into a global pandemic. SARS-CoV-2 is the etiological agent of coronavirus disease 2019 (COVID-19) which has a broad range of respiratory illnesses. As the virus circulates, it acquires nucleotide changes. These mutations are potentially due to the inherent differences in the selection pressures within the human population compared to the original zoonotic reservoir of SARS-CoV-2 and formerly naïve humans. The acquired mutations will most likely be neutral, but some may have implications for viral transmission, disease severity, and resistance to therapies or vaccines. This is a follow-up study from our early report (Hartley et al. J Genet Genomics. 01202021;48(1):40-51) which detected a rare variant (nsp12, RdRp P323F) circulating within Nevada in mid 2020 at high frequency. The primary goals of the current study were to determine the phylogenetic relationship of the SARS-CoV-2 genomes within Nevada and to determine if there are any unusual variants within Nevada compared to the current database of SARS-CoV-2 sequences. Whole genome sequencing and analysis of SARS-CoV-2 from 425 positively identified nasopharyngeal/nasal swab specimens were performed from October 2020 to August 2021 to determine any variants that could result in potential escape from current therapeutics. Our analysis focused on nucleotide mutations that generated amino acid variations in the viral Spike (S) protein, Receptor binding domain (RBD), and the RNA-dependent RNA-polymerase (RdRp) complex. The data indicate that SARS-CoV-2 sequences from Nevada did not contain any unusual variants that had not been previously reported. Additionally, we did not detect the previously identified the RdRp P323F variant in any of the samples. This suggests that the rare variant we detected before was only able to circulate because of the stay-at-home orders and semi-isolation experience during the early months of the pandemic. IMPORTANCE: SARS-COV-2 continues to circulate in the human population. In this study, SARS-CoV-2 positive nasopharyngeal/nasal swab samples were used for whole genome sequencing to determine the phylogenetic relationship of SARS-CoV-2 sequences within Nevada from October 2020 to August 2021. The resulting data is being added to a continually growing database of SARS-CoV-2 sequences that will be important for understanding the transmission and evolution of the virus as it spreads around the globe., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Being the second most populous country in the world, India presents valuable lessons for the world about dealing with the SARS-CoV-2 pandemic. From this perspective, we attempted a retrospective evaluation of India's SARS-CoV-2 genomic surveillance strategy and also gave some recommendations for undertaking effective genomic surveillance. The dynamics of the COVID-19 pandemic are continuously evolving, and there is a dire need to modulate the genomic surveillance strategy accordingly. The pandemic is now settling towards a low positivity rate scenario, so it is required to revise the practices and policies formulated for a high positivity rate scenario. The perspective also recommends adopting a decentralised approach for SARS-CoV-2 genomic surveillance with a focus on optimising the workflow of SARS-CoV-2 genomic surveillance to ensure early detection of emerging variants, especially in the low positivity rate scenario. The perspective emphasises a key observation that the SARS-CoV-2 genomic surveillance is an important mitigation effort during the pandemic, the guards of such mitigation efforts should not be lowered during the low positivity rate scenario. We attempt to highlight the limitations faced by the Indian healthcare administration during the SARS-CoV-2 genomic surveillance and, simultaneously, suggest policy interventions derived from our first-hand experience, which may be implementable in a vast, populated country like India., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tomar and Khairnar.)

Backgrounds: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a global threat since 2020. The emergence of the Omicron variant in 2021, which replaced Delta as the dominant variant of concern, has had a significant adverse impact on the global economy and public health. During this period, Zhejiang Province implemented dynamic zeroing and focused on preventing imported cases. This study aimed to gain clear insight into the characteristics of imported COVID-19 cases in Zhejiang Province., Methods: We conducted a systematic molecular epidemiological analysis of 146 imported cases between July 2021 and November 2022 in Zhejiang Province. Virus samples with cycle threshold (Ct) value less than 32 were performed next generation sequencing. Basing the whole genome sequence obtained after quality control and assembly of reads, the whole genome variation map and phylogenetic tree were constructed and further analyzed., Results: Our study identified critical months and populations for surveillance, profiled the variation of various lineages, determined the evolutionary relationships among various lineages of SARS-CoV-2, and compared the results in Zhejiang with those obtained worldwide during this period., Conclusion: The continuous molecular epidemiological surveillance of imported cases of COVID-19 in Zhejiang Province during 2021 to 2022 is consistent with the global epidemic trend., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhou, Sun, Mao, Su, Lou, Yan, Yao, Chen and Zhang.)

BackgroundSuccessive epidemic waves of COVID-19 illustrated the potential of SARS-CoV-2 variants to reshape the pandemic. Detecting and characterising emerging variants is essential to evaluate their public health impact and guide implementation of adapted control measures.AimTo describe the detection of emerging variant, B.1.640, in France through genomic surveillance and present investigations performed to inform public health decisions.MethodsIdentification and monitoring of SARS-CoV-2 variant B.1.640 was achieved through the French genomic surveillance system, producing 1,009 sequences. Additional investigation of 272 B.1.640-infected cases was performed between October 2021 and January 2022 using a standardised questionnaire and comparing with Omicron variant-infected cases.ResultsB.1.640 was identified in early October 2021 in a school cluster in Bretagne, later spreading throughout France. B.1.640 was detected at low levels at the end of SARS-CoV-2 Delta variant's dominance and progressively disappeared after the emergence of the Omicron (BA.1) variant. A high proportion of investigated B.1.640 cases were children aged under 14 (14%) and people over 60 (27%) years, because of large clusters in these age groups. B.1.640 cases reported previous SARS-CoV-2 infection (4%), anosmia (32%) and ageusia (34%), consistent with data on pre-Omicron SARS-CoV-2 variants. Eight percent of investigated B.1.640 cases were hospitalised, with an overrepresentation of individuals aged over 60 years and with risk factors.ConclusionEven though B.1.640 did not outcompete the Delta variant, its importation and continuous low-level spread raised concerns regarding its public health impact. The investigations informed public health decisions during the time that B.1.640 was circulating.