Direct Dengue Virus Genome Sequencing from Antigen NS1 Rapid Diagnostic Tests: A Proof-of-Concept with the Standard Q Dengue Duo Assay.

With nearly half of the world's population being at risk of infection, dengue virus represents a major global health issue. The use of dengue antigen rapid diagnostic tests (Ag-RDTs) represents an alternative to PCR methods for the diagnosis of acute infections since they display excellent sensitivities and specificities and can be performed outside the laboratory. The high genetic diversity of the dengue virus genome represents a challenge for vaccine development, and the progressive expansion of this virus into previously nonendemic regions justifies the implementation of a genomic surveillance program. In this proof-of-concept study, we show the feasibility of sequencing dengue virus genomes directly from positive Ag-RDT (Standard Q Dengue Duo Test assay, n = 7) cassettes stored up to 31 days at room temperature after testing. For 5 of the 7 samples, a high number of reads were obtained allowing phylogenetic analyses to be carried out to determine not only the serotypes (dengue 1, 2, 3 and 4 were detected) but also the genotypes. Furthermore, in one sample, our unbiased metagenomic next-generation sequencing approach made it possible to detect epizootic hemorrhagic disease virus sequences, an arthropod-transmitted virus in ruminants. To conclude, as such an approach requires no cold storage or freezing of samples, dengue Ag-RDTs represent a very pragmatic and robust alternative for the genomic surveillance of dengue virus.

SCANellome: Analysis of the Genomic Diversity of Human and Non-Human Primate Anelloviruses from Metagenomics Data.

Anelloviruses are extremely prevalent in the human population and are considered to be commensal parts of the human virome. The best-known member in humans is the Torque teno virus. Recent metagenomic next-generation sequencing investigations have helped reveal the considerable number of species and genotypes from the same genus that can be co-detected within a single individual and that this diversity increases as a function of age during the first months/years of life. As a result, to date, the bioinformatics analysis of this genetic diversity remains complex and constraining for researchers. Here, we present SCANellome, a user-friendly tool to investigate the anellome composition at the genus, species, and genotype levels of samples from metagenomics data generated by the Illumina and Nanopore platforms. SCANellome is based on an in-house up-to-date database that includes all human and non-human primate anellovirus reference sequences available on GenBank and meets the latest classification criteria established by the International Committee on Taxonomy of Viruses.

Swiss public health measures associated with reduced SARS-CoV-2 transmission using genome data.

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020-the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures decoupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 to 98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred using a phylodynamic model. We found that transmission slowed 35 to 63% upon outbreak detection in summer 2020 but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.

Epidemiological, virological and serological investigation of a SARS-CoV-2 outbreak (Alpha variant) in a primary school: A prospective longitudinal study.

OBJECTIVES: To report a prospective epidemiological, virological and serological investigation of a SARS-CoV-2 outbreak in a primary school. METHODS: As part of a longitudinal, prospective, school-based surveillance study, this investigation involved repeated testing of 73 pupils, 9 teachers, 13 non-teaching staff and 26 household members of participants who tested positive, with rapid antigen tests and/or RT-PCR (Day 0-2 and Day 5-7), serologies on dried capillary blood samples (Day 0-2 and Day 30), contact tracing interviews and SARS-CoV-2 whole genome sequencing. RESULTS: We identified 20 children (aged 4 to 6 years from 4 school classes), 2 teachers and a total of 4 household members who were infected by the Alpha variant during this outbreak. Infection attack rates were between 11.8 and 62.0% among pupils from the 4 school classes, 22.2% among teachers and 0% among non-teaching staff. Secondary attack rate among household members was 15.4%. Symptoms were reported by 63% of infected children, 100% of teachers and 50% of household members. All analysed sequences but one showed 100% identity. Serological tests detected 8 seroconversions unidentified by SARS-CoV-2 virological tests. CONCLUSIONS: This study confirmed child-to-child and child-to-adult SARS-CoV-2 transmission and introduction into households. Effective measures to limit transmission in schools have the potential to reduce the overall community circulation.

Genomic Diversity of Torque Teno Virus in Blood Samples from Febrile Paediatric Outpatients in Tanzania: A Descriptive Cohort Study.

Torque teno virus (TTV) is considered to be an ubiquitous member of the commensal human blood virome commonly reported in mixed genotype co-infections. This study investigates the genomic diversity of TTV in blood samples from 816 febrile Tanzanian children. Metagenomic next-generation sequencing was used to screen for TTV in individual blood samples from a cohort of 816 febrile Tanzanian paediatric outpatients. For positive samples, the number of TTV species and genotypes present were evaluated. We investigate the linear relationship between individual TTV diversity and the patient age by linear regression. TTV was detected in 97.2% of sera. ORF1 analysis revealed the presence of 149 genotypes from 38 species, suggesting the presence of 13 new species. These genotypes were mostly present as co-infections with a median of 11 genotypes/subject (range: 1−71). In terms of species, we found a median of nine species/subject (range: 1−29). We further show a significant association between the diversity of co-detected TTV and the age of the subjects (p value < 0.0001). This study shows that significant TTV genomic diversity is acquired by the age of five and that this diversity tends to increase with age, which indicates a repetitive TTV acquisition during the first months/years of life.

Reconstruction of transmission chains of SARS-CoV-2 amidst multiple outbreaks in a geriatric acute-care hospital: a combined retrospective epidemiological and genomic study.

Background: There is ongoing uncertainty regarding transmission chains and the respective roles of healthcare workers (HCWs) and elderly patients in nosocomial outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in geriatric settings. Methods: We performed a retrospective cohort study including patients with nosocomial coronavirus disease 2019 (COVID-19) in four outbreak-affected wards, and all SARS-CoV-2 RT-PCR positive HCWs from a Swiss university-affiliated geriatric acute-care hospital that admitted both Covid-19 and non-Covid-19 patients during the first pandemic wave in Spring 2020. We combined epidemiological and genetic sequencing data using a Bayesian modelling framework, and reconstructed transmission dynamics of SARS-CoV-2 involving patients and HCWs, to determine who infected whom. We evaluated general transmission patterns according to case type (HCWs working in dedicated Covid-19 cohorting wards: HCWcovid; HCWs working in non-Covid-19 wards where outbreaks occurred: HCWoutbreak; patients with nosocomial Covid-19: patientnoso) by deriving the proportion of infections attributed to each case type across all posterior trees and comparing them to random expectations. Results: During the study period (1 March to 7 May 2020), we included 180 SARS-CoV-2 positive cases: 127 HCWs (91 HCWcovid, 36 HCWoutbreak) and 53 patients. The attack rates ranged from 10% to 19% for patients, and 21% for HCWs. We estimated that 16 importation events occurred with high confidence (4 patients, 12 HCWs) that jointly led to up to 41 secondary cases; in six additional cases (5 HCWs, 1 patient), importation was possible with a posterior probability between 10% and 50%. Most patient-to-patient transmission events involved patients having shared a ward (95.2%, 95% credible interval [CrI] 84.2%-100%), in contrast to those having shared a room (19.7%, 95% CrI 6.7%-33.3%). Transmission events tended to cluster by case type: patientnoso were almost twice as likely to be infected by other patientnoso than expected (observed:expected ratio 2.16, 95% CrI 1.17-4.20, p=0.006); similarly, HCWoutbreak were more than twice as likely to be infected by other HCWoutbreak than expected (2.72, 95% CrI 0.87-9.00, p=0.06). The proportion of infectors being HCWcovid was as expected as random. We found a trend towards a greater proportion of high transmitters (≥2 secondary cases) among HCWoutbreak than patientnoso in the late phases (28.6% vs. 11.8%) of the outbreak, although this was not statistically significant. Conclusions: Most importation events were linked to HCW. Unexpectedly, transmission between HCWcovid was more limited than transmission between patients and HCWoutbreak. This finding highlights gaps in infection control and suggests the possible areas of improvements to limit the extent of nosocomial transmission. Funding: This study was supported by a grant from the Swiss National Science Foundation under the NRP78 funding scheme (Grant no. 4078P0_198363).

Occupational and community risk of SARS-CoV-2 infection among employees of a long-term care facility: an observational study.

BACKGROUND: We investigated the contribution of both occupational and community exposure for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among employees of a university-affiliated long-term care facility (LTCF), during the 1st pandemic wave in Switzerland (March-June 2020). METHODS: We performed a nested analysis of a seroprevalence study among all volunteering LTCF staff to determine community and nosocomial risk factors for SARS-CoV-2 seropositivity using modified Poison regression. We also combined epidemiological and genetic sequencing data from a coronavirus disease 2019 (COVID-19) outbreak investigation in a LTCF ward to infer transmission dynamics and acquisition routes of SARS-CoV-2, and evaluated strain relatedness using a maximum likelihood phylogenetic tree. RESULTS: Among 285 LTCF employees, 176 participated in the seroprevalence study, of whom 30 (17%) were seropositive for SARS-CoV-2. Most (141/176, 80%) were healthcare workers (HCWs). Risk factors for seropositivity included exposure to a COVID-19 inpatient (adjusted prevalence ratio [aPR] 2.6; 95% CI 0.9-8.1) and community contact with a COVID-19 case (aPR 1.7; 95% CI 0.8-3.5). Among 18 employees included in the outbreak investigation, the outbreak reconstruction suggests 4 likely importation events by HCWs with secondary transmissions to other HCWs and patients. CONCLUSIONS: These two complementary epidemiologic and molecular approaches suggest a substantial contribution of both occupational and community exposures to COVID-19 risk among HCWs in LTCFs. These data may help to better assess the importance of occupational health hazards and related legal implications during the COVID-19 pandemic.

SARS-CoV-2 Evolution among Oncological Population: In-Depth Virological Analysis of a Clinical Cohort.

BACKGROUND: Oncological patients have a higher risk of prolonged SARS-CoV-2 shedding, which, in turn, can lead to evolutionary mutations and emergence of novel viral variants. The aim of this study was to analyze biological samples of a cohort of oncological patients by deep sequencing to detect any significant viral mutations. METHODS: High-throughput sequencing was performed on selected samples from a SARS-CoV-2-positive oncological patient cohort. Analysis of variants and minority variants was performed using a validated bioinformatics pipeline. RESULTS: Among 54 oncological patients, we analyzed 12 samples of 6 patients, either serial nasopharyngeal swab samples or samples from the upper and lower respiratory tracts, by high-throughput sequencing. We identified amino acid changes D614G and P4715L as well as mutations at nucleotide positions 241 and 3037 in all samples. There were no other significant mutations, but we observed intra-host evolution in some minority variants, mainly in the ORF1ab gene. There was no significant mutation identified in the spike region and no minority variants common to several hosts. CONCLUSIONS: There was no major and rapid evolution of viral strains in this oncological patient cohort, but there was minority variant evolution, reflecting a dynamic pattern of quasi-species replication.

Blood virosphere in febrile Tanzanian children.

Viral infections are the leading cause of childhood acute febrile illnesses motivating consultation in sub-Saharan Africa. The majority of causal viruses are never identified in low-resource clinical settings as such testing is either not part of routine screening or available diagnostic tools have limited ability to detect new/unexpected viral variants. An in-depth exploration of the blood virome is therefore necessary to clarify the potential viral origin of fever in children. Metagenomic next-generation sequencing is a powerful tool for such broad investigations, allowing the detection of RNA and DNA viral genomes. Here, we describe the blood virome of 816 febrile children (<5 years) presenting at outpatient departments in Dar es Salaam over one-year. We show that half of the patients (394/816) had at least one detected virus recognized as causes of human infection/disease (13.8% enteroviruses (enterovirus A, B, C, and rhinovirus A and C), 12% rotaviruses, 11% human herpesvirus type 6). Additionally, we report the detection of a large number of viruses (related to arthropod, vertebrate or mammalian viral species) not yet known to cause human infection/disease, highlighting those who should be on the radar, deserve specific attention in the febrile paediatric population and, more broadly, for surveillance of emerging pathogens.Trial registration: ClinicalTrials.gov identifier: NCT02225769.

Clinical, virologic and immunologic features of a mild case of SARS-CoV-2 reinfection.

OBJECTIVES: To report a case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection 6 months after the first infection in a young healthy female physician. Both episodes led to mild coronavirus disease 2019 (COVID-19). METHODS: SARS-CoV-2 infections were detected by real-time reverse transcriptase PCR (RT-PCR) on nasopharyngeal specimens. Reinfection was confirmed by whole-genome sequencing. Kinetics of total anti-S receptor binding domain immunoglobulins (Ig anti-S RBD), anti-nucleoprotein (anti-N) and neutralizing antibodies were determined in serial serum samples retrieved during both infection episodes. Memory B-cell responses were assessed at day 12 after reinfection. RESULTS: Whole-genome sequencing identified two different SARS-CoV-2 genomes both belonging to clade 20A, with only one nonsynonymous mutation in the spike protein and clustered with viruses circulating in Geneva (Switzerland) at the time of each of the corresponding episodes. Seroconversion was documented with low levels of total Ig anti-S RBD and anti-N antibodies at 1 month after the first infection, whereas neutralizing antibodies quickly declined after the first episode and then were boosted by the reinfection, with high titres detectable 4 days after symptom onset. A strong memory B-cell response was detected at day 12 after onset of symptoms during reinfection, indicating that the first episode elicited cellular memory responses. CONCLUSIONS: Rapid decline of neutralizing antibodies may put medical personnel at risk of reinfection, as shown in this case. However, reinfection leads to a significant boosting of previous immune responses. Larger cohorts of reinfected subjects with detailed descriptions of their immune responses are needed to define correlates of protection and their duration after infection.

Puumala Virus Infection in Family, Switzerland.

We report 3 cases of Puumala virus infection in a family in Switzerland in January 2019. Clinical manifestations of the infection ranged from mild influenza-like illness to fatal disease. This cluster illustrates the wide range of clinical manifestations of Old World hantavirus infections and the challenge of diagnosing travel-related hemorrhagic fevers.

Virosaurus A Reference to Explore and Capture Virus Genetic Diversity.

The huge genetic diversity of circulating viruses is a challenge for diagnostic assays for emerging or rare viral diseases. High-throughput technology offers a new opportunity to explore the global virome of patients without preconception about the culpable pathogens. It requires a solid reference dataset to be accurate. Virosaurus has been designed to offer a non-biased, automatized and annotated database for clinical metagenomics studies and diagnosis. Raw viral sequences have been extracted from GenBank, and cleaned up to remove potentially erroneous sequences. Complete sequences have been identified for all genera infecting vertebrates, plants and other eukaryotes (insect, fungus, etc.). To facilitate the analysis of clinically relevant viruses, we have annotated all sequences with official and common virus names, acronym, genotypes, and genomic features (linear, circular, DNA, RNA, etc.). Sequences have been clustered to remove redundancy at 90% or 98% identity. The analysis of clustering results reveals the state of the virus genetic landscape knowledge. Because herpes and poxviruses were under-represented in complete genomes considering their potential diversity in nature, we used genes instead of complete genomes for those in Virosaurus.

Daily Viral Kinetics and Innate and Adaptive Immune Response Assessment in COVID-19: a Case Series.

Viral shedding patterns and their correlations with immune responses are still poorly characterized in mild coronavirus (CoV) disease 2019 (COVID-19). We monitored shedding of viral RNA and infectious virus and characterized the immune response kinetics of the first five patients quarantined in Geneva, Switzerland. High viral loads and infectious virus shedding were observed from the respiratory tract despite mild symptoms, with isolation of infectious virus and prolonged positivity by reverse transcriptase PCR (RT-PCR) until days 7 and 19 after symptom onset, respectively. Robust innate responses characterized by increases in activated CD14+ CD16+ monocytes and cytokine responses were observed as early as 2 days after symptom onset. Cellular and humoral severe acute respiratory syndrome (SARS)-CoV-2-specific adaptive responses were detectable in all patients. Infectious virus shedding was limited to the first week after symptom onset. A strong innate response, characterized by mobilization of activated monocytes during the first days of infection and SARS-CoV-2-specific antibodies, was detectable even in patients with mild disease.IMPORTANCE This work is particularly important because it simultaneously assessed the virology, immunology, and clinical presentation of the same subjects, whereas other studies assess these separately. We describe the detailed viral and immune profiles of the first five patients infected by SARS-CoV-2 and quarantined in Geneva, Switzerland. Viral loads peaked at the very beginning of the disease, and infectious virus was shed only during the early acute phase of disease. No infectious virus could be isolated by culture 7 days after onset of symptoms, while viral RNA was still detectable for a prolonged period. Importantly, we saw that all patients, even those with mild symptoms, mount an innate response sufficient for viral control (characterized by early activated cytokines and monocyte responses) and develop specific immunity as well as cellular and humoral SARS-CoV-2-specific adaptive responses, which already begin to decline a few months after the resolution of symptoms.

Viral Metagenomics in the Clinical Realm: Lessons Learned from a Swiss-Wide Ring Trial.

Shotgun metagenomics using next generation sequencing (NGS) is a promising technique to analyze both DNA and RNA microbial material from patient samples. Mostly used in a research setting, it is now increasingly being used in the clinical realm as well, notably to support diagnosis of viral infections, thereby calling for quality control and the implementation of ring trials (RT) to benchmark pipelines and ensure comparable results. The Swiss NGS clinical virology community therefore decided to conduct a RT in 2018, in order to benchmark current metagenomic workflows used at Swiss clinical virology laboratories, and thereby contribute to the definition of common best practices. The RT consisted of two parts (increments), in order to disentangle the variability arising from the experimental compared to the bioinformatics parts of the laboratory pipeline. In addition, the RT was also designed to assess the impact of databases compared to bioinformatics algorithms on the final results, by asking participants to perform the bioinformatics analysis with a common database, in addition to using their own in-house database. Five laboratories participated in the RT (seven pipelines were tested). We observed that the algorithms had a stronger impact on the overall performance than the choice of the reference database. Our results also suggest that differences in sample preparation can lead to significant differences in the performance, and that laboratories should aim for at least 5-10 Mio reads per sample and use depth of coverage in addition to other interpretation metrics such as the percent of coverage. Performance was generally lower when increasing the number of viruses per sample. The lessons learned from this pilot study will be useful for the development of larger-scale RTs to serve as regular quality control tests for laboratories performing NGS analyses of viruses in a clinical setting.

Nearly Complete Genome Sequence of a Novel Phlebovirus-Like Virus Detected in a Human Plasma Sample by High-Throughput Sequencing.

Here, we report a novel phlebovirus-like virus sequence detected in a plasma sample from a febrile adult patient collected in the United Republic of Tanzania in 2014. A nearly complete RNA sequence was generated by high-throughput sequencing on a HiSeq 2500 instrument and further confirmed after repeating the analysis, starting from the initial sample.

Viral Sequences Detection by High-Throughput Sequencing in Cerebrospinal Fluid of Individuals with and without Central Nervous System Disease.

: Meningitis, encephalitis, and myelitis are various forms of acute central nervous system (CNS) inflammation, which can coexist and lead to serious sequelae. Known aetiologies include infections and immune-mediated processes. Despite advances in clinical microbiology over the past decades, the cause of acute CNS inflammation remains unknown in approximately 50% of cases. High-throughput sequencing was performed to search for viral sequences in cerebrospinal fluid (CSF) samples collected from 26 patients considered to have acute CNS inflammation of unknown origin, and 10 patients with defined causes of CNS diseases. In order to better grasp the clinical significance of viral sequence data obtained in CSF, 30 patients without CNS disease who had a lumbar puncture performed during elective spinal anaesthesia were also analysed. One case of human astrovirus (HAstV)-MLB2-related meningitis and disseminated infection was identified. No other viral sequences that can easily be linked to CNS inflammation were detected. Viral sequences obtained in all patient groups are discussed. While some of them reflect harmless viral infections, others result from reagent or sample contamination, as well as index hopping. Altogether, this study highlights the potential of high-throughput sequencing in identifying previously unknown viral neuropathogens, as well as the interpretation issues related to its application in clinical microbiology.

Detection of dicistroviruses RNA in blood of febrile Tanzanian children.

Fever is the leading cause of paediatric outpatient consultations in Sub-Saharan Africa. Although most are suspected to be of viral origin, a putative causative pathogen is not identified in over a quarter of these febrile episodes. Using a de novo assembly sequencing approach, we report the detection (15.4%) of dicistroviruses (DicV) RNA in sera collected from 692 febrile Tanzanian children. In contrast, DicV RNA was only detected in 1/77 (1.3%) plasma samples from febrile Tanzanian adults, suggesting that children could represent the primary susceptible population. Estimated viral load by specific quantitative real-time RT-PCR assay ranged from < 1.32E3 to 1.44E7 viral RNA copies/mL serum. Three DicV full-length genomes were obtained, and a phylogenetic analyse on the capsid region showed the presence of two clusters representing tentative novel genus. Although DicV-positive cases were detected throughout the year, a significantly higher positivity rate was observed during the rainy season. This study reveals that novel DicV RNA is frequently detected in the blood of Tanzanian children, paving the way for further investigations to determine if DicV possibly represent a new agent in humans.

Investigation of the Plasma Virome from Cases of Unexplained Febrile Illness in Tanzania from 2013 to 2014: a Comparative Analysis between Unbiased and VirCapSeq-VERT High-Throughput Sequencing Approaches.

High-throughput sequencing can provide insights into epidemiology and medicine through comprehensive surveys of viral genetic sequences in environmental and clinical samples. Here, we characterize the plasma virome of Tanzanian patients with unexplained febrile illness by using two high-throughput sequencing methods: unbiased sequencing and VirCapSeq-VERT (a positive selection system). Sequences from dengue virus 2, West Nile virus, human immunodeficiency virus type 1, human pegivirus, and Epstein-Barr virus were identified in plasma. Both sequencing strategies recovered nearly complete genomes in samples containing multiple viruses. Whereas VirCapSeq-VERT had better sensitivity, unbiased sequencing provided better coverage of genome termini. Together, these data demonstrate the utility of high-throughput sequencing strategies in outbreak investigations.IMPORTANCE Characterization of the viruses found in the blood of febrile patients provides information pertinent to public health and diagnostic medicine. PCR and culture have historically played an important role in clinical microbiology; however, these methods require a targeted approach and may lack the capacity to identify novel or mixed viral infections. High-throughput sequencing can overcome these constraints. As the cost of running multiple samples continues to decrease, the implementation of high-throughput sequencing for diagnostic purposes is becoming more feasible. Here we present a comparative analysis of findings from an investigation of unexplained febrile illness using two strategies: unbiased high-throughput sequencing and VirCapSeq-VERT, a positive selection high-throughput sequencing system.