Targeted whole-genome recovery of single viral species in a complex environmental sample.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. We present an experimental method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A unique gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. We demonstrate this method's efficacy by spiking two known viral genomes, Simian virus 40 (SV40, 5,243 bp) and Human Adenovirus 5 (HAd5, 35,938 bp), into a sewage sample with a final abundance in the droplets of around 0.1% and 0.015%, respectively. We achieve 100% recovery of the complete sequence of the spiked-in SV40 genome with uniform coverage distribution. For the larger HAd5 genome, we cover approximately 99.4% of its sequence. Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables single-genome whole-genome amplification and targeting characterizations of rare viral species and will facilitate our ability to access the mutational profile in single-virus genomes and contribute to an improved understanding of viral ecology.

High-sensitivity whole-genome recovery of single viral species in environmental samples.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method's efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

Novel hepatitis B virus reverse transcriptase mutations in patients with sustained viremia despite long-term tenofovir treatment.

BACKGROUND: Chronic hepatitis B virus (HBV) treatment consists of nucleos(t)ide analogues to suppress viral replication. The HBV inhibitor tenofovir has a high barrier to resistance, however, evidence of virus-escape is emerging. This study investigates HBV evolution in patients undergoing tenofovir treatment with the primary aim to assess the emergence of putative resistance mutations. METHODS: HBV DNA was extracted from blood samples of two patients with HBeAg-positive chronic HBV infection and persistent viremia despite tenofovir treatment, and subsequently amplified by PCR before full-length HBV genomes were assembled by deep sequencing. The mutation linkage within the viral population was evaluated by clonal analysis of amplicons. RESULTS: Sequence analysis of HBV, derived from 11 samples collected 2010-2020 from one patient, identified 12 non-synonymous single-nucleotide polymorphisms (SNPs) emerging during a tenofovir treatment interruption from 2014 to 2017. Two of the SNPs were in the reverse transcriptase (RT; H35Q and D263E). The two RT mutations were linked and persisted despite restarting tenofovir treatment in 2017. For the second patient, we analyzed HBV derived from six samples collected 2014-2020 following 10 years of tenofovir treatment, and identified five non-synonymous SNPs, that confer resistance towards entecavir and/or lamivudine. Two RT mutations (H35N and P237T) emerged during subsequent 5-year entecavir treatment. H35N was maintained during final tenofovir treatment. CONCLUSIONS: Our findings indicate that changes at the conserved residue 35 (H35N/Q) in the HBV RT may be associated with tenofovir resistance. These variants have not previously been described, and further studies are warranted to assess resistance in vitro and in vivo.

The Evolving Faces of the SARS-CoV-2 Genome.

Surveillance of the evolving SARS-CoV-2 genome combined with epidemiological monitoring and emerging vaccination became paramount tasks to control the pandemic which is rapidly changing in time and space. Genomic surveillance must combine generation and sharing sequence data with appropriate bioinformatics monitoring and analysis methods. We applied molecular portrayal using self-organizing maps machine learning (SOM portrayal) to characterize the diversity of the virus genomes, their mutual relatedness and development since the beginning of the pandemic. The genetic landscape obtained visualizes the relevant mutations in a lineage-specific fashion and provides developmental paths in genetic state space from early lineages towards the variants of concern alpha, beta, gamma and delta. The different genes of the virus have specific footprints in the landscape reflecting their biological impact. SOM portrayal provides a novel option for 'bioinformatics surveillance' of the pandemic, with strong odds regarding visualization, intuitive perception and 'personalization' of the mutational patterns of the virus genomes.

Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome.

Anelloviruses are a ubiquitous component of healthy human viromes and remain highly prevalent after being acquired early in life. The full extent of "anellome" diversity and its evolutionary dynamics remain unexplored. We employed in-depth sequencing of blood-transfusion donor(s)-recipient pairs coupled with public genomic resources for a large-scale assembly of anellovirus genomes and used the data to characterize global and personal anellovirus diversity through time. The breadth of the anellome is much greater than previously appreciated, and individuals harbor unique anellomes and transmit lineages that can persist for several months within a diverse milieu of endemic host lineages. Anellovirus sequence diversity is shaped by extensive recombination at all levels of divergence, hindering traditional phylogenetic analyses. Our findings illuminate the transmission dynamics and vast diversity of anelloviruses and set the foundation for future studies to characterize their biology.

Simultaneous detection and mutation surveillance of SARS-CoV-2 and multiple respiratory viruses by rapid field-deployable sequencing.

BACKGROUND: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner. METHODS: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed nanopore sequencing of isothermal rapid viral amplification for near real-time analysis (NIRVANA). It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. FINDINGS: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per µL of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2-positive samples mirror the epidemiology of coronavirus disease 2019 (COVID-19). Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and pepper mild mottle virus (PMMoV) (an omnipresent virus and water-quality indicator) in municipal wastewater samples. CONCLUSIONS: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses. FUNDING: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01; M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436).

VirusLab: A Tool for Customized SARS-CoV-2 Data Analysis.

Since the beginning of 2020, the COVID-19 pandemic has posed unprecedented challenges to viral data analysis and connected host disease diagnostic methods. We propose VirusLab, a flexible system for analysing SARS-CoV-2 viral sequences and relating them to metadata or clinical information about the host. VirusLab capitalizes on two existing resources: ViruSurf, a database of public SARS-CoV-2 sequences supporting metadata-driven search, and VirusViz, a tool for visual analysis of search results. VirusLab is designed for taking advantage of these resources within a server-side architecture that: (i) covers pipelines based on approaches already in use (ARTIC, Galaxy) but entirely cutomizable upon user request; (ii) predigests analysis of raw sequencing data from different platforms (Oxford Nanopore and Illumina); (iii) gives access to public archives datasets; (iv) supplies user-friendly reporting - making it a tool that can also be integrated into a business environment. VirusLab can be installed and hosted within the premises of any organization where information about SARS-CoV-2 sequences can be safely integrated with information about hosts (e.g., clinical metadata). A system such as VirusLab is not currently available in the landscape of similar providers: our results show that VirusLab is a powerful tool to generate tabular/graphical and machine readable reports that can be integrated in more complex pipelines. We foresee that the proposed system can support many research-oriented and therapeutic scenarios within hospitals or the tracing of viral sequences and their mutational processes within organizations for viral surveillance.

Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia.

BACKGROUND: Dengue virus (DENV) infects hundreds of thousands of people annually in Indonesia. However, DENV sequence data from the country are limited, as samples from outbreaks must be shipped across long-distances to suitably equipped laboratories to be sequenced. This approach is time-consuming, expensive, and frequently results in failure due to low viral load or degradation of the RNA genome. METHODS: We evaluated a method designed to address this challenge, using the 'Primal Scheme' multiplex PCR tiling approach to rapidly generate short, overlapping amplicons covering the complete DENV coding-region, and sequencing the amplicons on the portable Nanopore MinION device. The resulting sequence data was assessed in terms of genome coverage, consensus sequence accuracy and by phylogenetic analysis. RESULTS: The multiplex approach proved capable of producing near complete coding-region coverage from all samples tested ([Formula: see text] = 99.96%, n = 18), 61% of which could not be fully amplified using the current, long-amplicon PCR, approach. Nanopore-generated consensus sequences were found to be between 99.17-99.92% identical to those produced by high-coverage Illumina sequencing. Consensus accuracy could be improved by masking regions below 20X coverage depth (99.69-99.92%). However, coding-region coverage was reduced at this depth ([Formula: see text] = 93.48%). Nanopore and Illumina consensus sequences generated from the same samples formed monophyletic clades on phylogenetic analysis, and Indonesian consensus sequences accurately clustered by geographical origin. CONCLUSION: The multiplex, short-amplicon approach proved superior for amplifying DENV genomes from clinical samples, particularly when the virus was present at low concentrations. The accuracy of Nanopore-generated consensus sequences from these amplicons was sufficient for identifying the geographic origin of the samples, demonstrating that the approach can be a useful tool for identifying and monitoring DENV clades circulating in low-resource settings across Indonesia. However, the inaccuracies in Nanopore-generated consensus sequences mean that the approach may not be appropriate for higher resolution transmission studies, particularly when more accurate sequencing technologies are available.

Travel Surveillance and Genomics Uncover a Hidden Zika Outbreak during the Waning Epidemic.

The Zika epidemic in the Americas has challenged surveillance and control. As the epidemic appears to be waning, it is unclear whether transmission is still ongoing, which is exacerbated by discrepancies in reporting. To uncover locations with lingering outbreaks, we investigated travel-associated Zika cases to identify transmission not captured by reporting. We uncovered an unreported outbreak in Cuba during 2017, a year after peak transmission in neighboring islands. By sequencing Zika virus, we show that the establishment of the virus was delayed by a year and that the ensuing outbreak was sparked by long-lived lineages of Zika virus from other Caribbean islands. Our data suggest that, although mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, such measures need to be maintained to be effective. Our study highlights how Zika virus may still be "silently" spreading and provides a framework for understanding outbreak dynamics. VIDEO ABSTRACT.

Improving risk assessment of the emergence of novel influenza A viruses by incorporating environmental surveillance.

Reassortment is an evolutionary mechanism by which influenza A viruses (IAV) generate genetic novelty. Reassortment is an important driver of host jumps and is widespread according to retrospective surveillance studies. However, predicting the epidemiological risk of reassortant emergence in novel hosts from surveillance data remains challenging. IAV strains persist and co-occur in the environment, promoting co-infection during environmental transmission. These conditions offer opportunity to understand reassortant emergence in reservoir and spillover hosts. Specifically, environmental RNA could provide rich information for understanding the evolutionary ecology of segmented viruses, and transform our ability to quantify epidemiological risk to spillover hosts. However, significant challenges with recovering and interpreting genomic RNA from the environment have impeded progress towards predicting reassortant emergence from environmental surveillance data. We discuss how the fields of genomics, experimental ecology and epidemiological modelling are well positioned to address these challenges. Coupling quantitative disease models and natural transmission studies with new molecular technologies, such as deep-mutational scanning and single-virus sequencing of environmental samples, should dramatically improve our understanding of viral co-occurrence and reassortment. We define observable risk metrics for emerging molecular technologies and propose a conceptual research framework for improving accuracy and efficiency of risk prediction. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Genotypes of hepatitis a virus in Turkey: first report and clinical profile of children infected with sub-genotypes IA and IIIA.

BACKGROUND: Hepatitis A virus (HAV) is a food and water-borne virus causing clinical (mainly hepatitis) and subclinical disease in humans. It is important to characterize circulating strains of HAV in order to prevent HAV infections using efficacious vaccines. The aim of this study was the detection and characterization of the circulating strains of HAV in Turkey by performing serology, RT-PCR, sequencing and phylogenetic analysis. METHODS: In this study, 355 HAV suspected cases were analysed by ELISA for the presence of antibodies to HAV. RNA was extracted from 54 HAV IgM positive human sera. None of the suspect cases were vaccinated against HAV and they never received blood transfusions. Samples found positive by RT-PCR using primers targeting the VP1/VP2A junction and VP1/VP3 capsid region of HAV, were subjected to sequencing and phylogenetic analyses. RESULTS: IgM type antibodies to HAV were detected in 54 patients. Twenty one of them were students. The age of IgM positive cases was between 3 and 60 years. IgM positivity differed in age groups and was higher in the age group 3 to 10 years. Phylogenetic analysis showed that the majority of HAV strains detected in this study belong to the "HAV 1B" cluster. In addition, the HAV sub-genotypes IA (KT874461.1) and IIIA (KT222963.1) were found in 2 children. These sub-genotypes were not previously reported in Turkey. The child who carried sub-genotype IIIA travelled to Afghanistan and presented with abdominal pain, icterus and vomitus. He was positive for anti-HAV IgM and IgG but negative for hepatitis B and C. Liver enzymes like aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and lactate dehydrogenase were severely elevated. Bilirubin levels were also increased. White blood cells, neutrophils and hemoglobin were decreased while lymphocytes and monocytes were increased. Similar clinical signs and laboratory findings were reported for the child infected with sub-genotype IA but aspartate aminotransferase and alanine aminotransferase were not severely elevated. CONCLUSIONS: The results indicate that molecular studies determining the HAV genotype variation in Turkey are timely and warranted. The majority of IgM positive cases in 3-10 year old patients indicate that childhood vaccination is important. Sub-genotype IB is the most prevalant genotype in Turkey. Surprisingly, sub-genotype IA and IIIA are also present in Turkey; future diagnostic efforts need to include diagnostic methods which can identify this emerging HAV genotypes. Our results also show that one important risk factor for contracting hepatitis A virus is international travel since genotype IIIA was detected in a child who had travelled to Afghanistan.

Perpetuation and reassortment of gull influenza A viruses in Atlantic North America.

Gulls are important hosts of avian influenza A viruses (AIVs) and gull AIVs often contain gene segments of mixed geographic and host lineage origins. In this study, the prevalence of AIV in gulls of Newfoundland, Canada from 2008 to 2011 was analyzed. Overall prevalence was low (30/1645, 1.8%) but there was a distinct peak of infection in the fall. AIV seroprevalence was high in Newfoundland gulls, with 50% of sampled gulls showing evidence of previous infection. Sequences of 16 gull AIVs were determined and analyzed to shed light on the transmission, reassortment and persistence dynamics of gull AIVs in Atlantic North America. Intercontinental and waterfowl lineage reassortment was prevalent. Of particular note were a wholly Eurasian AIV and another with an intercontinental reassortant waterfowl lineage virus. These patterns of geographic and inter-host group transmission highlight the importance of characterization of gull AIVs as part of attempts to understand global AIV dynamics.

Influenza virus A(H3N2) strain isolated from cerebrospinal fluid from a patient presenting myelopathy post infectious.

BACKGROUND: Neurological involvement during influenza infection has been described during epidemics and is often consistent with serious sequelae or death. OBJECTIVE: To investigate the etiologic agent involved in myelopathy post influenza-like syndrome. STUDY DESIGN: This investigation focuses on virus isolation from the cerebrospinal fluid (CSF) collected from a 19-year-old male student presenting with clinical diagnosis of myelopathy post influenza-like syndrome. To achieve this goal, different cell cultures and molecular methodologies were carried out. RESULTS: Influenza virus A(H3N2) strain was isolated in MDCK cell culture; virus particles were observed under electron microscopy. Phylogenetics analyses showed that the Brazilian influenza A(H3N2) strains were closely related to the A/Perth/16/2009-like. CONCLUSION: This study demonstrates that influenza virus A(H3N2) strain was the cause of illness of the students. According to the Brazilian influenza virus sentinel surveillance data A/Perth/16/2009-LIKE (H3N2) strain has predominated during the 2010 influenza virus season in Brasília-DF.