Viral Abundance Research Articles

SARS-CoV-2 RNA Detection in Wastewater and Its Effective Correlation with Clinical Data during the Outbreak of COVID-19 in Salamanca.

Wastewater treatment plants (WWTPs) are the final stage of the anthropogenic water cycle where a wide range of chemical and biological markers of human activity can be found. In COVID-19 disease contexts, wastewater surveillance has been used to infer community trends based on viral abundance and SARS-CoV-2 RNA variant composition, which has served to anticipate and establish appropriate protocols to prevent potential viral outbreaks. Numerous studies worldwide have provided reliable and robust tools to detect and quantify SARS-CoV-2 RNA in wastewater, although due to the high dilution and degradation rate of the viral RNA in such samples, the detection limit of the pathogen has been a bottleneck for the proposed protocols so far. The current work provides a comprehensive and systematic study of the different parameters that may affect the detection of SARS-CoV-2 RNA in wastewater and hinder its quantification. The results obtained using synthetic viral RNA as a template allow us to consider that 10 genome copies per µL is the minimum RNA concentration that provides reliable and consistent values for the quantification of SARS-CoV-2 RNA. RT-qPCR analysis of wastewater samples collected at the WWTP in Salamanca (western Spain) and at six pumping stations in the city showed that below this threshold, positive results must be confirmed by sequencing to identify the specific viral sequence. This allowed us to find correlations between the SARS-CoV-2 RNA levels found in wastewater and the COVID-19 clinical data reported by health authorities. The close match between environmental and clinical data from the Salamanca case study has been confirmed by similar experimental approaches in four other cities in the same region. The present methodological approach reinforces the usefulness of wastewater-based epidemiology (WBE) studies in the face of future pandemic outbreaks.

Aedes Mosquito Virome in Southwestern Cameroon: Lack of Core Virome, But a Very Rich and Diverse Virome in Ae. africanus Compared to Other Aedes Species.

In Cameroon, Aedes mosquitoes transmit various arboviruses, posing significant health risks. We aimed to characterize the Aedes virome in southwestern Cameroon and identify potential core viruses which might be associated with vector competence. A total of 398 Aedes mosquitoes were collected from four locations (Bafoussam, Buea, Edea, and Yaounde). Aedes albopictus dominated all sites except for Bafoussam, where Aedes africanus prevailed. Metagenomic analyses of the mosquitoes grouped per species into 54 pools revealed notable differences in the eukaryotic viromes between Ae. africanus and Ae. albopictus, with the former exhibiting greater richness and diversity. Thirty-seven eukaryotic virus species from 16 families were identified, including six novel viruses with near complete genome sequences. Seven viruses were further quantified in individual mosquitoes via qRT-PCR. Although none of them could be identified as core viruses, Guangzhou sobemo-like virus and Bafoussam mosquito solemovirus, were highly prevalent regionally in Ae. albopictus and Ae. africanus, respectively. This study highlights the diverse eukaryotic virome of Aedes species in southwestern Cameroon. Despite their shared genus, Aedes species exhibit limited viral sharing, with varying viral abundance and prevalence across locations. Ae. africanus, an understudied vector, harbors a rich and diverse virome, suggesting potential implications for arbovirus vector competence.

Sacbrood viruses and select Lake Sinai virus variants dominated Apis mellifera colonies symptomatic for European foulbrood.

European foulbrood (EFB) is a prevalent disease in the European honey bee (Apis mellifera) in the United States, which can lead to colony decline and collapse. The bacterial components of EFB are well-studied, but the diversity of viral infections within infected colonies has not been explored. In this study, we use meta-transcriptomics sequencing of 12 honey bee hives, symptomatic (+, n = 6) and asymptomatic (-, n = 6) for EFB, to investigate viral infection associated with the disease. We assembled 41 viral genomes, belonging to three families (Iflaviridae, Dicistroviridae, and Sinhaliviridae), all previously reported in honey bees, including Lake Sinai virus, deformed wing virus, sacbrood virus, Black queen cell virus, and Israeli acute paralysis virus. In colonies with severe EFB, we observed a higher occurrence of viral genomes (34 genomes) in contrast to fewer recovered from healthy colonies (seven genomes) and a complete absence of Dicistroviridae genomes.We observed specific Lake Sinai virus clades associated exclusively with EFB + or EFB - colonies, in addition to EFB-afflicted colonies that exhibited an increase in relative abundance of sacbrood viruses. Multivariate analyses highlighted that a combination of site and EFB disease status influenced RNA virome composition, while EFB status alone did not significantly impact it, presenting a challenge for comparisons between colonies kept in different yards. These findings contribute to the understanding of viral dynamics in honey bee colonies compromised by EFB and underscore the need for future investigations to consider viral composition when investigating EFB.IMPORTANCEThis study on the viromes of honey bee colonies affected by European foulbrood (EFB) sheds light on the dynamics of viral populations in bee colonies in the context of a prevalent bacterial brood disease. The identification of distinct Lake Sinai virus and sacbrood virus clades associated with colonies affected by severe EFB suggests a potential connection between viral composition and disease status, emphasizing the need for further investigation into the role of viruses during EFB infection. The observed increase in sacbrood viruses during EFB infection suggests a potential viral dysbiosis, with potential implications for honey bee brood health. These findings contribute valuable insights related to beekeeping practices, offering a foundation for future research aimed at understanding and mitigating the impact of bacterial and viral infection in commercial honey bee operations and the management of EFB.

A metagenomic investigation of the faecal RNA virome structure of asymptomatic chickens obtained from a commercial farm in Durban, KwaZulu-Natal province, South Africa

BackgroundVirome studies on birds, including chickens are relatively scarce, particularly from the African continent. Despite the continuous evolution of RNA viruses and severe losses recorded in poultry from seasonal viral outbreaks, the information on RNA virome composition is even scantier as a result of their highly unstable nature, genetic diversity, and difficulties associated with characterization. Also, information on factors that may modulate the occurrence of some viruses in birds is limited, particularly for domesticated birds. Viral metagenomics through advancements in sequencing technologies, has enabled the characterization of the entire virome of diverse host species using various samples.MethodsThe complex RNA viral constituents present in 27 faecal samples of asymptomatic chickens from a South African farm collected at 3-time points from two independent seasons were determined, and the impact of the chicken’s age and collection season on viral abundance and diversity was further investigated. The study utilized the non-invasive faecal sampling method, mRNA viral targeted enrichment steps, a whole transcriptome amplification strategy, Illumina sequencing, and bioinformatics tools.ResultsThe results obtained revealed a total of 48 viral species spanning across 11 orders, 15 families and 21 genera. Viral RNA families such as Coronaviridae, Picornaviridae, Reoviridae, Astroviridae, Caliciviridae, Picorbirnaviridae and Retroviridae were abundant, among which picornaviruses, demonstrated a 100% prevalence across the three age groups (2, 4 and 7 weeks) and two seasons (summer and winter) of the 27 faecal samples investigated. A further probe into the extent of variation between the different chicken groups investigated indicated that viral diversity and abundance were significantly influenced by age (P = 0.01099) and season (P = 0.00099) between chicken groups, while there was no effect on viral shedding within samples in a group (alpha diversity) for age (P = 0.146) and season (P = 0.242).ConclusionThe presence of an exceedingly varied chicken RNA virome, encompassing avian, mammalian, fungal, and dietary-associated viruses, underscores the complexities inherent in comprehending the causation, dynamics, and interspecies transmission of RNA viruses within the investigated chicken population. Hence, chickens, even in the absence of discernible symptoms, can harbour viruses that may exhibit opportunistic, commensal, or pathogenic characteristics.

Using an adaptive modeling framework to identify avian influenza spillover risk at the wild-domestic interface

The wild to domestic bird interface is an important nexus for emergence and transmission of highly pathogenic avian influenza (HPAI) viruses. Although the recent incursion of HPAI H5N1 Clade 2.3.4.4b into North America calls for emergency response and planning given the unprecedented scale, readily available data-driven models are lacking. Here, we provide high resolution spatial and temporal transmission risk models for the contiguous United States. Considering virus host ecology, we included weekly species-level wild waterfowl (Anatidae) abundance and endemic low pathogenic avian influenza virus prevalence metrics in combination with number of poultry farms per commodity type and relative biosecurity risks at two spatial scales: 3 km and county-level. Spillover risk varied across the annual cycle of waterfowl migration and some locations exhibited persistent risk throughout the year given higher poultry production. Validation using wild bird introduction events identified by phylogenetic analysis from 2022 to 2023 HPAI poultry outbreaks indicate strong model performance. The modular nature of our approach lends itself to building upon updated datasets under evolving conditions, testing hypothetical scenarios, or customizing results with proprietary data. This research demonstrates an adaptive approach for developing models to inform preparedness and response as novel outbreaks occur, viruses evolve, and additional data become available.

Coinfecting phages impede each other’s entry into the cell

The developmental choice made by temperate phages, between cell death (lysis) and viral dormancy (lysogeny), is influenced by the relative abundance of viruses and hosts in the environment. The paradigm for this abundance-driven decision is phage lambda of E.coli, whose propensity to lysogenize increases with the number of viruses coinfecting the same bacterium. It is believed that lambda uses this number to infer whether phages or bacteria outnumber each other. However, this interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). Here, we show this premise to be faulty. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, performed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of phage entry decrease with the number of adsorbed phages. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage attachment, as evidenced by compromised membrane integrity and loss of membrane potential. The dependence of entry dynamics on the surrounding medium results in a strong impact on the infection outcome, while the protracted entry of coinfecting phages increases the heterogeneity in infection outcome at a given MOI. Our findings in lambda, and similar results we obtained for phages T5 and P1, demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection.

Distinct characteristics of the gut virome in patients with osteoarthritis and gouty arthritis

Background/purpose(s)The gut microbiota and its metabolites play crucial roles in pathogenesis of arthritis, highlighting gut microbiota as a promising avenue for modulating autoimmunity. However, the characterization of the gut virome in arthritis patients, including osteoarthritis (OA) and gouty arthritis (GA), requires further investigation.MethodsWe employed virus-like particle (VLP)-based metagenomic sequencing to analyze gut viral community in 20 OA patients, 26 GA patients, and 31 healthy controls, encompassing a total of 77 fecal samples.ResultsOur analysis generated 6819 vOTUs, with a considerable proportion of viral genomes differing from existing catalogs. The gut virome in OA and GA patients differed significantly from healthy controls, showing variations in diversity and viral family abundances. We identified 157 OA-associated and 94 GA-associated vOTUs, achieving high accuracy in patient-control discrimination with random forest models. OA-associated viruses were predicted to infect pro-inflammatory bacteria or bacteria associated with immunoglobulin A production, while GA-associated viruses were linked to Bacteroidaceae or Lachnospiraceae phages. Furthermore, several viral functional orthologs displayed significant differences in frequency between OA-enriched and GA-enriched vOTUs, suggesting potential functional roles of these viruses. Additionally, we trained classification models based on gut viral signatures to effectively discriminate OA or GA patients from healthy controls, yielding AUC values up to 0.97, indicating the clinical utility of the gut virome in diagnosing OA or GA.ConclusionOur study highlights distinctive alterations in viral diversity and taxonomy within gut virome of OA and GA patients, offering insights into arthritis etiology and potential treatment and prevention strategies.Graphical

Viral Dynamics in the Tropical Pacific Ocean: A Comparison between Within and Outside a Warm Eddy.

In mesoscale eddies, the chemical properties and biological composition are different from those in the surrounding water due to their unique physical processes. The mechanism of physical-biological coupling in warm-core eddies is unclear, especially because no studies have examined the effects of environmental factors on bacteria and viruses. The purpose of the present study was to examine the influence of an anticyclonic warm eddy on the relationship between bacterial and viral abundances, as well as viral activity (viral production), at different depths. At the core of the warm eddy, the bacterial abundance (0.48 to 2.82 × 105 cells mL-1) fluctuated less than that outside the eddy (1.12 to 7.03 × 105 cells mL-1). In particular, there was a four-fold higher viral-bacterial abundance ratio (VBR) estimated within the eddy, below the layer of the deep chlorophyll maximum, than outside the eddy. An anticyclonic warm eddy with downwelling at its center may contribute to viruses being transmitted directly into the deep ocean through adsorption on particulate organic matter while sinking. Overall, our findings provide valuable insights into the interaction between bacterial and viral abundances and their ecological mechanisms within a warm eddy.

Known and novel viruses in Belgian honey bees: yearly differences, spatial clustering, and associations with overwintering loss.

The western honey bee (Apis mellifera) is a highly effective pollinator of flowering plants, including many crops, which gives honey bees an outstanding importance both ecologically and economically. Alarmingly high annual loss rates of managed honey bee colonies are a growing concern for beekeepers and scientists and have prompted a significant research effort toward bee health. Several detrimental factors have been identified, such as varroa mite infestation and disease from various bacterial and viral agents, but annual differences are often not elucidated. In this study, we utilize the viral metagenomic survey of the EPILOBEE project, a European research program for bee health, to elaborate on the most abundant bee viruses of Flanders. We complement the existing metagenomic data with absolute viral loads and their spatial and temporal distributions. Furthermore, we identify Apis orthomyxovirus 1 as a potentially emerging pathogen, as we find evidence for its active replication honey bees.

Integrative genomic analysis of the lung tissue microenvironment in SARS-CoV-2 and NL63 patients

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus has affected over 700 million people, and caused over 7 million deaths throughout the world as of April 2024, and continues to affect people through seasonal waves. While over 675 million people have recovered from this disease globally, the lingering effects of the disease are still under study. Long term effects of SARS-CoV-2 infection, known as 'long COVID,' include a wide range of symptoms including fatigue, chest pain, cellular damage, along with a strong innate immune response characterized by inflammatory cytokine production. Three years after the pandemic, data about long covid studies are finally emerging. More clinical studies and clinical trials are needed to understand and determine the factors that predispose individuals to these long-term side effects.In this methodology paper, our goal was to apply data driven approaches in order to explore the multidimensional landscape of infected lung tissue microenvironment to better understand complex interactions between viral infection, immune response and the lung microbiome of patients with (a) SARS-CoV-2 virus and (b) NL63 coronavirus. The samples were analyzed with several machine learning tools allowing simultaneous detection and quantification of viral RNA amount at genome and gene level; human gene expression and fractions of major types of immune cells, as well as metagenomic analysis of bacterial and viral abundance. To contrast and compare specific viral response to SARS-COV-2, we analyzed deep sequencing data from additional cohort of patients infected with NL63 strain of corona virus.Our correlation analysis of three types of RNA-seq based measurements in patients i.e. fraction of viral RNA (at genome and gene level), Human RNA (transcripts and gene level) and bacterial RNA (metagenomic analysis), showed significant correlation between viral load as well as level of specific viral gene expression with the fractions of immune cells present in lung lavage as well as with abundance of major fractions of lung microbiome in COVID-19 patients.Our methodology-based proof-of-concept study has provided novel insights into complex regulatory signaling interactions and correlative patterns between the viral infection, inhibition of innate and adaptive immune response as well as microbiome landscape of the lung tissue. These initial findings could provide better understanding of the diverse dynamics of immune response and the side effects of the SARS-CoV-2 infection and demonstrates the possibilities of the various types of analyses that could be performed from this type of data.

Next-generation sequencing technology reveals the viruses carried by poultry in the live poultry market of Guangdong, China

This study aimed to analyze the species and abundance of viruses carried by avian species in live poultry markets. In 2022, we collected 196 bird samples from two representative live poultry markets in Guangdong, China, of which 147 were randomly selected for metatranscriptome sequencing to construct a metatranscriptome library. This analysis yielded 17 viral families. Statistical analysis of the virus abundance of the six libraries showed that Picornaviridae, Retroviridae, Coronaviridae, and Othomyxoviridae were more abundant in the J1, J2, and J3 libraries, and Coronaviridae, Retroviridae, and Faviviridae were more abundant in the Y1, Y2, and E1 libraries. Finally, samples were screened using nested PCR and three viruses were identified. The positive results combined with high-throughput sequencing abundance data showed a positive correlation between virus abundance and the number of positive samples. This study provides scientific data to support the diagnosis and prevention of avian viral diseases.

The diverse liver viromes of Australian geckos and skinks are dominated by hepaciviruses and picornaviruses and reflect host taxonomy and habitat

Abstract Lizards have diverse ecologies and evolutionary histories, and represent a promising group to explore how hosts shape virome structure and virus evolution. Yet, little is known about the viromes of these animals. In Australia, squamates (lizards and snakes) comprise the most diverse order of vertebrates, and Australia hosts the highest diversity of lizards globally, with the greatest breadth of habitat use. We used meta-transcriptomic sequencing to determine the virome of nine co-distributed, tropical lizard species from three taxonomic families in Australia and analyzed these data to identify host traits associated with viral abundance and diversity. We show that lizards carry a large diversity of viruses, identifying more than thirty novel, highly divergent vertebrate-associated viruses. These viruses were from nine viral families, including several that contain well known pathogens, such as the Flaviviridae, Picornaviridae, Bornaviridae, Iridoviridae, and Rhabdoviridae. Members of the Flaviviridae were particularly abundant across species sampled here, largely belonging to the genus Hepacivirus: fourteen novel hepaciviruses were identified, broadening the known diversity of this group and better defining its evolution by uncovering new reptilian clades. The evolutionary histories of the viruses studied here frequently aligned with the biogeographic and phylogenetic histories of the hosts, indicating that exogenous viruses may help infer host evolutionary history if sampling is strategic and sampling density high enough. Notably, analysis of alpha and beta diversity revealed that virome composition and richness in the animals sampled here was shaped by host taxonomy and habitat. In sum, we identified a diverse range of reptile viruses that broadly contributes to our understanding of virus-host ecology and evolution.

Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS).A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline.Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively.A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Target enrichment metaviromics enables comprehensive surveillance of coronaviruses in environmental and animal samples

The COVID-19 pandemic has underscored the importance of understanding the role of animals in the transmission of coronaviruses (CoVs) and their impact on human health. A One Health approach, integrating human, animal, and environmental health, is essential for effective CoVs control. Next-generation sequencing has played a pivotal role in identifying and monitoring the evolution of novel CoVs strains, like SARS-CoV-2. However, viral occurrence and diversity studies in environmental and animal samples are challenging because of the complexity of viral communities and low abundance of viruses in these samples.Target enrichment sequencing (TES) has emerged as a valuable tool for investigating viral families in challenging samples. This approach involves the specific capture and enrichment of viral genomes using sequence-specific probes, thereby enhancing the efficiency of detection and characterization.In this study, we aimed to develop and validate a TES panel to study CoVs in various complex environmental and animal derived samples. The results demonstrated the panel's effectiveness in capturing and sequencing a wide diversity of CoVs providing valuable insights into their abundance and host diversity in urban wastewater, farm animal corpses lixiviates and bat guano samples. In sewage samples, CoVs were detected solely when TES was employed while in guano samples, sequencing of CoVs species was achieved in 2 out of 4 samples showing an almost three-logarithmic increase in the number of reads obtained in comparison with the untargeted approach. For animal lixiviates, only the TES application enabled the acquisition of CoVs reads. The information obtained can significantly contribute to early detection, surveillance, and control measures for CoVs, including viral discovery and potential spillover events. Additionally, this sequencing panel shows potential for studying other significant viral families and monitoring viral diversity in different animal populations.

Effect of the supplementation of exogenous complex non-starch polysaccharidases on the growth performance, rumen fermentation and microflora of fattening sheep.

The objective of this study was to evaluate the effects of exogenous non-starch polysaccharidases (a mixture of cellulase, xylanase, β-glucanase and mannanase) on the growth performance and nutrient digestibility, rumen fermentation, and rumen microflora of sheep. The animal trial was conducted using 36 5-month-old female fattening hybrid sheep (Duolang♂ × Hu♀) who were randomly assigned into four groups comprising nine sheep per treatment: CON, T1, T2, and T3, with 0, 0.1, 0.3, and 0.5% NSPases/kg DM of TMR, respectively. This complex enzyme product was screened for optimal ratios based on previous in vitro tests and responded positively to the in vitro fermentation of the TMR. When treated with NSPases, there was a non-linear effect of average daily gain and feed conversion rate, with the greatest improvement observed in the T2 group. There were no significant differences (p > 0.05) in nutrient intake or apparent digestibility among the NSPase-supplemented groups. In addition, T2 group had a significantly higher acetate to propionate ratio and pH (p < 0.05) than the other groups, and NH3-N and microbial protein concentrations showed a quadratic curve. The results revealed that both immunoglobulins and serum hormones increased linearly with addition (p < 0.05). As the T2 group showed the best growth performance, the CON and T2 groups were subjected to rumen metagenomic analysis. The results showed higher abundance of bacteria and lower abundance of Viruses in the rumen microbiota of the T2 group compared to the CON group. In addition, Uroviricota and Proteobacteria abundance was significantly lower in the T2 group than in the CON group at the phylum level (p < 0.05). These results suggest that the supplementation of high-concentrate rations with NSPases enhance immunity, reduces virus abundance in the rumen, improves rumen health, and promotes rumen fermentation. Our findings provide novel insights for improving growth performance and alleviating inflammatory responses arising from high concentrate feeding patterns in ruminants. However, the biological mechanisms cannot be elucidated by exploring the composition of rumen microbe alone, and further studies are required.

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

A serotype-specific and tiled amplicon multiplex PCR method for whole genome sequencing of dengue virus

Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1–4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1–4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks.

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater.

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

Host influence on the eukaryotic virome of sympatric mosquitoes and abundance of diverse viruses with a broad host range.

Mosquitoes harbor a large diversity of eukaryotic viruses. Those viromes probably influence mosquito physiology and the transmission of human pathogens. Nevertheless, their ecology remains largely unstudied. Here, we address two key questions in virome ecology. First, we assessed the influence of mosquito species on virome taxonomic diversity and relative abundance. Contrary to most previous studies, the potential effect of the habitat was explicitly included. Thousands of individuals of Culex poicilipes and Culex tritaeniorhynchus, two vectors of viral diseases, were concomitantly sampled in three habitats over two years. A total of 95 viral taxa from 25 families were identified with meta-transcriptomics, with 75% of taxa shared by both mosquitoes. Viromes significantly differed by mosquito species but not by habitat. Differences were largely due to changes in relative abundance of shared taxa. Then, we studied the diversity of viruses with a broad host range. We searched for viral taxa shared by the two Culex species and Aedes vexans, another disease vector, present in one of the habitats. Twenty-six out of the 163 viral taxa were found in the three mosquitoes. These taxa encompassed 14 families. A database analysis supported broad host ranges for many of those viruses, as well as a widespread geographical distribution. Thus, the viromes of mosquitoes from the same genera mainly differed in the relative abundance of shared taxa, whereas differences in viral diversity dominated between mosquito genera. Whether this new model of virome diversity and structure applies to other mosquito communities remains to be determined.

Snow viruses and their implications on red snow algal blooms.

Algal blooms can give snowmelt a red color, reducing snow albedo and creating a runaway effect that accelerates snow melting. The occurrence of red snow is predicted to grow in polar and subpolar regions with increasing global temperatures. We hypothesize that these algal blooms affect virus-bacteria interactions in snow, with potential effects on snowmelt dynamics. A genomic analysis of double-stranded DNA virus communities in red and white snow from the Whistler region of British Columbia, Canada, identified 792 putative viruses infecting bacteria. The most abundant putative snow viruses displayed low genomic similarity with known viruses. We recovered the complete circular genomes of nine putative viruses, two of which were classified as temperate. Putative snow viruses encoded genes involved in energy metabolisms, such as NAD+ synthesis and salvage pathways. In model phages, these genes facilitate increased viral particle production and lysis rates. The frequency of temperate phages was positively correlated with microbial abundance in the snow samples. These results suggest the increased frequency of temperate virus-bacteria interactions as microbial densities increase during snowmelt. We propose that this virus-bacteria dynamic may facilitate the red snow algae growth stimulated by bacteria.IMPORTANCEMicrobial communities in red snow algal blooms contribute to intensifying snowmelt rates. The role of viruses in snow during this environmental shift, however, has yet to be elucidated. Here, we characterize novel viruses extracted from snow viral metagenomes and define the functional capacities of snow viruses in both white and red snow. These results are contextualized using the composition and functions observed in the bacterial communities from the same snow samples. Together, these data demonstrate the energy metabolism performed by viruses and bacteria in a snow algal bloom, as well as expand the overall knowledge of viral genomes in extreme environments.