Novel members of the order Picornavirales identified in freshwater from Guarapiranga reservoir in São Paulo.

The global challenge of water resource availability is exacerbated by anthropogenic influences that promote the emergence of pollutants. Among these pollutants are microbiological agents, including viruses, which are ubiquitous in the biosphere and play a pivotal role in both ecological balance and the occurrence of diseases in animals and plants. Consequently, monitoring viruses in water sources becomes indispensable for the establishment of effective prevention, promotion, and control strategies. Within this context, the study focuses on the identification of novel viruses belonging to the Picornavirales order in freshwater from the Guarapiranga Reservoir in the state of São Paulo, Brazil. The samples were subjected to viral metagenomics. Our analysis led to the characterization of four distinct sequences (GinkV-05, AquaV_10, MarV_14, and MarV_64), which exhibited significant divergence compared to other members of the Picornavirales order. This remarkable diversity prompted the identification of a potential new genus within the Marnaviridae family, tentatively named Ginkgonavirus. Additionally, we characterized four sequences in a very distinct clade and propose the recognition of a novel family (named Aquaviridae) within the Picornavirales order. Our findings contribute valuable insights into the previously uncharted diversity of Picornavirales present in water sources, shedding light on an important facet of viral ecology and evolution in aquatic environments.

Cyanobacteria-cyanophage interactions between freshwater and marine ecosystems based on large-scale cyanophage genomic analysis.

The disparities in harmful algal blooms dynamics are largely attributed to variations in cyanobacteria populations within aquatic ecosystems. However, cyanobacteria-cyanophage interactions and their role in shaping cyanobacterial populations has been previously underappreciated. To address this knowledge gap, we isolated and sequenced 42 cyanophages from diverse water sources in China, with the majority (n = 35) originating from freshwater sources. We designated these sequences as the "Novel Cyanophage Genome sequence Collection" (NCGC). NCGC displayed notable genetic variations, with 95 % (40/42) of the sequences representing previously unidentified taxonomic ranks. By integrating NCGC with public data of cyanophages and cyanobacteria, we found evidence for more frequent historical cyanobacteria-cyanophage interactions in freshwater ecosystems. This was evidenced by a higher prevalence of prophage integrase-related genes in freshwater cyanophages (37.97 %) than marine cyanophages (7.42 %). In addition, freshwater cyanophages could infect a broader range of cyanobacteria orders (n = 4) than marine ones (n = 0). Correspondingly, freshwater cyanobacteria harbored more defense systems per million base pairs in their genomes, indicating more frequent phage infections. Evolutionary and cyanophage epidemiological studies suggest that interactions between cyanobacteria and cyanophages in freshwater and marine ecosystems are interconnected, and that brackish water can act as a transitional zone for freshwater and marine cyanophages. In conclusion, our research significantly expands the genetic information database of cyanophage, offering a wider selection of cyanophages to control harmful cyanobacterial blooms. Additionally, we represent a pioneering large-scale and comprehensive analysis of cyanobacteria and cyanophage sequencing data, and it provides theoretical guidance for the application of cyanophages in different environments.

Risk-based critical concentrations of enteric pathogens for recreational water criteria and recommended minimum sample volumes for routine water monitoring.

Concerns are rising about the contamination of recreational waters from human and animal waste, along with associated risks to public health. However, existing guidelines for managing pathogens in these environments have not yet fully integrated risk-based pathogen-specific criteria, which, along with recent advancements in indicators and markers, are essential to improve the protection of public health. This study aimed to establish risk-based critical concentration benchmarks for significant enteric pathogens, i.e., norovirus, rotavirus, adenovirus, Cryptosporidium spp., Giardia lamblia, Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7. Applying a 0.036 risk benchmark to both marine and freshwater environments, the study identified the lowest critical concentrations for children, who are the most susceptible group. Norovirus, C. jejuni, and Cryptosporidium presented lowest median critical concentrations for virus, bacteria, and protozoa, respectively: 0.74 GC, 1.73 CFU, and 0.39 viable oocysts per 100 mL in freshwater for children. These values were then used to determine minimum sample volumes corresponding to different recovery rates for culture method, digital polymerase chain reaction and quantitative PCR methods. The results indicate that for children, norovirus required the largest sample volumes of freshwater and marine water (52.08 to 178.57 L, based on the 5th percentile with a 10 % recovery rate), reflecting its low critical concentration and high potential for causing illness. In contrast, adenovirus and rotavirus required significantly smaller volumes (approximately 0.24 to 1.33 L). C. jejuni and Cryptosporidium, which required the highest sampling volumes for bacteria and protozoa, needed 1.72 to 11.09 L and 4.17 to 25.51 L, respectively. Additionally, the presented risk-based framework could provide a model for establishing pathogen thresholds, potentially guiding the creation of extensive risk-based criteria for various pathogens in recreational waters, thus aiding public health authorities in decision-making, strengthening pathogen monitoring, and improving water quality testing accuracy for enhanced health protection.

Diversity of Picorna-Like Viruses in the Teltow Canal, Berlin, Germany.

The viromes of freshwater bodies are underexplored. The Picornavirales order, with 371 acknowledged species, is one of the most expansive and diverse groups of eukaryotic RNA viruses. In this study, we add 513 picorna-like viruses to the assemblage of more than 2000 unassigned picorna-like viruses. Our set of the aquatic Picornavirales virome of the Teltow Canal in Berlin, Germany, consists of 239 complete and 274 partial genomes. This urban freshwater body is characterized by the predominance of marna-like viruses (30.8%) and dicistro-like viruses (19.1%), whereas picornaviruses, iflaviruses, solinvi-like viruses, polycipi-like viruses, and nora-like viruses are considerably less prevalent. Caliciviruses and secoviruses were absent in our sample. Although presenting characteristic domains of Picornavirales, more than 100 viruses (20.8%) could not be assigned to any of the 9 Picornavirales families. Thirty-three viruses of the Marnaviridae-mostly locarna-like viruses-exhibit a monocistronic genome layout. Besides a wealth of novel virus sequences, viruses with peculiar features are reported. Among these is a clade of untypeable marna-like viruses with dicistronic genomes, but with the capsid protein-encoding open reading frame located at the 5' part of their RNA. A virus with a similar genome layout but clustering with dicistroviruses was also observed. We further detected monocistronic viruses with a polymerase gene related to aparaviruses. The detection of Aichi virus and five novel posa-like viruses indicates a slight burden in municipal wastewater.

A Novel Freshwater Cyanophage, Mae-Yong924-1, Reveals a New Family.

Cyanobacterial blooms are a worldwide ecological issue. Cyanophages are aquatic viruses specifically infecting cyanobacteria. Little is known about freshwater cyanophages. In this study, a freshwater cyanophage, Mae-Yong924-1, was isolated by the double-layer agar plate method using Microcystis aeruginosa FACHB-924 as an indicator host. Mae-Yong924-1 has several unusual characteristics: a unique shape, cross-taxonomic order infectivity and a very unique genome sequence. Mae-Yong924-1 contains a nearly spherical head of about 100 nm in diameter. The tail or tail-like structure (approximately 40 nm in length) is like the tassel of a round Chinese lantern. It could lyse six diverse cyanobacteria strains across three orders including Chroococcales, Nostocales and Oscillatoriales. The genome of the cyanophage is 40,325 bp in length, with a G + C content of 48.32%, and 59 predicted open reading frames (ORFs), only 12 (20%) of which were functionally annotated. Both BLASTn and BLASTx scanning resulted in "No significant similarity found", i.e., the Mae-Yong924-1 genome shared extremely low homology with sequences in NCBI databases. Mae-Yong924-1 formed a root node alone and monopolized a root branch in the proteomic tree based on genome-wide sequence similarities. The results suggest that Mae-Yong924-1 may reveal a new unknown family apparently distinct from other viruses.

A Novel Wide-Range Freshwater Cyanophage MinS1 Infecting the Harmful Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.

Polymer monoliths for the concentration of viruses from environmental waters: A review.

Even at low concentrations in environmental waters, some viruses are highly infective, making them a threat to human health. They are the leading cause of waterborne enteric diseases. In agriculture, plant viruses in irrigation and runoff water threat the crops. The low concentrations pose a challenge to early contamination detection. Thus, concentrating the virus particles into a small volume may be mandatory to achieve reliable detection in molecular techniques. This paper reviews the organic monoliths developments and their applications to concentrate virus particles from waters (waste, surface, tap, sea, and irrigation waters). Free-radical polymerization and polyaddition reactions are the most common strategies to prepare the monoliths currently used for virus concentration. Here, the routes for preparing and functionalizing both methacrylate and epoxy-based monoliths will be shortly described, following a revision of their retention mechanisms and applications in the concentration of enteric and plant viruses in several kinds of waters.

Contamination of Human Bocavirus Genotypes 1, 2, 3, and 4 in Environmental Waters in Thailand.

Human bocavirus (HBoV) has been recognized as one of the common pathogens which cause respiratory disease and acute gastroenteritis in children worldwide. Recently, our studies reported the detection of HBoV in children with acute gastroenteritis and in oysters in Thailand. However, studies on the presence of HBoV in environmental waters in Thailand have not yet been conducted. In this study, 126 environmental water samples collected from November 2016 to July 2018 were investigated. Detection of HBoV was based on amplification of the VP1/VP2 region of the HBoV genome by nested PCR followed by nucleotide sequencing and phylogenetic analysis. HBoV was detected in 34 out of 126 samples (27.0%). All four HBoV genotypes, HBoV1 to HBoV4, were detected. HBoV2 was the most frequently detected genotype (61.8%), followed by HBoV1 (23.5%), HBoV4 (8.8%), and HBoV3 (5.9%). The highest detection rate of HBoV was observed during the warmest months in Thailand: April 2017 and March 2018. Phylogenetic analysis of VP1/VP2 nucleotide sequences of HBoV genotypes revealed that all four of the genotypes detected in environmental waters were closely related to genotypes detected in patients with acute gastroenteritis, which had been reported previously in the same geographical area. This study reports the existence of multiple HBoV genotypes in environmental waters and provides evidence of a considerably high magnitude of HBoV contamination in these waters. These findings demonstrate the potential risk of waterborne transmission of HBoV to humans. IMPORTANCE Recently, we reported the detection of HBoV genotypes 1, 2, and 3 in pediatric patients with acute gastroenteritis, and the detection of HBoV1 and 2 in oysters in Thailand. In this study, we reported the detection of HBoV1, 2, 3, and 4 contamination in environmental waters within the same geographic area. Phylogenetic analysis demonstrated that the HBoV genotypes detected in environmental waters and in oysters were closely related to HBoV detected in patients. These findings imply that HBoV contamination in oysters and in environmental waters could be a potential sources of foodborne and waterborne transmission to humans.

Novel Freshwater Cyanophages Provide New Insights into Evolutionary Relationships between Freshwater and Marine Cyanophages.

Cyanobacteria and cyanophages are present widely in both freshwater and marine environments. However, freshwater cyanophages remain unknown largely due to the small numbers of cyanophage isolates despite their ecological and environmental significance. In this study, we present the characterization of two novel lytic freshwater cyanophages isolated from a tropical inland lake in Singapore, namely, cyanopodovirus S-SRP01 and cyanomyovirus S-SRM01, infecting two different strains of Synechococcus spp. Functional annotation of S-SRP01 and S-SRM01 genomes revealed a high degree of homology with marine cyanophages. Phylogenetic trees of concatenated genes and whole-genome alignment provided further evidence that S-SRP01 is close evolutionarily to marine cyanopodoviruses, while S-SRM01 is evolutionarily close to marine cyanomyoviruses. Few genetic similarities between freshwater and marine cyanophages have been identified in previous studies. The isolation of S-SRP01 and S-SRM01 expand current knowledge on freshwater cyanophages infecting Synechococcus spp. Their high degree of gene sharing provides new insights into the evolutionary relationships between freshwater and marine cyanophages. This relatedness is further supported by the discovery of similar phenomenon from other freshwater viral metagenomes. IMPORTANCE This study expands the current knowledge on freshwater cyanophage isolates and cyanophage genetic diversity, indicating that freshwater and marine cyanophages infecting Synechococcus spp. may share close genetic similarity and evolutionary relationships.

Occurrence and diversity of viruses associated with cyanobacterial communities in a Brazilian freshwater reservoir.

As part of the phytoplankton of marine and freshwater environments around the world, cyanobacteria interact with viruses (cyanophages) that affect their abundance and diversity. Investigations focusing on cyanophages co-occurring with freshwater cyanobacteria are scarce, particularly in Brazil. The aim of this study was to assess the diversity of cyanophages associated with a Microcystis-dominated cyanobacterial bloom in a tropical reservoir. Samples were processed as viral fractions of water and cellular fractions, and temporal fluctuations in the abundance of Ma-LMM01-type cyanophages and their Microcystis hosts were determined by qPCR. We applied shotgun metagenomics to obtain a wider characterization of the cyanophage community. During the study period, Microcystis gene copies were quantified in all cellular fractions, and the copy number of the Ma-LMM01 phage gene tended to increase with host abundance. Metagenomic analysis demonstrated that Caudovirales was the major viral order associated with the cyanophage families Myoviridae (34-88%), Podoviridae (3-42%), and Siphoviridae (6-23%). The metagenomic analysis results confirmed the presence of Microcystis cyanophages in both viral and cellular fractions and demonstrated a high relative abundance of picocyanobacteria-related viruses and Prochlorococcus (36-52%) and Synechococcus (37-50%) phages. For other main cyanobacterial genera, no related cyanophages were identified, which was probably due to the scarce representation of cyanophage sequences in databanks. Thus, the studied reservoir hosted a diverse cyanophage community with a remarkable contribution of phages related to picoplanktonic cyanobacteria. These results provide insights that motivate future sequencing efforts to assess cyanophage diversity and recover complete genomes.

Marseilleviridae Lineage B Diversity and Bunch Formation Inhibited by Galactose.

Marseilleviridae is a family of large double-stranded DNA viruses that is currently divided into five subgroups, lineages A-E. Hokutovirus and kashiwazakivirus, both of which belong to lineage B, have been reported to induce host acanthamoeba cells to form aggregations called "bunches". This putatively results in increased opportunities to infect acanthamoeba cells, in contrast to lineage A, which has been reported to not form "bunches". In the present study, we isolated 14 virus strains of the family Marseilleviridae from several Japanese water samples, 11 of which were identified as lineage B viruses. All 11 lineage B strains caused infected amoeba cells to form bunches. We then investigated the involvement of monosaccharides in bunch formation by amoeba cells infected with hokutovirus. Galactose inhibited bunch formation, thereby allowing amoeba cells to delay the process, whereas mannose and glucose did not. A kinetic image analysis of hokutovirus-infected amoeba cells confirmed the inhibition of bunch formation by galactose. The number of hokutovirus-infected amoeba cells increased more rapidly than that of tokyovirus-infected cells, which belongs to lineage A. This result suggests that bunch formation by infected amoeba cells is advantageous for lineage B viruses.

Detection and evaluation of rotavirus surveillance methods as viral indicator in the aquatic environments.

Group A rotaviruses (RVAs) have been introduced as the most important causative agents of acute gastroenteritis in the young children. One of every 260 children born globally will die due to rotavirus (RV) before 5 years old. The RV is widely known as a viral indicator for health (fecal contamination) because this pathogen has a high treatment resistance nature, which has been listed as a relevant waterborne pathogen by the World Health Organization (WHO). Therefore, monitoring of environmental is important, and RV is one of the best-known indicators for monitoring. It has been proved that common standards for microbiological water quality do not guarantee the absence of viruses. On the other hand, in order to recover and determine RV quantity within water, standard methods are scarce. Therefore, dependable prediction of RV quantities in water sample is crucial to be able to improve supervision efficiency of the treatment procedure, precise quantitative evaluation of the microbial risks as well as microbiological water safety. Hence, this study aimed to introduce approaches to detecting and controlling RV in environmental waters, and discussed the challenges faced to enable a clear perception on the ubiquity of the RV within different types of water across the world.

Interaction dynamics and virus-host range for estuarine actinophages captured by epicPCR.

Viruses impact microbial diversity, gene flow and function through virus-host interactions. Although metagenomics surveys are rapidly cataloguing viral diversity, methods are needed to capture specific virus-host interactions in situ. Here, we leveraged metagenomics and repurposed emulsion paired isolation-concatenation PCR (epicPCR) to investigate viral diversity and virus-host interactions in situ over time in an estuarine environment. The method fuses a phage marker, the ribonucleotide reductase gene, with the host 16S rRNA gene of infected bacterial cells within emulsion droplets providing single-cell resolution for dozens of samples. EpicPCR captured in situ virus-host interactions for viral clades with no closely related database representatives. Abundant freshwater Actinobacteria lineages, in particular Rhodoluna sp., were the most common hosts for these poorly characterized viruses, with interactions correlated with environmental factors. Multiple methods used to identify virus-host interactions, including epicPCR, identified different and largely non-overlapping interactions within the vast virus-host interaction space. Tracking virus-host interaction dynamics also revealed that multi-host viruses had significantly longer periods with observed virus-host interactions, whereas single-host viruses were observed interacting with hosts at lower minimum abundances, suggesting more efficient interactions. Capturing in situ interactions with epicPCR revealed environmental and ecological factors shaping virus-host interactions, highlighting epicPCR as a valuable technique in viral ecology.

Identifying septic pollution exposure routes during a waterborne norovirus outbreak - A new application for human-associated microbial source tracking qPCR.

In June 2017, the Pennsylvania Department of Health (PADOH) was notified of multiple norovirus outbreaks associated with 179 ill individuals who attended separate events held at an outdoor venue and campground over a month period. Epidemiologic investigations were unable to identify a single exposure route and therefore unable to determine whether there was a persistent contamination source to target for exposure mitigation. Norovirus was detected in a fresh recreational water designated swimming area and a drinking water well. A hydrogeological site evaluation suggested a nearby septic leach field as a potential contamination source via ground water infiltration. Geological characterization revealed a steep dip of the bedrock beneath the septic leach field toward the well, providing a viral transport pathway in a geologic medium not previously documented as high risk for viral ground water contamination. The human-associated microbial source tracking (MST) genetic marker, HF183, was used as a microbial tracer to demonstrate the hydrogeological connection between the malfunctioning septic system, drinking water well, and recreational water area. Based on environmental investigation findings, venue management and local public health officials implemented a series of outbreak prevention strategies including discontinuing the use of the contaminated well, issuing a permit for a new drinking water well, increasing portable toilet and handwashing station availability, and promoting proper hand hygiene. Despite the outbreaks at the venue and evidence of ground water contamination impacting nearby recreational water and the drinking water well, no new norovirus cases were reported during a large event one week after implementing prevention practices. This investigation highlights a new application for human-associated MST methods to trace hydrological connections between multiple fecal pollutant exposure routes in an outbreak scenario. In turn, pollutant source information can be used to develop effective intervention practices to mitigate exposure and prevent future outbreaks associated with human fecal contaminated waters.

Rotavirus Surveillance in Tap Water, Recycled Water, and Sewage Sludge in Thailand: A Longitudinal Study, 2007-2018.

The objective of this study was to describe the epidemiological and molecular surveillance of rotaviruses in tap water, recycled water, and sewage sludge in Thailand from 2007 to 2018. Three hundred and seventy tap water, 202 recycled water, and 72 sewage sludge samples were collected and processed to detect the rotavirus VP7 gene using RT-nested PCR. Rotavirus G genotypes were identified by DNA sequencing and phylogenetic analysis. The frequency of rotavirus detection was 0.54% of the tap water samples, 30.2% of the recycled water samples, and 50.0% of the sewage sludge samples. During the 12-year surveillance, G1 was prevalent most years and constantly predominant in recycled water and sewage sludge. G2 was identified in a tap water sample and in recycled water samples. G3 and G9 were observed in both recycled water and sewage sludge samples. The uncommon G6 rotavirus strain was identified in one recycled water sample. The rotavirus VP4 gene was detected in rotavirus strains with an identified G genotype using RT-multiplex nested PCR. The unusual P[6] genotype was the most frequently detected, followed by mixed P[6]/[4] and P[4] genotypes. Phylogenetic analysis of both G and P genotypes showed a close genetic relationship with sequences of human rotavirus strains. The high nucleotide identity of the rotavirus strains found in this study to human rotavirus strains suggests that the rotaviruses are derived from human source. These results represent useful epidemiological and molecular information for evaluating rotavirus distribution in water for consumption and irrigation, and in biosolids for agricultural application.

Detection of Human Adenovirus, Rotavirus, and Enterovirus in Tap Water and Their Association with the Overall Quality of Water in Karachi, Pakistan.

Drinking water supplies in the developing world often serve as a biosphere for various organisms. Viral gastroenteritis is a neglected area of research in Pakistan, there are no data for the prevalence of enteric viruses in drinking water of the largest city of Karachi. The present study aimed to provide a survey of the existence of enteric viruses: human adenovirus (HAdV), human enteroviruses (hEV), and genotype A rotavirus (GARV) in tap water. Using a simple PCR approach, we detected 20%, 43%, and 23% of HAdV, hEV, and GARV in tap water samples, respectively. We have also shown an overall quality deficit of tap water at the pumping station and consumer tap. We have found no sample free from bacterial contaminations. The ranges for a total number of the heterotrophic plate count and coliform were found 8.7 × 102-4.5 × 106 CFU/mL and 210 to uncountable coliforms/100 mL, respectively. Moreover, we assessed the efficiency of small-scale water treatment methods for the removal of viruses.

A Novel RNA Virus, Macrobrachium rosenbergii Golda Virus (MrGV), Linked to Mass Mortalities of the Larval Giant Freshwater Prawn in Bangladesh.

Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single­stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of Macrobrachium rosenbergii golda virus (MrGV) suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 Stability in Multiple Water Matrices.

The advent of the global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates a thorough study of the stability and transmissibility in the environment. We characterized the stability of SARS-CoV-2 in three water matrices: fresh, tap, and seawater. The minimum infective dose of SARS-CoV-2 in Vero cells was confirmed to be 10³ PFU/mL. The stability of SARS-CoV-2 varied according to the water matrix: infective SARS-CoV-2 was undetectable after treatment with fresh water and seawater, but remained detectable for 2 days in tap water, when starting with an initial concentration of 104 PFU/mL. When the starting concentration was increased to 105 PFU/mL, a similar trend was observed. In addition, viral RNA persisted longer than infectious virus in all water matrices. This study was conducted in stagnant water containing a significantly high titer of virus, thus, human-to-human transmission of SARS-CoV-2 through the actual aquatic environment is expected to be rare.

Viruses of freshwater bloom-forming cyanobacteria: genomic features, infection strategies and coexistence with the host.

Freshwater bloom-forming cyanobacteria densely grow in the aquatic environments, leading to an increase in the viral-contact rate. They possess numerous antiviral genes, as well as cell differentiation- and physiological performance-related genes, owing to genome expansion. Their genomic features and unique lifestyles suggest that they coexist with cyanoviruses in ways different from marine cyanobacteria. Furthermore, genome contents of isolated freshwater bloom-forming cyanobacterial viruses have little in common with those of marine cyanoviruses studied to date. They lack the marine cyanoviral hallmark genes that sustain photosynthetic activity and redirect host metabolism to viral reproduction; therefore, they are predicted to share metabolisms and precursor pools with host cyanobacteria to ensure efficient viral reproduction and avoid nutrient deficiencies and antiviral response. Additionally, cyanovirus-cyanobacteria coexistence strategies may change as bloom density increases. Diverse genotypic populations of cyanoviruses and hosts coexist and fluctuate under high viral-contact rate conditions, leading to their rapid coevolution through antiviral responses. The ancestral and newly evolved genotypes coexist, thereby expanding the diversity levels of host and viral populations. Bottleneck events occurring due to season-related decreases in bloom-forming species abundance provide each genotype within cyanobacterial population an equal chance to increase in prevalence during the next bloom and enhance further diversification.