Metagenomic perspectives on antibiotic resistance genes in tap water: The environmental characteristic, potential mobility and health threat.

As an emerging environmental contaminant, antibiotic resistance genes (ARGs) in tap water have attracted great attention. Although studies have provided ARG profiles in tap water, research on their abundance levels, composition characteristics, and potential threat is still insufficient. Here, 9 household tap water samples were collected from the Guangdong-Hong Kong-Macao Greater Bay Area (GBA) in China. Additionally, 75 sets of environmental sample data (9 types) were downloaded from the public database. Metagenomics was then performed to explore the differences in the abundance and composition of ARGs. 221 ARG subtypes consisting of 17 types were detected in tap water. Although the ARG abundance in tap water was not significantly different from that found in drinking water plants and reservoirs, their composition varied. In tap water samples, the three most abundant classes of resistance genes were multidrug, fosfomycin and MLS (macrolide-lincosamide-streptogramin) ARGs, and their corresponding subtypes ompR, fosX and macB were also the most abundant ARG subtypes. Regarding the potential mobility, vanS had the highest abundance on plasmids and viruses, but the absence of key genes rendered resistance to vancomycin ineffective. Generally, the majority of ARGs present in tap water were those that have not been assessed and are currently not listed as high-threat level ARG families based on the World Health Organization Guideline. Although the current potential threat to human health posed by ARGs in tap water is limited, with persistent transfer and accumulation, especially in pathogens, the potential danger to human health posed by ARGs should not be ignored.

Prevalência de Influenza A, vírus sincicial respiratório eSARS-CoV-2 em pacientes com síndrome respiratóriaaguda grave em Passo Fundo, Rio Grande do Sul / Prevalence of Influenza A, respiratory syncytial virus andSARS-CoV-2 in patients with severe acute respiratorysyndrome in Passo Fundo, Rio Grande do Sul

A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.

Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.

Influenza A(H5N1) Virus Resilience in Milk after Thermal Inactivation.

Highly pathogenic avian influenza A(H5N1) detected in dairy cows raises concerns about milk safety. The effects of pasteurization-like temperatures on influenza viruses in retail and unpasteurized milk revealed virus resilience under certain conditions. Although pasteurization contributes to viral inactivation, influenza A virus, regardless of strain, displayed remarkable stability in pasteurized milk.

Innate lymphoid cells and infectious diseases.

Innate lymphoid cells (ILCs) are the main resident lymphocytes that mostly reside in tissues owing to the lack of adaptive antigen receptors. These cells are involved in early anti-infective immunity, antitumour immunity, regulation of tissue inflammation, and maintenance of homeostasis in the internal environment of tissues and have been referred to as the "first armies stationed in the human body". ILCs are widely distributed in the lungs, colon, lymph nodes, oral mucosa and even embryonic tissues. Due to the advantage of their distribution location, they are often among the first cells to come into contact with pathogens.Relevant studies have demonstrated that ILCs play an early role in the defence against a variety of pathogenic microorganisms, including bacteria, viruses, fungi and helminths, before they intervene in the adaptive immune system. ILCs can initiate a rapid, nonspecific response against pathogens prior to the initiation of an adaptive immune response and can generate a protective immune response against specific pathogens, secreting different effectors to play a role.There is growing evidence that ILCs play an important role in host control of infectious diseases. In this paper, we summarize and discuss the current known infectious diseases in which ILCs are involved and ILC contribution to the defence against infectious diseases. Further insights into the mechanisms of ILCs action in different infectious diseases will be useful in facilitating the development of therapeutic strategies for early control of infections.

Co-Circulation of 2 Oropouche Virus Lineages, Amazon Basin, Colombia, 2024.

In early 2024, explosive outbreaks of Oropouche virus (OROV) linked to a novel lineage were documented in the Amazon Region of Brazil. We report the introduction of this lineage into Colombia and its co-circulation with another OROV lineage. Continued surveillance is needed to prevent further spread of OROV in the Americas.

Conventional cytology scheme: adherence and associated factors.

OBJECTIVES: This study analyzed adherence rates to conventional cytology and associated factors in a cohort of women at a health service provider institution in Medellin, Colombia. STUDY DESIGN: Observational cohort study with repeated measures. METHODS: Clinical and sociodemographic data were obtained from databases for screenings between January 2018 and December 2022. Adherence, defined as undergoing 1, 2, or 3 cytology tests according to national guidelines, was the outcome. Statistical analysis involved a Poisson model with robust errors to identify factors associated with adherence. RESULTS: In total, 26,445 women were included, with a median age of 25 years (IQR: 22-27). Adherence rate was 20.4%. Having just high school education (RR = 0.51; 95% CI: 0.49-0.55), a history of pregnancy (RR = 0.63; 95% CI: 0.54-0.75), and a history of sexually transmitted infections (RR = 0.88; 95% CI: 0.78-0.99) decreased adherence. Conversely, the human papillomavirus (HPV) vaccination history increased adherence (RR = 2.11; 95% CI: 1.60-2.80). CONCLUSION: It is vital to monitor cytology programs to improve demand-induced and spontaneous consultations. Diligent follow-up, focusing on patients with factors linked to low adherence, along with appointment reminders, can enhance adherence to the screening protocol.

Epidemiological survey of calf diarrhea related viruses in several areas of Guangdong Province.

Introduction: Bovine torovirus (BToV), Bovine enterovirus (BEV), Bovine norovirus (BNoV), Bovine coronavirus (BCoV), Bovine rotavirus (BRV), and Bovine viral diarrhea virus (BVDV) are significant pathogens causing diarrhea in calves, characterized by their high prevalence and challenging prevention and control measures. Methods: We analyzed 295 calf diarrhea samples, amplifying the M gene from BToV-positive samples, the 5'UTR gene from BEV-positive samples, the RdRp gene from BNoV-positive samples, the VP7 gene from BRV-positive samples, the S gene from BCoV-positive samples, and the 5'UTR gene from BVDV-positive samples. Subsequent homology analysis and phylogenetic tree construction were performed. Results: The overall viral positive rate in Guangdong Province was 21.36%. Specific detection rates were as follows: Foshan City at 50.00% (18/36), Guangzhou City at 43.90% (36/82), Huizhou City at 21.21% (7/33), Yangjiang City at 2.08% (1/48), Meizhou City at 1.39% (1/72), and Heyuan City at 0.00% (0/24). The detection rates for BToV, BEV, BNoV, BCoV, BRV, and BVDV were 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, the highest overall virus detection rate was observed in the Guangzhou-Foshan region, with BRV and BEV showing the highest detection rates among the six viruses. This study marks the first report of BToV and BNoV in Guangdong Province. Phylogenetic analysis revealed that the BToV strain belonged to type II, sharing genetic similarities with epidemic strains from various provinces in China. The BEV strains were categorized into E and F types, with the F type being the predominant strain in Guangdong Province and exhibiting the closest genetic relationship to strains from Heilongjiang and Guangxi. The BNoV strains, along with Hebei strains, were identified as GIII.2 subgenotype. BCoV strains showed the highest genetic similarity to strains from Sichuan. All BRV strains were classified under the G6 subtype and had the closest genetic relationship with human rotavirus strains. BVDV strains were identified as subtype 1b, closely related to the Beijing strain. In conclusion, this study investigated the prevalence and evolutionary characteristics of diarrhea-associated viruses in calves in specific areas of Guangdong Province, providing a valuable reference for establishing effective prevention and control measures in cattle farms.

Enhancing T-cell recruitment in renal cell carcinoma with cytokine-armed adenoviruses.

Immunotherapy has emerged as a promising approach for cancer treatment, with oncolytic adenoviruses showing power as immunotherapeutic agents. In this study, we investigated the immunotherapeutic potential of an adenovirus construct expressing CXCL9, CXCL10, or IL-15 in clear cell renal cell carcinoma (ccRCC) tumor models. Our results demonstrated robust cytokine secretion upon viral treatment, suggesting effective transgene expression. Subsequent analysis using resistance-based transwell migration and microfluidic chip assays demonstrated increased T-cell migration in response to chemokine secretion by infected cells in both 2D and 3D cell models. Flow cytometry analysis revealed CXCR3 receptor expression across T-cell subsets, with the highest percentage found on CD8+ T-cells, underscoring their key role in immune cell migration. Alongside T-cells, we also detected NK-cells in the tumors of immunocompromised mice treated with cytokine-encoding adenoviruses. Furthermore, we identified potential immunogenic antigens that may enhance the efficacy and specificity of our armed oncolytic adenoviruses in ccRCC. Overall, our findings using ccRCC cell line, in vivo humanized mice, physiologically relevant PDCs in 2D and patient-derived organoids (PDOs) in 3D suggest that chemokine-armed adenoviruses hold promise for enhancing T-cell migration and improving immunotherapy outcomes in ccRCC. Our study contributes to the development of more effective ccRCC treatment strategies by elucidating immune cell infiltration and activation mechanisms within the tumor microenvironment (TME) and highlights the usefulness of PDOs for predicting clinical relevance and validating novel immunotherapeutic approaches. Overall, our research offers insights into the rational design and optimization of viral-based immunotherapies for ccRCC.

Corrigendum: Distribution characteristics of human herpes viruses in the lower respiratory tract and their impact on 30-day mortality in community-acquired pneumonia patients.

[This corrects the article DOI: 10.3389/fcimb.2024.1436509.].

Ampullaviruses: From Extreme Environments to Biotechnological Innovation.

Ampullaviruses are unique among viruses. They live in extreme environments and have special bottle-shaped architecture. These features make them useful tools for biotechnology. These viruses have compact genomes. They encode a range of enzymes and proteins. Their natural environment highlights their suitability for industrial applications. Ongoing research explores ways in which these viruses can improve enzyme stability. They are also employed in the creation of new biosensors and the development of new bioremediation techniques. High coinfection rates and the ecology of ampullaviruses at larger scales can also reveal new viral vectors. They can also help improve phage therapy. Here, we have explored the structure and function of ampullaviruses. We have focused on their use in biotechnology. We have also identified their characteristics that could prove to be useful. We have also pointed out key knowledge gaps and bridging them could further extend the biotechnological uses.

Complete genome sequence of the avian paramyxovirus serotype 9 strain duck/Miyazaki/128/2021.

Here, we report the complete genome sequence of the avian paramyxovirus serotype 9 strain duck/Miyazaki/128/2021, which was determined using the Illumina MiSeq platform. The position of the hemagglutinin-neuraminidase stop codon differed from that of the only other available completely sequenced prototype strain, duck/New York/22/1977.

Molecular mechanisms of emerging inflammasome complexes and their activation and signaling in inflammation and pyroptosis.

Inflammasomes are multi-protein complexes that assemble within the cytoplasm of mammalian cells in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), driving the secretion of the pro-inflammatory cytokines IL-1ß and IL-18, and pyroptosis. The best-characterized inflammasome complexes are the NLRP3, NAIP-NLRC4, NLRP1, AIM2, and Pyrin canonical caspase-1-containing inflammasomes, and the caspase-11 non-canonical inflammasome. Newer inflammasome sensor proteins have been identified, including NLRP6, NLRP7, NLRP9, NLRP10, NLRP11, NLRP12, CARD8, and MxA. These inflammasome sensors can sense PAMPs from bacteria, viruses and protozoa, or DAMPs in the form of mitochondrial damage, ROS, stress and heme. The mechanisms of action, physiological relevance, consequences in human diseases, and avenues for therapeutic intervention for these novel inflammasomes are beginning to be realized. Here, we discuss these emerging inflammasome complexes and their putative activation mechanisms, molecular and signaling pathways, and physiological roles in health and disease.

MVP: a modular viromics pipeline to identify, filter, cluster, annotate, and bin viruses from metagenomes.

While numerous computational frameworks and workflows are available for recovering prokaryote and eukaryote genomes from metagenome data, only a limited number of pipelines are designed specifically for viromics analysis. With many viromics tools developed in the last few years alone, it can be challenging for scientists with limited bioinformatics experience to easily recover, evaluate quality, annotate genes, dereplicate, assign taxonomy, and calculate relative abundance and coverage of viral genomes using state-of-the-art methods and standards. Here, we describe Modular Viromics Pipeline (MVP) v.1.0, a user-friendly pipeline written in Python and providing a simple framework to perform standard viromics analyses. MVP combines multiple tools to enable viral genome identification, characterization of genome quality, filtering, clustering, taxonomic and functional annotation, genome binning, and comprehensive summaries of results that can be used for downstream ecological analyses. Overall, MVP provides a standardized and reproducible pipeline for both extensive and robust characterization of viruses from large-scale sequencing data including metagenomes, metatranscriptomes, viromes, and isolate genomes. As a typical use case, we show how the entire MVP pipeline can be applied to a set of 20 metagenomes from wetland sediments using only 10 modules executed via command lines, leading to the identification of 11,656 viral contigs and 8,145 viral operational taxonomic units (vOTUs) displaying a clear beta-diversity pattern. Further, acting as a dynamic wrapper, MVP is designed to continuously incorporate updates and integrate new tools, ensuring its ongoing relevance in the rapidly evolving field of viromics. MVP is available at https://gitlab.com/ccoclet/mvp and as versioned packages in PyPi and Conda.IMPORTANCEThe significance of our work lies in the development of Modular Viromics Pipeline (MVP), an integrated and user-friendly pipeline tailored exclusively for viromics analyses. MVP stands out due to its modular design, which ensures easy installation, execution, and integration of new tools and databases. By combining state-of-the-art tools such as geNomad and CheckV, MVP provides high-quality viral genome recovery and taxonomy and host assignment, and functional annotation, addressing the limitations of existing pipelines. MVP's ability to handle diverse sample types, including environmental, human microbiome, and plant-associated samples, makes it a versatile tool for the broader microbiome research community. By standardizing the analysis process and providing easily interpretable results, MVP enables researchers to perform comprehensive studies of viral communities, significantly advancing our understanding of viral ecology and its impact on various ecosystems.

[Current vaccination and immunization strategies in dermatology]. / Aktuelles zu Impfungen und Immunisierungsstrategien in der Dermatologie.

Vaccinations are an important pillar of public health. They have high benefits for individuals and society as a whole by specifically preventing or mitigating infectious diseases. In many cases, they offer benefits that go beyond protection against the disease in question, e.g., protective cardiovascular effects. Vaccination recommendations in Germany are drawn up by the Standing Committee on Vaccination (STIKO), while the European Medicines Agency (EMA) is responsible for the approval of vaccines in the EU. Vaccinations may be carried out by physicians regardless of their specialty. In dermatology, vaccinations against varicella (chickenpox), herpes zoster, and human papillomavirus are established. The development of vaccines against other dermatologically relevant diseases and cancer vaccines is the subject of intensive research. Particularly in the case of immunosuppression, the physician must also take into consideration which vaccinations are possible and useful or contraindicated. Type I or type IV allergies to components of vaccinations are very rare, but reactions at the injection site often occur as a dermatological side effect. Urticarial reactions are also possible, as does the worsening of underlying dermatological conditions such as psoriasis vulgaris.

Reemergence of Oropouche Virus in the Americas and Risk for Spread in the United States and Its Territories, 2024.

Oropouche virus has recently caused outbreaks in South America and the Caribbean, expanding into areas to which the virus was previously not endemic. This geographic range expansion, in conjunction with the identification of vertical transmission and reports of deaths, has raised concerns about the broader threat this virus represents to the Americas. We review information on Oropouche virus, factors influencing its spread, transmission risk in the United States, and current status of public health response tools. On the basis of available data, the risk for sustained local transmission in the continental United States is considered low because of differences in vector ecology and in human-vector interactions when compared with Oropouche virus-endemic areas. However, more information is needed about the drivers for the current outbreak to clarify the risk for further expansion of this virus. Timely detection and control of this emerging pathogen should be prioritized to mitigate disease burden and stop its spread.

Strategies for engineering oncolytic viruses to enhance cancer immunotherapy.

Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer and is characterized by rapid metastasis and high mortality, presenting a challenge for early-stage treatment modalities. The heterogeneity of NSCLC's tumor microenvironment (TME) significantly influences the efficacy of anti-PD-1 immune checkpoint inhibitors (ICIs) therapy, leading to varied patient responses. This review characterized different strains of oncolytic viruses in NSCLC and the different gene edits in pre-existing oncolytic viruses. This study also aimed to provide strategies to enhance anti-PD-1 therapy in NSCLC by engineering oncolytic viruses (OVs). This study offers insights into the genomic adaptations necessary for OVs targeting NSCLC, identify genetic determinants of anti-PD-1 response variability, and propose genomic edits to bolster therapy effectiveness. The primary goal of this study is to present a theoretically designed OV with a detailed genomic framework capable of enhancing the response to anti-PD-1 therapy, thereby advancing the field of cancer immunotherapy.

Versatility of the Zinc-Finger Antiviral Protein (ZAP) As a Modulator of Viral Infections.

The zinc-finger antiviral protein (ZAP) is a restriction factor that proficiently impedes the replication of a variety of RNA and DNA viruses. In recent years, the affinity of ZAP's zinc-fingers for single-stranded RNA (ssRNA) rich in CpG dinucleotides was uncovered. High frequencies of CpGs in RNA may suggest a non-self origin, which underscores the importance of ZAP as a potential cellular sensor of (viral) RNA. Upon binding viral RNA, ZAP recruits cellular cofactors to orchestrate a finely tuned antiviral response that limits virus replication via distinct mechanisms. These include promoting degradation of viral RNA, inhibiting RNA translation, and synergizing with other immune pathways. Depending on the viral species and experimental set-up, different isoforms and cellular cofactors have been reported to be dominant in shaping the ZAP-mediated antiviral response. Here we review how ZAP differentially affects viral replication depending on distinct interactions with RNA, cellular cofactors, and viral proteins to discuss how these interactions shape the antiviral mechanisms that have thus far been reported for ZAP. Importantly, we zoom in on the unknown aspects of ZAP's antiviral system and its therapeutic potential to be employed in vaccine design.

Synonymous variant of TLR7 at restriction site rs864058 identified in Covid 19 Pakistani patients.

BACKGROUND: TLR7, the receptor accountable for immune response to RNA viruses, has been studied extensively to identify its variants related to the severity of Covid-19 in different populations worldwide. However, the genotype of Pakistani population is still unknown. This study aimed to determine the TLR7 genotypes and their relation with severity in our population. METHODS: This cross sectional study collected data on 151 Covid-19 positive patients (aged 18-80 years), from June 2022 to May 2023, after an informed consent, from Ziauddin University and Hospital. Prior to that approval from ethics review committee was taken. The demographic variables and comorbidities were recorded along with health status till LAMA (Leave Against Medical Advise), recovery or death. The DNA was extracted from collected blood samples, PCR and Sanger sequencing was done for identification of TLR7 variants. SPSS was used for data analyses and Chi-Square for categorical variables. P-values of <0.05 was considered significant. RESULTS: Out of 151 patients' sequencing was done for 59 samples. The restriction site, rs864058 of TLR7 gene, identified G/A and G/G variants. This missense variant of TLR7 identified at rs864058 of TLR7 gene, has not been previously reported in population control databases. The genotype G/G was main variant of 49 (83%) patients, whereas, G/A was found in 10 (17%). Majority, 25 (51%) of patients with mild covid-19 had GG genotype but results were not significant (P=0.684). Among female patients the main genotype was GA 8 (80%) while male had G/G 29 (59.2%) with significant results (P=0.024). Since G/G genotype was the major genotype, high percentage was found in hypertensives [20 (40.8%)], Diabetics [13 (26.5%)], depression [24 (49%)] and pneumonia patients [20 (40.8%)]. However, significant association (P=0.023) was only found with pneumonia. Males, in majority had severe [17 (68%)] infection and death [40 (26.4%)], whereas, females had mild [14 (25%)] with [12 (7.9%)] deaths. CONCLUSION: A variant rs864058 "G/A" of TLR7, in relation to covid-19 were found in our population. Males were found more at risk of morbidity and mortality due to covid-19. Larger studies are required to further confirm these results.

Antiviral drugs in animal-derived matrices: A review.

The ban of antiviral drugs in food-producing animals in several parts of the world, latest by Commission Delegated Regulation (EU) 2022/1644, has increased the need for food control laboratories to develop analytical methods and perform official controls. However, little is known about antiviral drugs, their use, and its analysis in food-producing animals in the EU. This review aims to provide insights into relevant viruses, antiviral drugs, and animal-derived matrices for analytical method development and monitoring purposes to implement in food control laboratories. For years, animal viruses, such as African swine fever and avian influenza, have caused many outbreaks. Besides, they led to large economic losses due to the applied control measures and a lack of available treatments. Considering these losses and the known effectiveness of authorized human antiviral drugs in different organisms, medicines such as amantadine in Chinese poultry have been misused. Various analytical methods, including screening assays and sensors (published 2016-2023), and mass spectrometry methods (published 2012-2023) have been outlined in this review for the monitoring of antiviral drugs in animal-derived matrices. However, pharmacokinetics information on metabolite formation and distribution of these substances in different animal-derived matrices is incomplete. Additionally, apart from a few countries, there is a lack of available data on the potential misuse of different antiviral drugs in animal-derived matrices. Although a handful of important antiviral drugs, such as influenza, broad-spectrum, antiretroviral, and herpes drugs, and animal-derived matrices, such as chicken muscle, are identified, the priority of the scope should be further specified by closing the aforementioned gaps.

Spatiotemporal dynamics of giant viruses within a deep freshwater lake reveal a distinct dark-water community.

Giant viruses significantly regulate the ecological dynamics of diverse ecosystems. Although metagenomics has expanded our understanding of their diversity and ecological roles played in marine environments, little is known about giant viruses of freshwater ecosystems. Most previous studies have employed short-read sequencing and therefore resulted in fragmented genomes, hampering accurate assessment of genetic diversity. We sought to bridge this knowledge gap and overcome previous technical limitations. We subjected spatiotemporal (2 depths × 12 months) samples from Lake Biwa to metagenome-assembled genome reconstruction enhanced by long-read metagenomics. This yielded 293 giant virus metagenome-assembled genomes. Of these, 285 included previously unknown species in five orders of nucleocytoviruses and the first representatives of freshwater mirusviruses, which exhibited marked divergence from marine-derived lineages. The good performance of our long-read metagenomic assembly was demonstrated by the detection of 42 (14.3%) genomes composed of single contigs with completeness values >90%. Giant viruses were partitioned across water depths, with most species specific to either the sunlit epilimnion or the dark hypolimnion. Epilimnion-specific members tended to be transient and exhibit short and intense abundance peaks, in line with the fact that they regulate the surface algal blooms. During the spring bloom, mirusviruses and members of three nucleocytovirus families were among the most abundant viruses. In contrast, hypolimnion-specific ones including a mirusvirus genome were typically more persistent in the hypolimnion throughout the water-stratified period, suggesting that they infect hosts specific to the hypolimnion and play previously unexplored ecological roles in dark water microbial ecosystems.