TarBase-v9.0 extends experimentally supported miRNA-gene interactions to cell-types and virally encoded miRNAs.

TarBase is a reference database dedicated to produce, curate and deliver high quality experimentally-supported microRNA (miRNA) targets on protein-coding transcripts. In its latest version (v9.0, https://dianalab.e-ce.uth.gr/tarbasev9), it pushes the envelope by introducing virally-encoded miRNAs, interactions leading to target-directed miRNA degradation (TDMD) events and the largest collection of miRNA-gene interactions to date in a plethora of experimental settings, tissues and cell-types. It catalogues ∼6 million entries, comprising ∼2 million unique miRNA-gene pairs, supported by 37 experimental (high- and low-yield) protocols in 172 tissues and cell-types. Interactions are annotated with rich metadata including information on genes/transcripts, miRNAs, samples, experimental contexts and publications, while millions of miRNA-binding locations are also provided at cell-type resolution. A completely re-designed interface with state-of-the-art web technologies, incorporates more features, and allows flexible and ingenious use. The new interface provides the capability to design sophisticated queries with numerous filtering criteria including cell lines, experimental conditions, cell types, experimental methods, species and/or tissues of interest. Additionally, a plethora of fine-tuning capacities have been integrated to the platform, offering the refinement of the returned interactions based on miRNA confidence and expression levels, while boundless local retrieval of the offered interactions and metadata is enabled.

The most exposed regions of SARS-CoV-2 structural proteins are subject to strong positive selection and gene overlap may locally modify this behavior.

The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic that emerged in 2019 has been an unprecedented event in international science, as it has been possible to sequence millions of genomes, tracking their evolution very closely. This has enabled various types of secondary analyses of these genomes, including the measurement of their sequence selection pressure. In this work, we have been able to measure the selective pressure of all the described SARS-CoV-2 genes, even analyzed by sequence regions, and we show how this type of analysis allows us to separate the genes between those subject to positive selection (usually those that code for surface proteins or those exposed to the host immune system) and those subject to negative selection because they require greater conservation of their structure and function. We have also seen that when another gene with an overlapping reading frame appears within a gene sequence, the overlapping sequence between the two genes evolves under a stronger purifying selection than the average of the non-overlapping regions of the main gene. We propose this type of analysis as a useful tool for locating and analyzing all the genes of a viral genome when an adequate number of sequences are available.IMPORTANCEWe have analyzed the selection pressure of all severe acute respiratory syndrome coronavirus 2 genes by means of the nonsynonymous (Ka) to synonymous (Ks) substitution rate. We found that protein-coding genes are exposed to strong positive selection, especially in the regions of interaction with other molecules (host receptor and genome of the virus itself). However, overlapping coding regions are more protected and show negative selection. This suggests that this measure could be used to study viral gene function as well as overlapping genes.

Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

Development of a new effective African swine fever virus vaccine candidate by deletion of the H240R and MGF505-7R genes results in protective immunity against the Eurasia strain.

IMPORTANCE: African swine fever (ASF) caused by ASF virus (ASFV) is a highly contagious and acute hemorrhagic viral disease in domestic pigs. Until now, no effective commercial vaccine and antiviral drugs are available for ASF control. Here, we generated a new live-attenuated vaccine candidate (ASFV-ΔH240R-Δ7R) by deleting H240R and MGF505-7R genes from the highly pathogenic ASFV HLJ/18 genome. Piglets immunized with ASFV-ΔH240R-Δ7R were safe without any ASF-related signs and produced specific antibodies against p30. Challenged with a virulent ASFV HLJ/18, the piglets immunized with high-dose group (105 HAD50) exhibited 100% protection without clinical symptoms, showing that low levels of virus replication with no observed pathogenicity by postmortem and histological analysis. Overall, our results provided a new strategy by designing live-attenuated vaccine candidate, resulting in protection against ASFV infection.

Deletion of DP148R, DP71L, and DP96R Attenuates African Swine Fever Virus, and the Mutant Strain Confers Complete Protection against Homologous Challenges in Pigs.

The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.

A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains.

While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel, enabling the pooled screening of entire viral genomes. Here, we applied this approach to Kaposi's sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

Genomic Landscapes of Epstein-Barr Virus in Pulmonary Lymphoepithelioma-Like Carcinoma.

Epstein-Barr virus (EBV) infection is associated with multiple malignancies, including pulmonary lymphoepithelioma-like carcinoma (pLELC), a particular subtype of primary lung cancer. However, the genomic characteristics of EBV related to pLELC remain unclear. Here, we obtained the whole-genome data set of EBV isolated from 78 pLELC patients and 37 healthy controls using EBV-captured sequencing. Compared with the reference genome (NC_007605), a total of 3,995 variations were detected across pLELC-derived EBV sequences, with the mutational hot spots located in latent genes. Combined with 180 published EBV sequences derived from healthy people in Southern China, we performed a genome-wide association study and identified 32 variations significantly related to pLELC (P < 2.56 × 10-05, Bonferroni correction), with the top signal of single nucleotide polymorphism (SNP) coordinate T7327C (OR = 1.22, P = 2.39 × 10-15) locating in the origin of plasmid replication (OriP). The results of population structure analysis of EBV isolates in East Asian showed the EBV strains derived from pLELC were more similar to those from nasopharyngeal carcinoma (NPC) than other EBV-associated diseases. In addition, typical latency type-II infection were recognized for EBV of pLELC at both transcription and methylation levels. Taken together, we defined the global view of EBV genomic profiles in pLELC patients for the first time, providing new insights to deepening our understanding of this rare EBV-associated primary lung carcinoma. IMPORTANCE Pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare, distinctive subtype of primary lung cancer closely associated with Epstein-Barr virus (EBV) infection. Here, we gave the first overview of pLELC-derived EBV at the level of genome, methylation and transcription. We obtained the EBV sequences data set from 78 primary pLELC patients, and revealed the sequences diversity across EBV genome and detected variability in known immune epitopes. Genome-wide association analysis combining 217 healthy controls identifies significant variations related to the risk of pLELC. Meanwhile, we characterized the integration landscapes of EBV at the genome-wide level. These results provided new insight for understanding EBV's role in pLELC tumorigenesis.

Molecular evolution and genomic characteristics of genotype H1 of measles virus.

Measles is one of the most infectious diseases of humans. It is caused by the measles virus (MeV) and can lead to serious illness, lifelong complications, and even death. Whole-genome sequencing (WGS) is now available to study molecular epidemiology and identify MeV transmission pathways. In the present study, WGS of 23 MeV strains of genotype H1, collected in Mainland China between 2006 and 2018, were generated and compared to 31 WGSs from the public domain to analyze genomic characteristics, evolutionary rates and date of emergence of H1 genotype. The noncoding region between M and F protein genes (M/F NCR) was the most variable region throughout the genome. Although the nucleotide substitution rate of H1 WGS was around 0.75 × 10-3 substitution per site per year, the M/F NCR had an evolutionary rate three times higher, with 2.44 × 10-3 substitution per site per year. Phylogenetic analysis identified three distinct genetic groups. The Time of the Most Recent Common Ancestor (TMRCA) of H1 genotype was estimated at approximately 1988, while the first genetic group appeared around 1995 followed by two other genetic groups in 1999-2002. Bayesian skyline plot showed that the genetic diversity of the H1 genotype remained stable even though the number of MeV cases decreased 50 times between 2014 (52 628) and 2020 (993). The current coronavirus disease 2019 (COVID-19) pandemic might have some effect on the measles epidemic and further studies will be necessary to assess the genetic diversity of the H1 genotype in a post-COVID area.

Recent Issues in Varicella-Zoster Virus Latency.

Varicella-zoster virus (VZV) is a human herpes virus which causes varicella (chicken pox) as a primary infection, and, following a variable period of latency in neurons in the peripheral ganglia, may reactivate to cause herpes zoster (shingles) as well as a variety of neurological syndromes. In this overview we consider some recent issues in alphaherpesvirus latency with special focus on VZV ganglionic latency. A key question is the nature and extent of viral gene transcription during viral latency. While it is known that this is highly restricted, it is only recently that the very high degree of that restriction has been clarified, with both VZV gene 63-encoded transcripts and discovery of a novel VZV transcript (VLT) that maps antisense to the viral transactivator gene 61. It has also emerged in recent years that there is significant epigenetic regulation of VZV gene transcription, and the mechanisms underlying this are complex and being unraveled. The last few years has also seen an increased interest in the immunological aspects of VZV latency and reactivation, in particular from the perspective of inborn errors of host immunity that predispose to different VZV reactivation syndromes.

Triple reassortment increases compatibility among viral ribonucleoprotein genes of contemporary avian and human influenza A viruses.

Compatibility among the influenza A virus (IAV) ribonucleoprotein (RNP) genes affects viral replication efficiency and can limit the emergence of novel reassortants, including those with potential pandemic risks. In this study, we determined the polymerase activities of 2,451 RNP reassortants among three seasonal and eight enzootic IAVs by using a minigenome assay. Results showed that the 2009 H1N1 RNP are more compatible with the tested enzootic RNP than seasonal H3N2 RNP and that triple reassortment increased such compatibility. The RNP reassortants among 2009 H1N1, canine H3N8, and avian H4N6 IAVs had the highest polymerase activities. Residues in the RNA binding motifs and the contact regions among RNP proteins affected polymerase activities. Our data indicates that compatibility among seasonal and enzootic RNPs are selective, and enzoosis of multiple strains in the animal-human interface can facilitate emergence of an RNP with increased replication efficiency in mammals, including humans.

Diagnostic evaluation of qRT-PCR-based kit and dPCR-based kit for COVID-19.

BACKGROUND: Coronavirus disease of 2019 (COVID-19) is well known as a fatal disease, first discovered at Wuhan in China, ranging from mild to death, such as shortness of breath and fever. Early diagnosis of COVID-19 is a crucial point in preventing global prevalence. OBJECTIVE: We aimed to evaluate the diagnostic competency and efficiency with the Allplex™ 2019-nCoV Assay kit and the Dr. PCR 20 K COVID-19 Detection kit, designed based on the qRT-PCR and dPCR technologies, respectively. METHODS: A total of 30 negative and 20 COVID-19 positive specimens were assigned to the diagnostic test by using different COVID-19 diagnosis kits. Diagnostic accuracy was measured by statistical testing with sensitivity, specificity, and co-efficiency calculations. RESULTS: Comparing both diagnostic kits, we confirmed that the diagnostic results of 30 negative and 20 positive cases were the same pre-diagnostic results. The diagnostic statistics test results were perfectly matched with value (1). Cohen's Kappa coefficient was demonstrated that the given kits in two different ways were "almost perfect" with value (1). In evaluating the detection capability, the dilutional linearity experiments substantiate that the Dr. PCR 20 K COVID-19 Detection kit could detect SARS-CoV-2 viral load at a concentration ten times lower than that of the Allplex™ 2019-nCoV Assay kit. CONCLUSIONS: In this study, we propose that the dPCR diagnosis using LOAA dPCR could be a powerful method for COVID-19 point-of-care tests requiring immediate diagnosis in a limited time and space through the advantages of relatively low sample concentration and small equipment size compared to conventional qRT-PCR.

Prokaryotic viruses impact functional microorganisms in nutrient removal and carbon cycle in wastewater treatment plants.

As one of the largest biotechnological applications, activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses, with 10-1,000-fold higher concentrations than in natural environments. However, the compositional variation and host-connections of AS viruses remain poorly explored. Here, we report a catalogue of ~50,000 prokaryotic viruses from six WWTPs, increasing the number of described viral species of AS by 23-fold, and showing the very high viral diversity which is largely unknown (98.4-99.6% of total viral contigs). Most viral genera are represented in more than one AS system with 53 identified across all. Viral infection widely spans 8 archaeal and 58 bacterial phyla, linking viruses with aerobic/anaerobic heterotrophs, and other functional microorganisms controlling nitrogen/phosphorous removal. Notably, Mycobacterium, notorious for causing AS foaming, is associated with 402 viral genera. Our findings expand the current AS virus catalogue and provide reference for the phage treatment to control undesired microorganisms in WWTPs.

Motif mutations in pseudoknot stem I upstream of start codon in Senecavirus A genome: Impacts on activity of viral IRES and on rescue of recombinant virus.

Senecavirus A (SVA), formerly known as Seneca Valley virus, is classified into the genus Senecavirus in the family Picornaviridae. Mature virion harbors an approximately 7 300-nt-long, positive-sense, and single-stranded RNA genome, which contains 5' and 3' untranslated regions (UTRs). Internal ribosome entry site (IRES) is identified in the SVA 5' UTR, and includes a RNA pseudoknot upstream of the start codon. This pseudoknot contains two stem structures, pseudoknot stem I and II (PKS-I and -II). The PKS-I is composed of two base-paired motifs (PKS-Ia and -Ib), between which there is an unpaired spacing (UpS). We reported previously that motif mutation in the PKS-II did not abolish the IRES activity, but interfered with SVA recovery from cDNA clone. In this study, we constructed five SVA minigenomes with point mutations in the PKS-I motif. Dual-luciferase reporter assay showed that motif mutations in PKS-I did not significantly interfere with the IRES activity to initiate protein expression. Correspondingly, we constructed five SVA cDNA clones with point mutations in the PKS-I motif. These genetically modified cDNA clones were separately transfected into BSR-T7/5 cells in attempting to rescue competent SVAs. However, only two viruses, namely PKS-Ia- and UpS-mutated recombinants, could be recovered from their individual cDNA clones. It can be concluded that the PKS-Ib is indispensable for viral growth.

African Swine Fever Virus Bearing an I226R Gene Deletion Elicits Robust Immunity in Pigs to African Swine Fever.

African swine fever (ASF) is a severe hemorrhagic infectious disease in pigs caused by African swine fever virus (ASFV), leading to devastating economic losses in epidemic regions. Its control currently depends on thorough culling and clearance of the diseased and surrounding suspected pigs. An ASF vaccine has been extensively explored for years worldwide, especially in hog-intensive areas where it is highly desired, but it is still unavailable for numerous reasons. Here, we report another ASF vaccine candidate, named SY18ΔI226R, bearing a deletion of the I226R gene with a replacement of an enhanced green fluorescent protein (eGFP) expression cassette at the right end of the viral genome. This deletion results in the complete loss of virulence of SY18 as the gene-deleted strain does not cause any clinical symptoms in all pigs inoculated with a dosage of either 104.0 or 107.0 50% tissue culture infective doses (TCID50). Apparent viremia with a gradual decline was monitored, while virus shedding was detected only occasionally in oral or anal swabs. ASFV-specific antibody appeared at 9 days postinoculation. After intramuscular challenge with its parental strain ASFV SY18 at 21 days postinoculation, all the challenged pigs survived, without obvious febrile or abnormal clinical signs. No viral DNA could be detected upon the dissection of any tissue when viremia disappeared. These results indicated that SY18ΔI226R is safe in swine and elicits robust immunity to virulent ASFV infection. IMPORTANCE Outbreaks of African swine fever have resulted in devastating losses to the swine industry worldwide, but there is currently no commercial vaccine available. Although several vaccine candidates have been reported, none has been approved for use for several reasons, especially ones concerning biosafety. Here, we identified a new undescribed functional gene, I226R. When deleted from the ASFV genome, the virus completely loses its virulence in swine. Importantly, pigs infected with this gene-deleted virus were resistant to infection by intramuscular challenge with 102.5 or 104.0 TCID50 of its virulent parental virus. Furthermore, the nucleic acid of the gene-deleted virus and its virulent parental virus was rarely detected from oral or anal swabs. Viruses could not be detected in any tissues after necropsy when viremia became negative, indicating that robust immunity was achieved. Therefore, SY18ΔI226R is a novel, ideal, and efficacious vaccine candidate for genotype II ASF.

Development and in vivo evaluation of MGF100-1R deletion mutant in an African swine fever virus Chinese strain.

African swine fever virus (ASFV) is a large nucleoplasmic DNA virus, in which the genome is around 170-198 kilobases (kb). More than 50 % genes have unknown functions. Here, MGF100-1R gene is chosen to study the primary function and sublocalization. The gene was located at the left variable region of the ASFV genome that belongs to MGF100 families. It located at the cytoplasm without cytotoxic activities. However, it related to induce the transcriptional levels of pro-inflammatory cytokines. A deletion mutant of MGF100-1R gene was constructed based on ASFV Chinese strain GZ201801. The recombinant deletion mutant (ASFV△MGF100-1R) was demonstrated in vitro that the gene is non-essential for virus replication with a similar replication kinetics in bone marrow-derived macrophages (BMDMs) cell cultures when compared to parental virus. In vivo evaluation, ASFV△MGF100-1R was inoculated intramuscularly and led to a similar pathogenesis that caused by the parental ASFV GZ201801, confirming that deletion of MGF100-1R gene from the ASFV genome does not impact virulence.

Phylogenetic and codon usage analysis for replicase and capsid genes of porcine circovirus 3.

Porcine circovirus type 3 (PCV3) is a highly contagious virus belonging to the family Circoviridae that causes the severe dermatitis and nephropathy syndrome. To date, PCV3 has a worldwide distribution and bring huge economic losses to swine industry. Replicase (Rep) and capsid (Cap) are two major coded proteins of PCV3. Considering the large number of new PCV3 isolates were reported in the past few years and the research for the codon usage pattern of Rep and Cap genes was still a gap, phylogenetic and codon usage analysis of these two genes was performed. Phylogenetic analyses showed that Rep genes in PCV3a were dispersed with no clear clusters while corresponding sequences in PCV3b clustered into two groups and Cap genes clustered into distinct clades according to different genotypes. Relative synonymous codon usage (RSCU) analysis revealed that the codon usage bias existed and effective number of codon (ENC) analysis showed that the bias was slight low. ENC-GC3s plot indicated that mutational pressure and other factors both played a role in PCV3 codon usage and neutrality plot analysis showed that natural selection was the main force influencing the codon usage pattern. The results presented here provided the important basic data on codon usage pattern of Rep and Cap genes, and a better understanding of the evolution and potential origin of PCV3.

The Baltimore Classification of Viruses 50 Years Later: How Does It Stand in the Light of Virus Evolution?

Fifty years ago, David Baltimore published a brief conceptual paper delineating the classification of viruses by the routes of genome expression. The six "Baltimore classes" of viruses, with a subsequently added 7th class, became the conceptual framework for the development of virology during the next five decades. During this time, it became clear that the Baltimore classes, with relatively minor additions, indeed cover the diversity of virus genome expression schemes that also define the replication cycles. Here, we examine the status of the Baltimore classes 50 years after their advent and explore their links with the global ecology and biology of the respective viruses. We discuss an extension of the Baltimore scheme and why many logically admissible expression-replication schemes do not appear to be realized in nature. Recent phylogenomic analyses allow tracing the complex connections between the Baltimore classes and the monophyletic realms of viruses. The five classes of RNA viruses and reverse-transcribing viruses share an origin, whereas both the single-stranded DNA viruses and double-stranded DNA (dsDNA) viruses evolved on multiple independent occasions. Most of the Baltimore classes of viruses probably emerged during the earliest era of life evolution, at the stage of the primordial pool of diverse replicators, and before the advent of modern-like cells with large dsDNA genomes. The Baltimore classes remain an integral part of the conceptual foundation of biology, providing the essential structure for the logical space of information transfer processes, which is nontrivially connected with the routes of evolution of viruses and other replicators.

CRISPR/Cas12a-mediated gold nanoparticle aggregation for colorimetric detection of SARS-CoV-2.

The trans-cleavage activity of the target-activated CRISPR/Cas12a liberated an RNA crosslinker from a molecular transducer, which facilitated the assembly of gold nanoparticles. Integration of the molecular transducer with isothermal amplification and CRISPR/Cas12a resulted in visual detection of the N gene and E gene of SARS-CoV-2 in 45 min.

Ecology of inorganic sulfur auxiliary metabolism in widespread bacteriophages.

Microbial sulfur metabolism contributes to biogeochemical cycling on global scales. Sulfur metabolizing microbes are infected by phages that can encode auxiliary metabolic genes (AMGs) to alter sulfur metabolism within host cells but remain poorly characterized. Here we identified 191 phages derived from twelve environments that encoded 227 AMGs for oxidation of sulfur and thiosulfate (dsrA, dsrC/tusE, soxC, soxD and soxYZ). Evidence for retention of AMGs during niche-differentiation of diverse phage populations provided evidence that auxiliary metabolism imparts measurable fitness benefits to phages with ramifications for ecosystem biogeochemistry. Gene abundance and expression profiles of AMGs suggested significant contributions by phages to sulfur and thiosulfate oxidation in freshwater lakes and oceans, and a sensitive response to changing sulfur concentrations in hydrothermal environments. Overall, our study provides fundamental insights on the distribution, diversity, and ecology of phage auxiliary metabolism associated with sulfur and reinforces the necessity of incorporating viral contributions into biogeochemical configurations.