Virus Research

Since its emergence in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been posing a serious threat to public health worldwide as the causative agent of coronavirus disease 2019 (COVID-19). Now distributed in a pandemic pattern, this disease still lacks an effective drug treatment with low toxicity, leading pharmaceutical companies and research labs to work against time to find a candidate molecule to efficiently treat the affected patients. Due to the well-known broad-spectrum antimicrobial activity of the lactoferrin protein, we sought to verify whether its bovine form (bLf) would also be effective in vitro against SARS-CoV-2. Using an antiviral assay based on quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found that bLf reduced progeny virus yield by up to [~]84,6% in African green monkey kidney epithelial cells (Vero E6) and [~]68,6% in adenocarcinomic human alveolar basal epithelial cells (A549) at 1 mg/mL, a concentration previously shown to have low cytotoxicity. Therefore, our preliminary data suggest that bLf has the potential to constitute a biochemical approach to fight the new coronavirus pandemic.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication. Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection - The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

Understanding the pathogenesis of SARS-CoV-2 is important for developing effective treatment strategies. Viruses hijack the host metabolism to redirect the resources for their replication and survival. How SARS-CoV-2 influences the host metabolism is still unclear. In this study, we analyzed transcriptomic data obtained from different human respiratory cell lines and patient samples (Swab, PBMC, lung biopsy, BALF) to understand the metabolic alterations in response to SARS-CoV-2 infection. For this purpose, the expression pattern of metabolic genes in the human genome-scale metabolic network model Recon3D was explored. We identified metabolic genes and pathways and reporter metabolites under each SARS-CoV-2-infected condition and compared them to identify common and unique changes in the metabolism. Our analysis revealed host-dependent dysregulation of glycolysis, mitochondrial metabolism, amino acid metabolism, glutathione metabolism, polyamine synthesis, and lipid metabolism. We observed different metabolic changes that are pro- and antiviral in nature. We generated hypotheses on how antiviral metabolism can be targeted/enhanced for reducing viral titers. These warrant further exploration with more samples and in vitro studies to test predictions.

BackgroundInfectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that currently represents a large problem for salmonid aquaculture worldwide. Prevention and treatment methods are only partially effective. Genetic selection and genome engineering strategies have potential to develop ISAV resistant salmon stocks. However, this requires a detailed understanding of the genomic regulation of ISAV pathogenesis. Here, we used single cell RNA sequencing on a salmonid cell line to provide a high dimensional insight into the transcriptional landscape that underpin host-virus interactions during ISAV infection at the single cell level. ResultsSalmon head kidney 1 (SHK-1) cells were single-cell RNA sequenced before challenge, and at 24h, 48h, and 96h post-ISAV challenge. The results revealed marked changes in the host transcriptome at 48h and 96h post-infection, even in uninfected cells, potentially suggesting paracrine signalling. This paracrine activation of uninfected cells seemed to be unspecific, involving pathways such as mRNA sensing, ubiquitination or proteasome, and also the up-regulation of the mitochondrial ribosome genes. At 24h post infection, cells showed expression signatures consistent with viral entry, with up-regulation of genes such as PI3K, FAK or JNK. At 48h and 96h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. ConclusionsThis study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection, and revealed potential host-virus interactions at the cellular level. The results highlight the value of single-cell sequencing to characterise cell culture models of viral infection, and the results can be exploited in future functional studies to increase the resistance of Atlantic salmon to ISAV.

The global virome is still largely unknown. In this study we describe the discovery of a new Betaflexivirus species in Ferraria crispa plants. The plant samples were collected in an experiment of 25 Asparagales plants, obtained from an urban botanic garden in the Netherlands, and analyzed by smallRNA-seq as well as RNA-seq. The new Betaflexivirus only occurred in four Ferraria cripsa plants and was tentatively named Ferraria Betaflexivirus 1 (FerBfV-1). This Betaflexivirus showed an RNA genome structure characteristic for the genus Capillovirus, a single-stranded (+) RNA virus. The closest known Capilloviruses in GenBank showed a protein similarity less than 49%, which is well below the proposed demarcation value of species in this genus. Further comparison analysis of the Capillovirus genus revealed the possible presence of two groups.

This study reports the discovery of a new Phenuivirus species, named Lachenalia Phenuivirus-1 (LacPhV-1), from Lachenalia plants in an urban botanic garden in Amsterdam. Using a combination of smallRNA-seq, RNA-seq, and advanced bioinformatics, we identified a segmented negative-strand RNA virus in the Phenuiviridae family. Our findings show significant divergence between this new virus and known Phenuiviruses, such as Tulip Streak Virus (TuSV) and Lactuca Big Vein associated Phlebovirus (LBVaPV), supporting its classification as a distinct species. Notably, the sequence differences found in the conserved 5 and 3 ends of these segments suggest potential co-evolution. Despite the observed genomic distances, there is significant conservation in the RdRp subdomain, underscoring evolutionary relationships among LacPhV-1, TuSV, and LBVaPV. Our findings expand the known global virome and highlight the importance of exploring plant viromes in diverse ecological settings to better understand virus evolution and diversity.

Cannabis sativa has a variety of industrial interest products, such as phytocannabinoids, terpenoids, phenylpropanoids, acyl sugars, and fibers. Several described pathogens, including viral species, will impact the current green cannabis revolution. The recent sequencing of its genome and transcriptome, allowing the optimization and understanding of the production of the metabolite, are a relevant tool for viruses presence analyzing. Using the cannabis transcriptome and Data mining analysis, we describe the first amalgavirus infecting Cannabis, the Cannabis sativa amalgavirus 1 (CSA1). The plant amalgaviruses has nine species infecting relevant crops. Like the other genus members, this cannabis virus has approximately 3.5 kb with two partially overlapping putative open reading frames with the characteristic +1 programmed ribosomal frameshifting. Mainly detected in the male plant, the CSA1 mapped reads were present in the flower and leaf tissues. Nevertheless, the possible impacts of viral replication on host metabolism and the production of secondary compounds are unknown.

Seaweeds are increasingly recognized as sustainable food sources; however, their large-scale cultivation faces challenges similar to land crops, including susceptibility to pathogens. Plant viruses pose a significant threat to global food security, yet little is known about the diversity of viruses in seaweeds. This study investigates virus-associated small interfering RNA (siRNA) responses in commercially relevant seaweed species to understand RNA virus diversity, particularly in edible varieties. Through small RNA sequencing of 16 samples from Saccharina latissima and Alaria esculenta, we identified three new RNA viruses Aev-NL1, Slv-NL2 and Slv-NL3, and one new DNA virus (phaeovirus). The partial genome of the new DNA virus was discovered in the A. esculenta samples and shared 67% DNA sequence identity with the major coat protein of the large double-stranded DNA phaeovirus Feldmannia irregularis virus a. In four out of five A. esculenta samples, a new bisegmented ormycovirus-like RNA virus (Aev-NL1) was identified. A similar new virus, Slv-NL1, was found in previously published S. latissima RNA-seq data, sharing 87% sequence identity with Aev-NL1. Lastly, two novel RNA viruses, Slv-NL2 and Slv-NL3, were discovered in all eight S. latissima samples sharing limited similarity at the genome level but high sequence identity at protein level of both ORFs (>94%). Further investigation of the novel viruses presence across our limited set of samples revealed no conclusive associations with diseased seaweed phenotypes. The discovery of four new viruses in only a limited set of samples highlights the presence of previously unrecognized viral diversity in seaweed, thereby underscoring the importance of understanding viral diversity in seaweed as its virome is currently understudied.

Human rhinovirus (RV) is the most frequent cause of the common cold, as well as severe exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Currently, there are no effective and accurate diagnostic tools or antiviral therapies. MicroRNAs (miRNAs) are small, non-coding sections of RNA involved in the regulation of gene expression and have been shown to be associated with different pathologies. However, the precise role of miRNAs in RV infection is not yet well established. This study aimed to analyse the impact of RV16 on miRNA expression across the viral life-cycle to identify a small panel with altered expression. We then aimed to specifically interrogate these results using our in-house developed modelling programme to identify specific genes regulated by these miRNAs during RV infection which can then be tested functionally. Our results first identified that three miRNAs, miR-155-5p, miR-140-3p, and miR-122-5p were potential biomarkers being differentially regulated at specific time points post infection. Our modelling program then linked these miRNAs to four genes (OLFML3, STAG2, SMARCA2, CD40LG) that play important roles in regulating the hosts antiviral responses and viral progression. Together, this work identifies a potential panel of biomarkers that could, along with our previous work, form clear diagnostic markers for RV16 infection and identifies specific targets that can be functionally interrogated helping to identify new cellular targets modified by RV16 infection that could inform future therapeutic design.

Monkeypox is a viral zoonosis with symptoms that are reminiscent to those experienced in previous smallpox cases. GSAID databases (Global Initiative on Sharing Avian Influenza Data) was used to assess 630 genomes of MPXV. Six primary clades were inferred from the phylogenetic study, coupled with a lesser percentage in radiating clades. Individual clades that make up various nationalities may have formed as a result of a particular SNP hotspot type, which may have mutated in a particular population type. The most significant mutation, based on a mutational hotspot analysis, was found at G3729A and G5143A. The gene ORF138, which encodes the Ankyrin repeat (ANK) protein, was found to have the most mutations. This protein is known to mediate molecular recognition via protein-protein interactions. It was shown that 243 host proteins interacted with 10 monkeypox proteins identified as the hub proteins E3, SPI2, C5, K7, E8, G6, N2, B14, CRMB, and A41 through 262 direct connections. The interaction with chemokine system-related proteins provides further evidence that the human protein is being suppressed by the monkey pox virus in order to facilitate its survival against innate immunity. A few FDA-approved molecules were likely used as possible inhibitors after being researched for blocking F13, a significant envelope protein on the membrane of extracellular versions of virus. A total of 2500 putative ligands were docked individually with the F13 protein. The F13 protein and these molecules interaction may help prevent the monkey pox virus from spreading. As a result, after being confirmed by experiments, these putative inhibitors might have an impact on the activity of these proteins and be utilised in monkeypox treatments.

Long-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. This technology is also used for the identification and annotation of genes of various organisms, including viruses. Bovine herpesvirus type 1 (BoHV-1) is an important pathogen of cattle worldwide. However, the transcriptome of this virus is still largely unannotated. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technology (ONT) MinION, and the Illumina LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Very long polycistronic and complex viral transcripts were also detected. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps formed by transcriptional read-throughs or overlapping the 5-untranslated regions of divergently-oriented transcripts. The impact of the viral infection on the host cell transcriptome was also assessed. Our results demonstrate that genes associated with antiviral response as well as viral transcription and translation are upregulated.

Although viruses play an important role in human health and plant health, most viruses are still undetected. One of the reasons is that they may evolve in distinct natural habitats that are not yet extensively sampled for virus analysis. An example of such a habitat is urban botanic gardens. We analyzed 25 Asparagales plants with a mild to severe disease phenotype from a Dutch urban botanic garden for the presence of known an unknown virus types and virus variants by smallRNA-seq and RNA-seq. We found in all samples evidence for (past) Potyviridae presence, mostly Ornithogalum virus (OV) and Ornithogalum mosaic virus (OrMV), as well as a new Iris mild mosaic virus variant (IMMV) and a yet unknown species of Potyvirus. Also, presence of a new Betaflexivirus, a new Polerovirus and a new Phenuivirus were detected. Most analyzed plants harbored multiple viruses, 18 out of 25 plants showed evidence for three to seven viruses and 12 out of 13 viruses were present in four to 11 samples. In this study, we describe the characteristics of a newly discovered Potyvirus and identify several variants of known potyviruses. We place these findings in the context of known viruses. However, we were unable to link these potyviruses to any specific disease phenotype.

COVID-19 disease has had a relatively less severe impact in Africa. To understand the role of SARS CoV2 mutations on COVID-19 disease in Africa, we analysed 282 complete nucleotide sequences from African isolates deposited in the NCBI Virus Database. Sequences were aligned against the prototype Wuhan sequence (GenBank accession: NC_045512.2) in BWA v. 0.7.17. SAM and BAM files were created, sorted and indexed in SAMtools v. 1.10 and marked for duplicates using Picard v. 2.23.4. Variants were called with mpileup in BCFtools v. 1.11. Phylograms were created using Mr. Bayes v 3.2.6. A total of 2,349 single nucleotide polymorphism (SNP) profiles across 294 sites were identified. Clades associated with severe disease in the United States, France, Italy, and Brazil had low frequencies in Africa (L84S=2.5%, L3606F=1.4%, L3606F/V378I/=0.35, G251V=2%). Sub Saharan Africa (SSA) accounted for only 3% of P323L and 4% of Q57H mutations in Africa. Comparatively low infections in SSA were attributed to the low frequency of the D614G clade in earlier samples (25% vs 67% global). Higher disease burden occurred in countries with higher D614G frequencies (Egypt=98%, Morocco=90%, Tunisia=52%, South Africa) with D614G as the first confirmed case. V367F, D364Y, V483A and G476S mutations associated with efficient ACE2 receptor binding and severe disease were not observed in Africa. 95% of all RdRp mutations were deaminations leading to CpG depletion and possible attenuation of virulence. More genomic and experimental studies are needed to increase our understanding of the temporal evolution of the virus in Africa, clarify our findings, and reveal hot spots that may undermine successful therapeutic and vaccine interventions.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is a major health burden. Volatile garlic organosulfur compounds, such as the thiol-reactive allicin (diallyl thiosulfinate) exert strong antimicrobial activity against various respiratory pathogens. Here, we investigated the antiviral activity of allicin against SARS-CoV-2 in infected Vero E6 and Calu-3 lung cells. Calu-3 cells showed greater allicin tolerance due >4-fold increased GSH levels compared to Vero E6. However, biocompatible allicin doses efficiently inhibited viral replication in both cell lines. Proteome analyses of SARS-CoV-2 infected Calu-3 cells revealed a strong induction of the antiviral interferon-stimulated gene (ISG) signature (e.g. cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, 25OAS and ISG15), pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTC1, ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin abrogated the ISG host response and reverted the host cellular pathways to levels of uninfected Calu-3 cells, confirming the antiviral and immunomodulatory activity of allicin in the host proteome. Thus, biocompatible doses of allicin could be promising for protection of lung cells against SARS-CoV-2.

The full-length genome of a novel Emaravirus has been identified and characterized from sycamore maple (Acer pseudoplatanus) - a tree species of significant importance in urban and forest areas - showing leaf mottle symptoms. RNA-Seq was performed using RNA preparations from a symptomatic and a symptomless maple tree. Purified double-stranded cDNA from each sample were used for RNA-Seq analysis on the Illumina HiSeq2500system and 14-198 MB data/sample of 100 bp-long paired-end sequence reads were generated. The sequence assembly and analysis revealed the presence of six RNA segments in the symptomatic sample (RNA1: 7,075 nt-long encoding the viral replicase; RNA2: 2,289 nt-long encoding the glycoprotein precursor; RNA3: 1,525 nt-long encoding the nucleocapsid protein; RNA4: 1,533 nt-long encoding the putative movement protein; RNA5: 1,825 nt-long encoding a hypothetical protein P5; RNA6: 1,179 nt-long encoding a hypothetical protein P6). Two independent HTS sequencing runs from the same symptomatic maple tree detected the same genome segments. For one of these sequencing runs the cDNA library was prepared using a primer targeting the conserved genome terminal region, known to be shared between emaraviruses genome segments and a high amount of sequence data was generated. We suggest, therefore, that the six identified genome segments represent the complete genome of a novel emaravirus from maple, which we tentatively name maple mottle-associated virus (MaMaV). RT-PCR assays were performed on symptomatic and non-symptomatic leaves of A. pseudoplatanus trees coming growing on two different locations in Berlin. MaMaV was only detected from symptomatic trees and all six RNAs were generally simultaneously detected. Non-symptomatic samples were consistently negative for MaMaV. These results suggest that MaMaV might be the symptom inducing virus in the sampled trees. In the present state of the art, this is the first time an Emaravirus is described from maple and is fully genetically characterized.

Surveillance of genetic diversity in the SARS-CoV-2 is extremely important to detect the emergence of more infectious and deadly strains of the virus. In this study, we monitored mutational events in the SARS-CoV-2 genome through whole genome sequencing. The samples (n=48) were collected from the hot spot regions of the metropolitan city Karachi, Pakistan during the four months (May 2020 to August 2020) of first wave of the COVID-19 pandemic. The data analysis highlighted 122 mutations, including 120 single nucleotide variations (SNV), and 2 deletions. Among the 122 mutations, there were 71 singletons, and 51 recurrent mutations. A total of 16 mutations, including 5 nonsynonymous mutations, were detected in spike protein. Notably, the spike protein missense mutation D614G was observed in 31 genomes. The phylogenetic analysis revealed majority of the genomes (36) classified as B lineage, where 2 genomes were from B.6 lineage, 5 genomes from B.1 ancestral lineage and remaining from B.1 sub-lineages. It was noteworthy that three clusters of B.1 sub-lineages were observed, including B.1.36 lineage (10 genomes), B.1.160 lineage (11 genomes), and B.1.255 lineage (5 genomes), which represent independent events of SARS-CoV-2 transmission within the city. The sub-lineage B.1.36 had higher representation from the Asian countries and the UK, B.1.160 correspond to the European countries with highest representation from the UK, Denmark, and lesser representation from India, Saudi Arabia, France and Switzerland, and the third sub-lineage (B.1.255) correspond to the USA. Collectively, our study provides meaningful insight into the evolution of SARS-CoV-2 lineages in spatio-temporal local transmission during the first wave of the pandemic.

Bird species are known to be the main reservoir of a range of respiratory viruses such as Influenza, Newcastle and Coronaviruses. Migratory birds are particularly important for the maintenance and long distance spread of the virus to wild bird and poultry species but eventually to mammal species as well. Antarcticas pristine environment and wildlife is of immense biological value, but the spread of such deadly viruses pose a substantial threat to the regions fragile ecosystems. To investigate the presence of respiratory viruses in the region we sampled feces of different wild migratory birds at various localities in the South Shetland Islands in the Antarctic summer of 2023 and screened them for coronaviruses (CoVs) and influenza A virus (IAV). Viral screening was performed by the conventional pancoronavirus RT-PCR protocol (CoVs), by quantitative one-step real-time RT-PCR (IAVs) followed by metatranscriptomic sequencing of positive samples. During January and February of 2023, we collected and examined a total of 243 fecal samples representing Stercorarius spp (N=5), Larus dominicanus (N=16), Phalacrocorax bransfieldensis (N=3), Pygoscelis adeliae (N=19), Pygoscelis antarcticus (N=38), Pygoscelis papua (N=139), Pygoscelis spp (N=23). All tested samples were negative for influenza A and one sample from the colony of L. dominicanus at Keller Peninsula, King George Island, tested positive for CoVs. Metatranscriptomic sequencing recovered a full deltaCoV genome. Nucleotide and amino acid distance analysis revealed that the deltacoronavirus detected belongs to subgenus Buldecovirus and to the novel wild bird deltaCoV clade previously identified infecting Antarctica penguins. The identified deltaCov is most closely related to a deltacoronavirus previously identified 2014 in P. papua penguin sampled at Isla Kopaitik, Base OHiggin suggesting a potential cross species transmission. The presence of CoVs in Antarctic migratory seabirds raises concerns about their impact on the wild bird population in Antarctica and their potential role in virus dispersion through intra and intercontinental migratory routes. These findings contribute valuable insights into virus dynamics among seabird populations, laying the groundwork for future investigations in this field and warning of the importance of viral surveillance on the Antarctic fauna. RepositoriesThe raw reads were submitted to NCBI Sequence Read Archive (SRA) database and are available under project number: PRJNA1160912 and Run Accessions: SRR30664529 and SRR30664530. Impact statementThis study provides insights into the presence of respiratory viruses, specifically coronaviruses (CoVs), in Antarctic seabirds. By detecting a novel wild bird deltacoronavirus in gulls population in the South Shetland Islands, our research contributes to the growing body of literature on viral transmission in remote ecosystems. The findings highlight the potential of migratory birds to act as reservoirs and vectors of viruses in the Antarctic region and underscores the urgent need for continued viral surveillance.

Even after two decades since the identification of the first giant virus, the Acanthamoeba polyphaga mimivirus (APMV), it still elude scientists. Their gigantic size and genome are unique in the whole virosphere, and many aspects of their biology are still unknown, including their possible hosts. They are cultivated in laboratories using Acanthamoeba cells as hosts, but little is known about the infectivity of these giant viruses in vertebrate cells. However, there is evidence of the possible involvement of APMV in pneumonia and activation of inflammatory pathways. Among the hundreds of prospected giant viruses members is Tupanvirus, isolated in Brazil. Its particles have a characteristically large size varying between 1.2 to 2 m and are covered by fibrils. In the present work, we aim to study the consequences of the incubation of APMV and Tupanvirus with mammalian cells. These cells express Toll-like receptors (TLR) that are capable of recognizing lipopolysaccharides, favoring the internalization of the antigen and activation of the inflammatory system. We used a lineage of human lung adenocarcinoma cells (A549) to evaluate possible effects of TLR activation by the giant viruses and if we could detect the probable cause of the said giant-virus dependent pneumonia. Our results show that APMV and Tupanvirus (TPV) activate cellular receptors related to the Toll-like 4 type-induced inflammatory response and that the A549 cells are capable of internalizing the latter virus. Therefore, this study brings new insights into the possible interactions established between mimiviruses (here represented by APMV and Tupanvirus) and members of the innate cellular immune response.

The mouse mammary tumor virus (MMTV) induces mammary tumors in mice. Recently, we have shown that MMTV perturbs expression of the host miR-17-92 cluster in MMTV-infected mammary glands and MMTV-induced mammary tumors. Known as oncomiR-1, this cluster is often dysregulated in a number of cancers, especially breast cancer. Therefore, we investigated whether there was a functional interaction between MMTV and the cluster. Our results reveal that MMTV expression led to dysregulation of the cluster in both mammary epithelial HC11 and HEK293T cells with the expression of miR-92a cluster member being affected the most. Conversely, over-expression of the whole or partial cluster significantly repressed MMTV expression. Interestingly, overexpression of miR-92a alone resulted in MMTV repression to the same extent as observed with the whole or partial cluster. Inhibition of miR-92a led to nearly a complete recovery of MMTV gene expression. Deletion/substitution of the miR-92a seed sequence within the cluster rescued MMTV expression. Dual luciferase assays identified MMTV genomic RNA as the target of miR-92a. These results show that the miR-17-92 cluster acts as part of the cells well-known miRNA-based anti-viral response to thwart incoming MMTV infection. Interestingly, miR-92a expression was the least amongst cluster members in MMTV-induced breast tumors, suggesting that the elevated level of MMTV expression in tumors is due perhaps to MMTV subverting miR-92a overexpression, restricting its ability to control MMTV replication during tumorigenesis. Thus, our study provides the first evidence demonstrating the biological significance of host miRNAs in controlling MMTV replication and influencing the process of tumorigenesis. ImportanceMMTV is a non-acute, slow-transforming retrovirus that causes breast cancer and lymphomas in mice. Consequently, understanding how MMTV interacts with its host can reveal critical aspects of cellular anti-viral responses and the process of tumorigenesis. This study reveals that the host miR-17-92 cluster plays an anti-viral role during MMTV infection which seems to be counteracted by MMTV during tumorigenesis. This observation is significant since MMTV-induced tumors share similarity with human breast cancer and the MMTV/mouse model is the best mammalian animal model to study breast cancer initiation, progression, and therapy. Moreover, MMTV is increasingly (though controversial) being implicated in human breast cancer, leukemias/lymphomas, and biliary cirrhosis due to its potential zoonosis into humans. Thus, if proven true, understanding how miRNAs modulate MMTV replication will be critical in appreciating how hosts miRNAs affect virus replication/tumorigenesis, and towards the development of novel miRNA-based therapeutics (e.g., anti-miRs) to thwart MMTV replication in humans.

After eight months of the pandemic declaration, COVID-19 has not been globally controlled. Several efforts to control SARS-CoV-2 dissemination are still running including vaccines and drug treatments. The effectiveness of these procedures depends, in part, that the regions to which these treatments are directed do not vary considerably. Although, it is known that the mutation rate of SARS-CoV-2 is relatively low it is necessary to monitor the adaptation and evolution of the virus in the different stages of the pandemic. Thus, identification, analysis of the dynamics, and possible functional and structural implication of mutations are relevant. Here, we first estimate the number of COVID-19 cases with a virus with a specific mutation and then calculate its global relative frequency (NRFp). Using this approach in a dataset of 100 924 genomes from GISAID, we identified 41 mutations to be present in viruses in an estimated number of 750 000 global COVID-19 cases (0.03 NRFp). We classified these mutations into three groups: high-frequent, low-frequent non-synonymous, and low-frequent synonymous. Analysis of the dynamics of these mutations by month and continent showed that high-frequent mutations appeared early in the pandemic, all are present in all continents and some of them are almost fixed in the global population. On the other hand, low-frequent mutations (non-synonymous and synonymous) appear late in the pandemic and seems to be at least partially continent-specific. This could be due to that high-frequent mutation appeared early when lockdown policies had not yet been applied and low-frequent mutations appeared after lockdown policies. Thus, preventing global dissemination of them. Finally, we present a brief structural and functional review of the analyzed ORFs and the possible implications of the 25 identified non-synonymous mutations.