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Influenza polymerase (FluPol) plays a key role in the viral infection cycle by transcribing and replicating the viral genome. FluPol is a multifunctional, heterotrimeric enzyme with cap-binding, endonuclease, RNA-dependent RNA polymerase and polyadenylation activities. It performs its functions in the context of the viral ribonucleoprotein particle (RNP), wherein the template viral RNA is coated by multiple copies of viral nucleoprotein. Moreover, it interacts with a number of host proteins that are essential cofactors and, consequently, adaptive mutations in the polymerase are required for crossing the avian-human species barrier. In this review, we show how mechanistic understanding of how FluPol performs its multiple functions has greatly advanced over the last decade through determination of high-resolution structures by X-ray crystallography and cryo-electron microscopy. These have revealed not only the detailed architecture of FluPol but highlighted the remarkably conformational flexibility that is inherent to its functioning as a dynamic RNA synthesis machine. Structural studies are also underpinning current attempts to develop next-generation anti-influenza drugs that directly target FluPol.

Background: The establishment of strategy to inhibit the virus replication is an attractive means in combating SARS-CoV-2 infection. Objective: We studied phyto-compounds from Strychnos nux-vomica (a poisonous plant) against SARS-CoV-2 RNA-dependent RNA polymerase by computational methods. Method: Molecular docking, molecular dynamics (MD) simulation and energetics calculations were employed to elucidate the role of the phyto-compounds. Results: Ergotamine with a binding free energy of -14.39 kcal/mol showed a promising capability in terms of binding affinity and the interaction to conserved motifs, especially the SDD signature sequence. The calculated dissociation constants for ATP, ergotamine, isosungucine and sungucine were 12 μM, 0.072 nM, 0.011 nM and 0.152 nM, respectively. The exhibited kd by these phyto-compounds reflected tens of thousands fold potency as compared to ATP. The binding free energies of sungucine and isosungucine were much lower (-13.93 and -15.55 kcal/mol, respectively) compared to that of ATP (-6.98 kcal/mol). Conclusion: Sharing the same binding location as that of ATP and having high binding affinities, Ergotamine, Isosungucine, Sungucine and Strychnine N-oxide could be effective in controlling the SARS-CoV-2 virus replication by blocking the ATP and inhibiting the enzyme function.

One victorivirus was detected in the isolate of Corynespora cassiicola strains 20180909-03, which was named Corynespora cassiicola victorivirus 1 (CcVV1). The whole-genome sequence of the virus was sequenced and identified. The CcVV1 genome is 5140 nt and contains 56.87%GC with two large open reading frames (ORFs) overlapping at the tetranucleotide AUGA. The two ORFs were predicted to encode coat protein (CP) and RNA-dependent RNA polymerase (RdRp) respectively, which were conservative in dsRNA fungal viruses of the family Totiviridae. Conservative domains comparison and phylogenetic analysis of the deduced amino acid sequence of RdRp and CP showed that CcVV1 was a new virus of the Victorivirus genus. As far as we know, it is the first report of a genomic sequence of the genus Victorivirus infecting Corynespora cassiicola.

As ZIKV continues to spread, many “unknowns” remain and research is needed to advance the understanding of this important pathogen. Viral RNA dependent-RNA polymerases (RdRp) are validated targets for inhibitors of the replication of several viruses. Several studies have set up in vitro enzymatic assays of the RdRp of the Zika virus for testing of candidate inhibitors. While most of these studies use short synthetic polymers, we have shown in a previous work that the Zika polymerase domain is capable of a de novo synthesis of the viral genome using the natural viral RNA as template. Here we have studied the role of the sequences at the 3′end of the minus-strand RNA in the initiation of the RNA synthesis by the Zika isolated RdRp. Our results strongly suggest that the region containing the 105 first nucleotides from the 3′ end of the minus-strand RNA is important for initiation of the positive RNA synthesis. This indicates that this region displays all the primary and secondary structures to be efficiently recognized by the recombinant RdRp in vitro. Moreover, we show that the 46 nucleotides are sufficient to initiate RNA synthesis. In addition, the ZIKV polymerase domain poorly replicated the RNA of other RNA viruses and appeared highly selective for its own RNA.

Introduction: The outburst of the novel coronavirus COVID-19, at the end of December 2019 has turned itself into a pandemic taking a heavy toll on human lives. The causal agent being SARS-CoV-2, a member of the long-known Coronaviridae family, is a positive sense single-stranded enveloped virus and quite closely related to SARS-CoV. It has become the need of the hour to understand the pathophysiology of this disease, so that drugs, vaccines, treatment regimens and plausible therapeutic agents can be produced. Methods: In this regard, recent studies uncovered the fact that the viral genome of SARS-CoV-2 encodes nonstructural proteins like RNA dependent RNA polymerase (RdRp) which is an important tool for its transcription and replication process. A large number of nucleic acid based anti-viral drugs are being repurposed for treating COVID-19 targeting RdRp. Few of them are in the advanced stage of clinical trials including Remdesivir. While performing close investigation of the large set of nucleic acid based drugs, we were surprised to find that the synthetic nucleic acid backbone is explored very little or rare. Results: We have designed scaffolds derived from peptide nucleic acid (PNA) and subjected them for in-silico screening systematically. These designed molecules have demonstrated excellent binding towards RdRp. Compound 12 was found to possess similar binding affinity as Remdesivir with comparable pharmacokinetics. However, the in-silico toxicity prediction indicates compound 12 may be a superior molecule which can be explored further due to its excellent safety-profile with LD50 (12,000mg/kg) as opposed to Remdesivir (LD50 =1000mg/kg). Conclusion: Compound 12 falls in the safe category of class 6. Synthetic feasibility, equipotent binding and very low toxicity of this peptide nucleic acid derived compounds can serve as a leading scaffold to design, synthesize and evaluate many of similar compounds for the treatment of COVID-19.

Public sequence data represents a major opportunity for viral discovery, but its exploration has been inhibited by a lack of efficient methods for searching this corpus, which is currently at the petabase scale and growing exponentially. To address the ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 and expand the known sequence diversity of viruses, we aligned pangenomes for coronaviruses (CoV) and other viral families to 5.6 petabases of public sequencing data from 3.8 million biologically diverse samples. To implement this strategy, we developed a cloud computing architecture, Serratus, tailored for ultra-high throughput sequence alignment at the petabase scale. From this search, we identified and assembled thousands of CoV and CoV-like genomes and genome fragments ranging from known strains to putatively novel genera. We generalise this strategy to other viral families, identifying several novel deltaviruses and huge bacteriophages. To catalyse a new era of viral discovery we made millions of viral alignments and family identifications freely available to the research community. Expanding the known diversity and zoonotic reservoirs of CoV and other emerging pathogens can accelerate vaccine and therapeutic developments for the current pandemic, and help us anticipate and mitigate future ones.

The RNA dependent RNA polymerase (RdRp) plays crucial role in virus life cycle by replicating the viral genome. The SARS-CoV-2 is an RNA virus that rapidly spread worldwide and acquired mutations. This study was carried out to identify mutations in RdRp as the SARS-CoV-2 spread in India. We compared 50217 RdRp sequences reported from India with the first reported RdRp sequence from Wuhan, China to identify 223 mutations acquired among Indian isolates. Our protein modelling study revealed that several mutants can potentially alter stability and flexibility of RdRp. We predicted the potential B cell epitopes contributed by RdRp and identified thirty-six linear continuous and twenty-five discontinuous epitopes. Among 223 RdRp mutants, 44% of them localises in the B cell epitopes region. Altogether, this study highlights the need to identify and characterize the variations in RdRp to understand the impact of these mutations on SARS-CoV-2.

International audience; Background and Aims: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and known as the viral replicase. The RNA-dependent RNA polymerase (RdRp) domain is responsible for the synthesis of viral RNA.Approach and Results: Here, we identified a highly conserved α-helix located in the RdRp thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RdRp palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release.Conclusions: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RdRp with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RdRp thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production

The emergence of fast-spreading SARS-CoV-2 mutants has sparked a new phase of COVID-19 pandemic. There is a dire necessity for antivirals targeting highly conserved genomic domains on SARS-CoV-2 that are less prone to mutation. The nsp12, also known as the RNA-dependent RNA-polymerase (RdRp), the core component of 'SARS-CoV-2 replication-transcription complex', is a potential well-conserved druggable antiviral target. Several FDA-approved RdRp 'nucleotide analog inhibitors (NAIs)' such as remdesivir, have been repurposed to treat COVID-19 infections. The NAIs target RdRp protein translation and competitively block the nucleotide insertion into the RNA chain, resulting in the inhibition of viral replication. However, the replication proofreading function of nsp14-ExoN could provide resistance to SARS-CoV-2 against many NAIs. Conversely, the 'non-nucleoside analog inhibitors (NNAIs)' bind to allosteric sites on viral polymerase surface, change the redox state; thereby, exert antiviral activity by altering interactions between the enzyme substrate and active core catalytic site of the RdRp. NNAIs neither require metabolic activation (unlike NAIs) nor compete with intracellular pool of nucleotide triphosphates (NTPs) for anti-RdRp activity. The NNAIs from phytonutrient origin are potential antiviral candidates compared to their synthetic counterparts. Several in-silico studies reported the antiviral spectrum of natural phytonutrient-NNAIs such as Suramin, Silibinin (flavonolignan), Theaflavin (tea polyphenol), Baicalein (5,6,7-trihydroxyflavone), Corilagin (gallotannin), Hesperidin (citrus bioflavonoid), Lycorine (pyrrolidine alkaloid), with superior redox characteristics (free binding energy, hydrogen-bonds, etc.) than antiviral drugs (i.e. remdesivir, favipiravir). These phytonutrient-NNAIs also exert anti-inflammatory, antioxidant, immunomodulatory and cardioprotective functions, with multifunctional therapeutic benefits in the clinical management of COVID-19.

The recent outbreak of SARS-CoV-2 across the globe and the absence of a specific cure against the disease lead the scientific community to investigate some alternative indigenous treatments. SARS-CoV-2 is the virus responsible for the coronavirus ailment 2019 (COVID-19). This virus has 4 auxiliary proteins namely the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The main proteases and RNA dependent RNA polymerase are also essential structures by which the virus replicates and survives. Each of these proteins are structures of the virus that are potential targets for drugs which are leads in the drug discovery process of any drug for the virus. Currently available treatments are not specific to the disease and therefore carry unwanted adverse effects that can be highly dangerous and sometimes fatal. Many of these treatments are supplementary in nature or based on repurposed drugs from other viral outbreaks. Alternatives of conventional drugs are required to control the spread and severity of the disease. Allicin, curcumin and their derivatives have been researched for their antiviral property and shown to have good binding affinity towards SARS-CoV-2 structures essential in their survival, especially the main proteases and RNA dependent RNA polymerases. The structural bioinformatics tools have elicited methods to predict the bioactivity of the natural product-based compounds. Apart from the beneficial medication that they offer, natural products carry along other advantages for the current pandemic situation in terms of supply, logistics, and affordability.

TAXONOMY Potato virus X is the type-member of the plant-infecting Potexvirus genus in the family Alphaflexiviridae. PHYSICAL PROPERTIES Potato virus X (PVX) virions are flexuous filaments 460-480 nm in length. Virions are 13 nm in diameter and have a helical pitch of 3.4 nm. The genome is approximately 6.4 kb with a 5' cap and 3' poly(A) terminus. PVX contains five open reading frames, four of which are essential for cell-to-cell and systemic movement. One protein encodes the viral replicase. Cellular inclusions, known as X-bodies, occur near the nucleus of virus-infected cells. HOSTS The primary host is potato, but it infects a wide range of dicots. Diagnostic hosts include Datura stramonium and Nicotiana tabacum. PVX is transmitted in nature by mechanical contact. USEFUL WEBSITE: https://talk.ictvonline.org/ictv-reports/ictv_online_report/positive-sense-rna-viruses/w/alphaflexiviridae/1330/genus-potexvirus.

Informational macromolecules in biology are composed of subunits of a single handedness, d-nucleotides in nucleic acids and l-amino acids in proteins. Although this chiral uniformity may be expedient, it is not a chemical necessity, as demonstrated by the recent example of an RNA enzyme that catalyzes the RNA-templated polymerization of RNA molecules of the opposite handedness. This reaction, when carried out iteratively, can provide the basis for exponential amplification of RNA molecules and the information they contain. By carrying out thermal cycling, analogous to the polymerase chain reaction, and supplying oligonucleotide building blocks that comprise both the functional strand of RNA and its complement, cross-chiral exponential amplification was achieved. This process was used to amplify the l-RNA form of the hammerhead ribozyme, catalyzed by the d-RNA form of the polymerase. The resulting l-hammerhead exhibits the expected activity in cleaving a corresponding l-RNA substrate. Exponential amplification was also carried out within individual droplets of a water-in-oil emulsion. The ability to amplify enantio-RNAs, both in bulk solution and within compartments, provides a means to evolve cross-chiral RNA polymerases based on the function of the RNAs they produce.

To further our understanding of the pro-viral roles of the host cytosolic heat shock protein 70 (Hsp70) family, we chose the conserved Arabidopsis thaliana Hsp70-2 and the unique Erd2 (early response to dehydration 2), which contain Hsp70 domains. Based on in vitro studies with purified components, we show that AtHsp70-2 and AtErd2 perform pro-viral functions equivalent to that of the yeast Ssa1 Hsp70. These functions include activation of the tombusvirus RdRp, and stimulation of replicase assembly. Yeast-based complementation studies demonstrate that AtHsp70-2 or AtErd2 are present in the purified tombusvirus replicase. RNA silencing and over-expression studies in Nicotiana benthamiana suggest that both Hsp70-2 and Erd2 are co-opted by tomato bushy stunt virus (TBSV). Moreover, we used allosteric inhibitors of Hsp70s to inhibit replication of TBSV and related plant viruses in plants. Altogether, interfering with the functions of the co-opted Hsp70s could be an effective antiviral approach against tombusviruses in plants.

Background & AimsAlthough Hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet and it is currently unknown whether HEV infection leads to cellular membrane modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms.MethodsWe generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC).ResultsOne of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC.ConclusionsOur study reveals that HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.Lay summaryHepatitis E virus (HEV) is the leading cause of acute hepatitis worldwide but many steps of its lifecycle are still elusive. Thanks to the development of new antibodies that recognize the different forms of the HEV capsid protein, we were able to visualize vesicular and tubular structures that were established by the virus in the host cell. In addition, extensive efforts to identify these structures led us to conclude that HEV hijacks the endocytic recycling compartment of the cell to form this network of vesicles and tubules, which might be the hallmark of HEV infection.

The entomopathogenic fungus Beauveria bassiana is used worldwide for biological control of insects. Seven dsRNA segments were detected in a single B. bassiana strain, RCEF1446. High-throughput sequencing indicated the presence of three mycoviruses in RCEF1446. Two were identified as the known mycoviruses Beauveria bassiana victorivirus 1 and Beauveria bassiana polymycovirus 1, and the novel mycovirus was designated as "Beauveria bassiana bipartite mycovirus 1" (BbBV1). The complete sequence of the BbBV1 is described here. The mycovirus contains two dsRNA segments. The RNA 1 (dsRNA 4) of BbBV1 is 2,026 bp in length, encoding a RNA-dependent RNA polymerase (RdRp) (68.54 kDa), while the RNA 2 (dsRNA 6) is 1,810 bp in length, encoding a hypothetical protein (35.55 kDa) with unknown function. Moreover, the amino acid sequence of RdRp showed the highest sequence identity of 62.31% to Botryosphaeria dothidea bipartite mycovirus 1. Phylogenetic analysis based on RdRp sequences revealed that BbBV1 represents a distinct lineage of unassigned dsRNA mycoviruses infecting fungi.

RNA-dependent RNA polymerases (RDR) generate double-stranded (ds)RNA triggers for RNA silencing across eukaryotes. Among the three clades, α-clade and β-clade members are key components of RNA silencing and mediators of stress responses across eukaryotes. However, γ-clade members are unusual in that they are represented in phylogenetically distant plants and fungi, and their functions are unknown. Using genetic, bioinformatic and biochemical methods, we show that γ-clade RDRs from Oryza sativa L. are involved in plant development as well as regulation of expression of coding and noncoding RNAs. Overexpression of γ-clade RDRs in transgenic rice and tobacco plants resulted in robust growth phenotype, whereas their silencing in rice displayed strong inhibition of growth. Small (s)RNA and RNA-seq analysis of OsRDR3 mis-expression lines suggested that it is specifically involved in the regulation of repeat-rich regions in the genome. Biochemical analysis confirmed that OsRDR3 has robust polymerase activities on both single stranded (ss)RNA and ssDNA templates similar to the activities reported for α-clade RDRs such as AtRDR6. Our results provide the first evidence of the importance of γ-clade RDRs in plant development, their atypical biochemical activities and their contribution to the regulation of gene expression.

Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19. Accumulation of mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp may facilitate antigenic drift, generating favipiravir resistance. Focussing on the chain-termination mechanism utilized by favipiravir, we used high-throughput interface-based protein design to generate > 100 000 designs of the favipiravir-binding site of RdRp and identify mutational hotspots. We identified several single-point mutants and designs having a sequence identity of 97%-98% with wild-type RdRp, suggesting that SARS-CoV-2 can develop favipiravir resistance with few mutations. Out of 134 mutations documented in the CoV-GLUE database, 63 specific mutations were already predicted as resistant in our calculations, thus attaining Eœ 47% correlation with the sequencing data. These findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir.

The causative agent of COVID-19 is a novel betacoronavirus or severe acute respiratory syndrome coronavirus (SARS-CoV-2), which has emerged as a pandemic of global concern. Considering its rapid transmission, WHO has declared public health emergency on 11th March 2020 worldwide. SARS-CoV-2 is a genetically diverse positive sense RNA virus that typically exhibit high rates of mutation than DNA viruses. Higher rates of mutation bring higher genomic variability which may lead to viral evolution and enabling viruses to evade the pre-existing immunity of host and quickly acquire drug resistance properties. The objective of our study was to compare the SARS-CoV-2 RdRp sequences of Indian SARS-CoV-2 isolates with those of Wuhan type virus. A total of 384 point mutations were detected from 488 sequence of the RdRp protein of Indian SARS-CoV-2 genome, out of which seven were used for subsequent study. Furthermore, prediction of secondary structure, protein modeling and its dynamics were performed which revealed that seven mutations (R118C, T148I, Y149C, E802A, Q822H, V880I and D893Y) significantly altered the stability and flexibility of RdRp protein. Present study was therefore, undertaken to analyze the variations occurring in RdRp due to multiple mutations leading to the alterations in the structure and function of RNA-dependent RNA polymerase which is essential for the replication /transcription of this virus and hence can be utilized as a promising therapeutic target to curb SARS-CoV-2 infections. Supplementary Information The online version contains supplementary material available at 10.1007/s00203-021-02527-9.

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain., Graphical Abstract Graphical AbstractCoronaviral RNA polymerase uses nucleoside triphosphates to make RNA and modify proteins, fueling viral replication. Acting as direct inhibitors or alternative substrates, nucleotide analogs can thwart SARS-CoV-2 and other viruses.

The RNA-dependent RNA polymerase (RdRp) of all known double-stranded RNA viruses is located within the viral particle and is responsible for the transcription and replication of the viral genome. Through an RT-PCR assay, we determined that purified virions, in vitro translated RdRp proteins, and purified recombinant RdRp proteins of partitiviruses also have reverse transcriptase (RT) function. We show that partitivirus RdRps 1) synthesized DNA from homologous and heterologous dsRNA templates; 2) are active using both ssRNA and dsRNA templates; and 3) are active at lower temperatures compared to an optimal reaction temperature of commercial RT enzymes. This finding poses an intriguing question: why do partitiviruses, with dsRNA genomes, have a polymerase with RT functions? In comparison, 3Dpol, the RdRp of poliovirus, did not show any RT activity. Our findings lead us to propose a new evolutionary model for RNA viruses where the RdRp of dsRNA viruses could be the ancestor of RdRps.

Here, we describe a novel mycovirus, tentatively designated as "Botryosphaeria dothidea mitovirus 3" (BdMV3), isolated from Botryosphaeria dothidea strain FJ, which causes pear ring rot disease in Fujian Province, China. The complete genome nucleotide sequence of BdMV3 is 2538 nt in length and contains a single 2070-nt open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) of 689 amino acids (aa) using the fungal mitochondrial genetic code. BLASTp analysis revealed that the RdRp of BdMV3 shares 28.91%-69.36% sequence identity (query sequence coverage more than 90%) with those of members of the genus Mitovirus, with the highest sequence identity of 69.36% and 68.79% to the corresponding RdRp aa sequences of Rhizoctonia solani mitovirus 10 and Macrophomina phaseolina mitovirus 4, respectively. Phylogenetic analysis based on RdRp aa sequences indicated that BdMV3 is a new member of the genus Mitovirus in the family Mitoviridae.

Background: The 2019 novel coronavirus (2019-nCoV), also known as coronavirus 2 (SARS-CoV-2) acute respiratory syndrome has recently emerged and continued to spread rapidly with high mortality and morbidity rates. Currently, no efficacious therapy is available to relieve coronavirus infections. As new drug design and development takes time, there is a possibility offindingan effective treatment from existing antiviral agents. Objective: The aim of this study is to find out the relationship between thepossible drug targets and themechanism of action of antiviral drugs. This review discusses the efforts indevelopingdrug from known or new molecules. Methods: Viruses usually have two structural integrities, proteins and nucleic acids, both of which can be possible drug targets. Herein, we systemically discuss the structural-functional relationships of the spike, 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro) and RNA-dependent RNA polymerase (RdRp), as these are prominent structural features of thecoronavirus. Certain antiviral drugs such as Remdesivir are RNA-dependent RNA polymerase inhibitorswiththe ability to terminate RNA replication by inhibiting ATP. Results: It is reported that ATP is involved in synthesis of coronavirus non-structural proteins from 3CLpro and PLpro. Similarly, mechanisms of action of many other antiviral agents havebeen discussed in this review. It will provide new insights into the mechanism of inhibition, and let us develop new therapeutic antiviral approaches against novel SARS-CoV-2 coronavirus. Conclusion: In conclusion, this review summarizes recent progress in developing protease inhibitors for SARS-CoV-2.

Viral RNA-dependent RNA polymerases (RdRPs) play central roles in the genome replication and transcription processes of RNA viruses. RdRPs initiate RNA synthesis either in primer-dependent or de novo mechanism, with the latter often assisted by a ‘priming element’ (PE) within the RdRP thumb domain. However, RdRP PEs exhibit high-level structural diversity, making it difficult to reconcile their conserved function in de novo initiation. Here we determined a 3.1-Å crystal structure of the Flaviviridae classical swine fever virus (CSFV) RdRP with a relative complete PE. Structure-based mutagenesis in combination with enzymology data further highlights the importance of a glycine residue (G671) and the participation of residues 665–680 in RdRP initiation. When compared with other representative Flaviviridae RdRPs, CSFV RdRP PE is structurally distinct but consistent in terminal initiation preference. Taken together, our work suggests that a conformational change in CSFV RdRP PE is necessary to fulfill de novo initiation, and similar ‘induced-fit’ mechanisms may be commonly taken by PE-containing de novo viral RdRPs.

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir., Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.

Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID50 /ml of 106.29 ± 0.23 at 6 days post-infection. The virions were non-enveloped with a diameter of 75-85 nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35 days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A. rostrata. The pathogenicity of the AEAdoV needs to be confirmed further.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.

A putative new virus with sequence similarity to members of the genus Cavemovirus in the family Caulimoviridae was identified in wild chicory (Cichorium intybus) by next-generation sequencing (NGS). The putative new virus was tentatively named “chicory mosaic cavemovirus” (ChiMV), and its genome was determined to be 7,775 nucleotides (nt) long with the typical genome organization of cavemoviruses. ORF1 encodes a putative coat protein/movement polyprotein (1,278 aa), ORF2 encodes a putative replicase (650 aa), and ORF3 encodes a putative transactivator factor (384 aa). The first two putative proteins have 46.2% and 68.7% amino acid sequence identity to the CP/MP protein (YP_004347414) and replicase (YP_004347415), respectively, of sweet potato collusive virus (SPCV). ORF3 encodes a protein with 38.5% amino acid sequence identity to the putative transactivator factor (NP_056849) of cassava vein mosaic virus (CsVMV). The new putative viral genome and those of three cavemoviruses (epiphyllum virus 4 [EpV-4], SPCV, and CsVMV) differ by 24-27% in the nt sequence of the replicase gene, which exceeds the species demarcation cutoff (>20%) for the family.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.

Sclerotinia sclerotiorum ourmiavirus 17 (SsOV17) was isolated from the hypovirulent strain GF3 of Sclerotinia sclerotiorum. The genome of SsOV17 is 2,802 nt in length and contains a single long open reading frame (ORF) flanked by a short structured 5'-untranslated region (5'-UTR) (28 nt) and a long 3'-UTR (788 nt), respectively. The ORF encodes a protein with 663 amino acids and a predicted molecular mass of 75.0 kDa. A BLASTp search indicated that the protein encoded by SsOV17 is closely related to the putative RNA-dependent RNA polymerase (RdRp) of Sclerotinia sclerotiorum ourmiavirus 13 (71% identity). A multiple sequence alignment indicated that eight conserved amino acid motifs were present in the RdRp conserved region of SsOV17. Phylogenetic analysis demonstrated that SsOV17 clustered with members of the genus Botoulivirus.

Mind your metals Iron–sulfur clusters are important cofactors for proteins involved in metabolism and electron transfer but are also sometimes found in enzymes involved in transcription and replication of DNA. In vitro expression of such enzymes can result in faulty cluster assembly and confusion about the composition of the functional enzyme. Using a careful anoxic purification scheme, Maio et al. found that the severe acute respiratory syndrome coronavirus 2 RNA–dependent RNA polymerase contains two iron–sulfur clusters at two sites previously observed to bind zinc ions. Mutation of the ligating cysteine residues resulted in loss of polymerase activity. A less severe loss of activity was seen in the zinc-containing enzyme. Treatment with the nitroxide drug TEMPOL resulted in degradation of the clusters, enzyme inhibition, and inhibition of viral replication in cell culture. Science , abi5224, this issue p. 236

The global COVID-19 pandemic has brought tremendous momentum to the field of messenger RNA (mRNA) vaccines. The advantages of this vaccine platform, such as rapid development and high efficacy, resulted in mRNA vaccines being the first approved vaccines against COVID-19. Looking forward to the development of future vaccines, how can we make RNA vaccines even better? While improvements in the stability of the formulation and cost of the vaccine are inevitable, one of the main challenges is lowering the dose of RNA in order to avoid side effects associated with high doses of RNA. One way to do this is by using self-amplifying RNA (saRNA), a type of mRNA that encodes a replicase that copies the original strand of RNA once it's in the cell. Here, we discuss the origins of saRNA, how it works in comparison to mRNA, current challenges in the field and the future of saRNA vaccines. © 2021 The Authors. Published by Portland Press Limited. All Rights Reserved.

Tomato brown rugose fruit virus (ToBRFV) is a novel tobamovirus initially reported in Jordan and Israel, and, more recently, in Europe, China, the USA and Mexico, which causes severe symptoms in tomato and pepper, as well as in other members of Solanaceae. ToBRFV can cause important losses so early detection is crucial to help thwart its dispersal, more so in countries where this virus is emerging as an important pathogen, as is the case in Mexico. Therefore, precise diagnostic is required to distinguish this virus from other members of this family that are not quarantined. We have analyzed the sequence of ToBRFV and compared to several other tobamoviruses and identified regions that are amenable for specific primer design. Regions in the replicase (p126 and p183), movement and coat proteins (MP and CP, respectively) genes were selected. Preliminary results indicated that CP primers provided the most consistent results. Furthermore, these primers were also used to detect ToBRFV via real-time qRT-PCR in leaf samples from tomato. The method was used successfully to detect 20 copies of viral transcript.

The involvement of the nucleus during flavivirus infection has been observed in only a small number of cases and can be limited to primarily two viral proteins; the structural protein C and the RNA polymerase NS5. Previously we observed that by blocking nuclear transport, WNV strain Kunjin (WNVKUN) replication is severely affected and through mutation of the identified NLS in WNVKUN NS5 protein. In this study, we interrogated the potential nuclear functions of WNVKUN NS5 has on the host transcriptome, by means of RNA sequencing (RNAseq). In a direct comparison between wild type and mutant NS5, it can also be determined that the nuclear translocation of NS5 results in a significant down-regulation of host genes involved in the innate immune response. When compared to published RNAseq data from WNV infection, many of these genes were overlapping indicting the role of NS5 induced transcription during infection.

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.

The D614G mutation in the Spike protein of the SARS-CoV-2 has effectively replaced the early pandemic-causing variant. Using pseudotyped lentivectors, we confirmed that the aspartate replacement by glycine in position 614 is markedly more infectious. Molecular modelling suggests that the G614 mutation facilitates transition towards an open state of the Spike protein. To explain the epidemiological success of D614G, we analysed the evolution of 27,086 high-quality SARS-CoV-2 genome sequences from GISAID. We observed striking coevolution of D614G with the P323L mutation in the viral polymerase. Importantly, the exclusive presence of G614 or L323 did not become epidemiologically relevant. In contrast, the combination of the two mutations gave rise to a viral G/L variant that has all but replaced the initial D/P variant. Our results suggest that the P323L mutation, located in the interface domain of the RNA-dependent RNA polymerase, is a necessary alteration that led to the epidemiological success of the present variant of SARS-CoV-2. However, we did not observe a significant correlation between reported COVID-19 mortality in different countries and the prevalence of the Wuhan versus G/L variant. Nevertheless, when comparing the speed of emergence and the ultimate predominance in individual countries, it is clear that the G/L variant displays major epidemiological supremacy over the original variant.

RNA‐dependent RNA polymerase 1 (RDR1) is essential for plant antiviral defence, but its role in plant defence against viroid infection remains unknown. The present study aimed to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Overexpression of Nicotiana tabacum RDR1 (NtRDR1) delayed the accumulation of potato spindle tuber viroid (PSTVd) genomic RNA and PSTVd‐derived small RNA (sRNA) in Nicotiana benthamiana plants at the early invasion stage, but not in the late stage of infection. Conversely, virus‐induced gene silencing of tomato RDR1 (SlRDR1a) increased the susceptibility to PSTVd infection (increased viroid accumulation). Salicylic acid (SA) pretreatment induced SlRDR1a expression and enhanced the defence against PSTVd infection in tomato plants. Our study demonstrated that RDR1 is involved in SA‐mediated defence and restricts the early systemic invasion by PSTVd in plants. The decreased PSTVd accumulation in N. benthamiana was not caused by efficient accumulation of PSTVd sRNAs. These results deepen our understanding of the mechanism of RDR1 in plant defence responses to viroid attack., Downregulation of RDR1a in tomato increased susceptibility to PSTVd infection; severe leaf curling with vein necrosis occurred in TRV:SlRDR1a tomato compared with TRV:00 tomatoes 28 days postinoculation (dpi) with PSTVd.

Junin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections., Junin virus (JUNV) causes Argentine hemorrhagic fever and encodes the large protein (L) of the RNA dependent RNA polymerase (RdRp) and its regulator, the matrix zinc-binding protein (Z). Here, the authors present the 3.54 Å cryo-EM structure of the complex of JUNV L with Z, and they propose a model of how JUNV L is regulated by Z during the viral life cycle and RNA synthesis.

Zoonotic arenaviruses can lead to life-threating diseases in humans. These viruses encode a large (L) polymerase that transcribes and replicates the viral genome. At the late stage of replication, the multifunctional Z protein interacts with the L polymerase to shut down RNA synthesis and initiate virion assembly. However, the mechanism by which the Z protein regulates the activity of L polymerase is unclear. Here, we used cryo-electron microscopy to resolve the structures of both Lassa and Machupo virus L polymerases in complex with their cognate Z proteins, and viral RNA, to 3.1–3.9 A resolutions. These structures reveal that Z protein binding induces conformational changes in two catalytic motifs of the L polymerase, and restrains their conformational dynamics to inhibit RNA synthesis, which is supported by hydrogen–deuterium exchange mass spectrometry analysis. Importantly, we show, by in vitro polymerase reactions, that Z proteins of Lassa and Machupo viruses can cross-inhibit their L polymerases, albeit with decreased inhibition efficiencies. This cross-reactivity results from a highly conserved determinant motif at the contacting interface, but is affected by other variable auxiliary motifs due to the divergent evolution of Old World and New World arenaviruses. These findings could provide promising targets for developing broad-spectrum antiviral drugs. Cryo-electron microscopy analyses show how Lassa and Machupo virus Z proteins bind the viral L polymerase and how this inhibits virus replication.

The outbreaks of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in 2019, have highlighted the concerns about the lack of potential vaccines or antivirals approved for inhibition of CoVs infection. SARS-CoV-2 RNA dependent RNA polymerase (RdRp) which is almost preserved across different viral species can be a potential target for development of antiviral drugs, including nucleoside analogues (NA). However, ExoN proofreading activity of CoVs leads to their protection from several NAs. Therefore, potential platforms based on the development of efficient NAs with broad-spectrum efficacy against human CoVs should be explored. This study was then aimed to present an overview on the development of NAs-based drug repurposing for targeting SARS-CoV-2 RdRp by computational analysis. Afterwards, the clinical development of some NAs including Favipiravir, Sofosbuvir, Ribavirin, Tenofovir, and Remdesivir as potential inhibitors of RdRp, were surveyed. Overall, exploring broad-spectrum NAs as promising inhibitors of RdRp may provide useful information about the identification of potential antiviral repurposed drugs against SARS-CoV-2.

Reverse transcription fluorescence resonance energy transfer‐polymerase chain reaction (FRET‐PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) (A23403G in the spike protein; C14408T in the RNA‐dependent RNA polymerase). Based on high‐resolution melting curve analysis, the reverse transcription (RT) FRET‐PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET‐PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available., Highlights A23403G in the spike protein and C14408T in the RNA‐dependent RNA polymerase are identified as two most common mutations in the SARS‐CoV‐2 variants. Reverse transcription FRET‐PCRs identified the mutations while no DNA sequencing required. Designed FRET‐PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is one of the RNA coronaviruses which share the highest mutation rates of RNA viruses when compared with that of their hosts. The collective mutation rate of RNA viruses is up to a million times higher than their hosts and is correlated with enhanced virulence of viruses. The RNA, genomic material of SARS-CoV-2, has the capacity of showing amplified fast changes as the infection spreads. These changes were frequently observed in genes for spike glycoprotein, nucleocapsid, ORF1ab, and ORF8, together with RNA dependent RNA polymerase. In contrast, genes for envelope, membrane, ORF6, ORF7a and ORF7b showed no observable changes in terms of amino acid substitutions. Mutated SARS COV-2 at these particular sites has been associated with viral infectivity, false laboratory results and viral genome mutation and interferes with therapeutic targets. Interferences with therapeutic targets is frequently observed in genes for RdRp. Additionally, mutated viral genes for RdRp render slow fidelity of RdRp protein, resulting in a high mutation rate. Such a high mutation rate might allow new virulent forms of the virus to emerge and influence the disease profile. This review aimed to elaborate on the effect of genomic and amino acid sequence mutations on the virulence and therapeutic targets of SARS COV-2. To achieve this objective, multiple literatures have been reviewed.

Background: COVID-19 is a life-threatening novel corona viral infection to our civilization and spreading rapidly. Tremendousefforts have been made by the researchers to search for a drug to control SARS-CoV-2. Methods: Here, a series of arsenical derivatives were optimized and analyzed with in silico study to search the inhibitor of RNA dependent RNA polymerase (RdRp), the major replication factor of SARS-CoV-2. All the optimized derivatives were blindly docked with RdRp of SARS-CoV-2 using iGEMDOCK v2.1. Results: Based on the lower idock score in the catalytic pocket of RdRp, darinaparsin (-82.52 kcal/- mol) was revealed to be the most effective among them. Darinaparsin strongly binds with both Nsp9 replicase protein (-8.77 kcal/mol) and Nsp15 endoribonuclease (-8.3 kcal/mol) of SARS-- CoV-2 as confirmed from the AutoDock analysis. During infection, the ssRNA of SARS-CoV-2 is translated into large polyproteins forming viral replication complex by specific proteases like 3CL protease and papain protease. This is also another target to control the virus infection where darinaparsin also performs the inhibitory role to proteases of 3CL protease (-7.69 kcal/mol) and papain protease (-8.43 kcal/mol). Conclusion: In the host cell, the furin protease serves as a gateway to the viral entry and darinaparsin docked with furin protease, which revealed a strong binding affinity. Thus, screening of potential arsenic drugs would help in providing the fast in-vitro to in-vivo analysis towards the development of therapeutics against SARS-CoV-2.

Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

Citrus aurantifolia (acid lime) trees exhibiting yellowing, stem pitting and decline symptoms in Oman were found to be infected by Citrus tristeza virus (CTV) (genus; Closterovirus, family; Closteroviridae). The sequence of the 3′ approximately 9.8 Kb genome, encompassing the partial RNA dependent RNA polymerase to P23 genes including the 3′ UTR region was determined. Pairwise nucleotide comparisons revealed that the Oman isolate had the highest nucleotide identity with those from Mexico and Egyptian isolates. Phylogenetic analysis showed that the Oman isolate clustered with the Egyptian isolate and was identified as T36 genotype. Recombination analysis showed the presence of three potential recombination events and the findings of this study are discussed. Further, extensive studies on CTV species mainly in Oman and as a whole in the Arabian Peninsula is needed, especially after investigating the recombinant 3′ CTV genome isolate.

The vampire bat (Desmodus rotundus) is a haematophagous animal that feeds exclusively on the blood of domestic mammals. Vampire bat feeding habits enable their contact with mammalian hosts and may enhance zoonotic spillover. Moreover, they may carry several pathogenic organisms, including coronaviruses (CoVs), for which they are important hosts. The human pathogens that cause severe acute respiratory syndrome (SARS‐CoV), Middle East respiratory syndrome (MERS‐CoV) and possibly coronavirus disease 2019 (SARS‐CoV‐2) all originated in bats but required bridge hosts to spread into human populations. To monitor the presence of potential zoonotic viruses in bats, the present work evaluated the presence of CoVs in vampire bats from southern Brazil. A total of 101 vampire bats were captured and euthanized between 2017 and 2019 in Rio Grande do Sul state, southern Brazil. The brain, heart, liver, lungs, kidneys and intestines were collected and macerated individually. The samples were pooled and submitted to high‐throughput sequencing (HTS) using the Illumina MiSeq platform and subsequently individually screened using a pancoronavirus RT‐PCR protocol. We detected CoV‐related sequences in HTS, but only two (2/101; 1.98%) animals had CoV detected in the intestines by RT‐PCR. Partial sequences of RdRp and spike genes were obtained in the same sample and the RdRp region in the other sample. The sequences were classified as belonging to Alphacoronavirus. The sequences were closely related to alphacoronaviruses detected in vampire bats from Peru. The continuous monitoring of bat CoVs may help to map and predict putative future zoonotic agents with great impacts on human health.

A novel mycovirus belonging to the proposed family "Fusariviridae" was discovered in Alternaria Solani by sequencing a double-stranded RNA extracted from this phytopathogenic fungus. The virus was tentatively named “Alternaria solani fusarivirus 1” (AsFV1). AsFV1 has a single-stranded positive-sense (+ssRNA) genome of 6,845 nucleotides containing three open reading frames (ORFs) and a poly(A) tail. The largest ORF, ORF1 encodes a large polypeptide of 1,556 amino acids (aa) with conserved RNA-dependent RNA polymerase and helicase domains. The ORF2 and ORF3 have overlapping regions, encoding a putative protein of 522 amino acids (aa) and a putative protein of 105 amino acids (aa) respectively, for which function is unknown now. Multiple sequence alignments and phylogenetic analysis revealed AsFV1 belonging to Fusariviridae. This is the first report of the full-length nucleotide sequence of a fusarivirus infected with Alternaria solani.

Efficient immune responses against viral infection are determined by sufficient activation of nucleic acid sensor-mediated innate immunity1,2. Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains an ongoing global pandemic. It is an urgent challenge to clarify the innate recognition mechanism to control this virus. Here we show that retinoic acid-inducible gene-I (RIG-I) sufficiently restrains SARS-CoV-2 replication in human lung cells in a type I/III interferon (IFN)-independent manner. RIG-I recognizes the 3' untranslated region of the SARS-CoV-2 RNA genome via the helicase domains, but not the C-terminal domain. This new mode of RIG-I recognition does not stimulate its ATPase, thereby aborting the activation of the conventional mitochondrial antiviral-signaling protein-dependent pathways, which is in accordance with lack of cytokine induction. Nevertheless, the interaction of RIG-I with the viral genome directly abrogates viral RNA-dependent RNA polymerase mediation of the first step of replication. Consistently, genetic ablation of RIG-I allows lung cells to produce viral particles that expressed the viral spike protein. By contrast, the anti-SARS-CoV-2 activity was restored by all-trans retinoic acid treatment through upregulation of RIG-I protein expression in primary lung cells derived from patients with chronic obstructive pulmonary disease. Thus, our findings demonstrate the distinctive role of RIG-I as a restraining factor in the early phase of SARS-CoV-2 infection in human lung cells.

SARS-CoV-2 is an enveloped positive-sense RNA virus, contain crown-like spikes on its surface, exceptional of large RNA genome, and a special replication machinery. Common symptoms of SARS-CoV-2 include cough, common cold, fever, sore throat, and a variety of severe acute respiratory disease (SARD) such as pneumonia. SARS-CoV-2 infects epithelial cells, T-cells, macrophages, and dendritic cells and also influences the production and implantation of pro-inflammatory cytokines and chemokines. Repurposing of various drugs during this emergency condition can reduce the rate of mortality as well as time and cost. Two druggable protein and enzyme targets have been selected in this review article due to their crucial role in the viral life cycle. The eukaryotic translation initiation factor (eIF4A), cyclophilin, nucleocapsid protein, spike protein, Angiotensin-converting enzyme 2 (ACE2), 3-chymotrypsin-like cysteine protease (3CLpro), and RNA-dependent RNA polymerase (RdRp) play significant role in early and late phase of SARS-CoV-2 replication and translation. This review paper is based on the rationale of inhibiting of various SARS-CoV-2 proteins and enzymes as novel therapeutic approaches for the management and treatment of patients with SARS-CoV-2 infection. We also discussed the structural and functional relationship of different proteins and enzymes to develop therapeutic approaches for novel coronavirus SARS-CoV-2.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel member of the genus betacoronavirus in the Coronaviridae family. It has been identified as the causative agent of coronavirus disease 2019 (COVID-19), spreading rapidly in Asia, America and Europe. Like some other RNA viruses, RNA replication and transcription of SARS-CoV-2 rely on its RNA-dependent RNA polymerase (RdRP), which is a therapeutic target of clinical importance. Crystal structure of SARS-CoV-2 was solved recently (PDB ID 6M71) with some missing residues. Objective: We used SARS-CoV-2 RdRP as a target protein to screen for possible chemical molecules with potential anti-viral effects. Methods: Here we modelled the missing residues 896-905 via homology modelling and then analysed the interactions of Hepatitis C virus allosteric non-nucleoside inhibitors (NNIs) in the reported NNIs binding sites in SARS-CoV-2 RdRP. Results: We found that MK-3281, filibuvir, setrobuvir and dasabuvir might be able to inhibit SARS-CoV-2 RdRP based on their binding affinities in the respective binding sites. Conclusion: Further in vitro and in vivo experimental research will be carried out to evaluate their effectiveness in COVID-19 treatment in the near future.