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A new positive-sense single-stranded RNA (+ssRNA) mycovirus, Verticillium dahliae magoulivirus 1 (VdMoV1), was isolated from two strains (2-19 and XLZ70) of Verticillium dahliae. The complete genome of VdMoV1 is 2303 nucleotides (nt) in length and has a large open reading frame (nt positions from 61 to 1938) encoding an RNA-dependent RNA polymerase (RdRp). A multiple sequence alignment indicated that the central region of the RdRp encoded by VdMoV1 contains eight typical viral RdRp motifs. BLASTp analysis demonstrated that VdMoV1 has the highest sequence identity (86.88%) to Bremia lactucae associated ourmia-like virus 2 (BlaOLV2). Phylogenetic analysis revealed that VdMoV1 is a new member of the genus Magoulivirus. As far as we know, VdMoV1 is the first reported member of the family Botourmiaviridae infecting V. dahliae.

Double filtration plasmapheresis (DFPP) is an apheretic technique that selectively removes high molecular weight substances using a plasma component filter. DFPP has been used to treat positive-sense RNA virus infections, mainly chronic hepatitis C virus (HCV) infection, because of its ability to directly eliminate viral particles from blood plasma from 2008 to about 2015, before direct-acting antiviral agents was marketed. This effect has been termed virus removal and eradication by DFPP. HCV is a positive-sense RNA virus similar to West Nile virus, dengue virus and the SARS and Middle East respiratory syndrome coronaviruses. SARS-CoV-2 is classified same viral species. These viruses are all classified in Family Flaviviridae which are family of single-stranded plus-stranded RNA viruses. Viral particles are 40–60 nm in diameter, enveloped and spherical in shape. We present a rare case of HCV removal where an RNA virus infection that copresented with virus-associated autoimmune hepatitis was eliminated using DFPP. Our results indicate that DFPP may facilitate prompt viraemia reduction and may have novel treatment applications for SARS-CoV-2, that is, use of therapeutic plasma exchange for fulminant COVID-19.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that are implicated in RNA metabolism, such as alternative splicing, mRNA stabilization and translational regulation. According to their different cellular localization, hnRNPs display multiple functions. Most hnRNPs were predominantly located in the nucleus, but some of them could redistribute to the cytoplasm during virus infection. HnRNPs consist of different domains and motifs that enable these proteins to recognize predetermined nucleotide sequences. In the virus-host interactions, hnRNPs specifically bind to viral RNA or proteins. And some of the viral protein-hnRNP interactions require the viral RNA or other host factors as the intermediate. Through various mechanisms, hnRNPs could regulate viral translation, viral genome replication, the switch of translation to replication and virion release. This review highlights the common features and the distinguish roles of hnRNPs in the life cycle of positive single-stranded RNA viruses.

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.

Virus infection is a process that requires combined contributions from both virus and host factors. For this process to be efficient within the crowded host environment, viruses have evolved ways to manipulate and reorganize host structures to produce cellular microenvironments. Positive-strand RNA virus replication and assembly occurs in association with cytoplasmic membranes, causing a reorganization of these membranes to create microenvironments that support viral processes. Similarities between virus-induced membrane domains and cellular organelles have led to the description of these structures as virus replication organelles (vRO). Electron microscopy analysis of vROs in positive-strand RNA virus infected cells has revealed surprising morphological similarities between genetically diverse virus species. For all positive-strand RNA viruses, vROs can be categorized into two groups: those that make invaginations into the cellular membranes (In-vRO), and those that cause the production of protrusions from cellular membranes (Pr-vRO), most often in the form of double membrane vesicles (DMVs). In this review, we will discuss the current knowledge on the structure and biogenesis of these two different vRO classes as well as comparing morphology and function of vROs between various positive-strand RNA viruses. Finally, we will discuss recent studies describing pharmaceutical intervention in vRO formation as an avenue to control virus infection.

A unique capsidless virus with a positive-sense, single-stranded RNA genome (hadakavirus 1, HadV1), a member of the extended picorna-like supergroup, was isolated previously from the phytopathogenic fungus Fusarium oxysporum. Here, we describe the molecular and biological characterisation of a second hadakavirus strain from Fusarium nygamai, which has not been investigated in detail previously as a virus host. This virus, hadakavirus 1 strain 1NL (HadV1-1NL), has features similar to the first hadakavirus, HadV1-7n, despite having a different number of segments (10 for HadV1-1NL vs. 11 for HadV1-7n). The 10 genomic RNA segments of HadV1-1NL range in size from 0.9 kb to 2.5 kb. All HadV1-1NL segments show 67% to 86% local nucleotide sequence identity to their HadV1-7n counterparts, whereas HadV1-1NL has no homolog of HadV1-7n RNA8, which encodes a zinc-finger motif. Another interesting feature is the possible coding incapability of HadV1-1NL RNA10. HadV1-1NL was predicted to be capsidless based on the RNase A susceptibility of its replicative form dsRNA. Phenotypic comparison of multiple virus-infected and virus-free single-spore isolates indicated asymptomatic infection by HadV1-1NL. Less-efficient vertical transmission via spores was observed as the infected fungal colonies from which the spores were derived became older, as was observed for HadV1-7n. This study shows a second example of a hadakavirus that appears to have unusual features.

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.

Reverse genetics is the prospective analysis of how genotype determines phenotype. In a typical experiment, a researcher alters a viral genome, then observes the phenotypic outcome. Among RNA viruses, this approach was first applied to positive-strand RNA viruses in the mid-1970s and over nearly 50 years has become a powerful and widely used approach for dissecting the mechanisms of viral replication and pathogenesis. During this time the global health importance of two virus groups, flaviviruses (genus Flavivirus, family Flaviviridae) and betacoronaviruses (genus Betacoronavirus, subfamily Orthocoronavirinae, family Coronaviridae), have dramatically increased, yet these viruses have genomes that are technically challenging to manipulate. As a result, several new techniques have been developed to overcome these challenges. Here I briefly review key historical aspects of positive-strand RNA virus reverse genetics, describe some recent reverse genetic innovations, particularly as applied to flaviviruses and coronaviruses, and discuss their benefits and limitations within the larger context of rigorous genetic analysis.

A marked reorganization of internal membranes occurs in the cytoplasm of cells infected by single stranded positive-sense RNA viruses. Most cell compartments change their asset to provide lipids for membrane rearrangement into replication organelles, where to concentrate viral proteins and enzymes while hiding from pathogen pattern recognition molecules. Because the endoplasmic reticulum is a central hub for lipid metabolism, when viruses hijack the organelle to form their replication organelles, a cascade of events change the intracellular environment. This results in a marked increase in lipid consumption, both by lipolysis and lipophagy of lipid droplets. In addition, lipids are used to produce energy for viral replication. At the same time, inflammation is started by signalling lipids, where lysosomal processing plays a relevant role. This review is aimed at providing an overview on what takes place after human class IV viruses have released their genome into the host cell and the consequences on lipid metabolism, including lysosomes.

Positive-strand RNA viruses, the largest genetic class of eukaryotic viruses, include coronaviruses and many other established and emerging pathogens. A major target for understanding and controlling these viruses is their genome replication, which occurs in virus-induced membrane vesicles that organize replication steps and protect double-stranded RNA intermediates from innate immune recognition. The structure of these complexes has been greatly illuminated by recent cryo-electron microscope tomography studies with several viruses. One key finding in diverse systems is the organization of crucial viral RNA replication factors in multimeric rings or crowns that among other functions serve as exit channels gating release of progeny genomes to the cytosol for translation and encapsidation. Emerging results suggest that these crowns serve additional important purposes in replication complex assembly, function, and interaction with downstream processes such as encapsidation. The findings provide insights into viral function and evolution and new bases for understanding, controlling, and engineering positive-strand RNA viruses.

Host IFNL4 haplotype status contributes to the development of chronic hepatitis C virus (HCV) infection in individuals who are acutely infected with the virus. In silico studies revealed that specific amino acid variants at multiple sites on the HCV polyprotein correlate with functional single-nucleotide polymorphisms (SNPs) in the IFNL4 locus. Thus, SNPs at the IFNL4 locus may select variants that influence virus replication and thereby the outcome of infection. Here, we examine the most significantly IFNL4-associated amino acid variants that lie in the ‘lambda (L) 2 loop’ of the HCV NS5B RNA polymerase. L2 loop variants were introduced into both sub-genomic replicon and full-length infectious clones of HCV and viral replication was examined in the presence and absence of exogenous IFNλ4. Our data demonstrate that while mutation of the NS5B L2 loop affects replication, individual IFNL4-associated variants have modest but consistent effects on replication in both the presence and absence of IFNλ4. Given the strong genetic association between these variants and IFNL4, these data suggest a nuanced effect of each individual position on viral replication, the combined effect of which might mediate resistance to the effects of IFNλ4.

Funding: This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/L003988/1 and 1645891, and strategic funding to The Pirbright Institute, BBS/E/I/00007035, BBS/E/I/00007034, BBS/E/I/00007037 and BBS/E/I/00007039. Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified. Publisher PDF

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of nonstructural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (Remdesivir, Alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative ‘furin-like cleavage site’ in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

The last two decades have seen the rise of viromics, the study of viral communities through the detection and characterization of virus genome sequences. Here we systematically review and summarize the scope and limitations of our current understanding of avian viromes, in both domesticated and wild-bird populations. We compare this viromic work to the broader literature on avian prokaryotic microbiomes, and highlight the growing importance of structured sampling and experimental design for testing explanatory hypotheses. We provide a number of recommendations for sample collection and preliminary data analysis to guide the development of avian viromics. Avian viromes have the potential to inform disease surveillance in poultry and improve our understanding of the risk of zoonotic viruses to human health.

The species Enterovirus A (EV-A) consists of two conventional clusters and one unconventional cluster. At present, sequence analysis shows no evidence of recombination between conventional and unconventional EV-A types. However, the factors underlying this genetic barrier are unclear. Here, we systematically dissected the genome components linked to these peculiar phenomena, using the viral reverse genetic tools. We reported that viral capsids of the unconventional EV-A types expressed poorly in human cells. The trans-encapsidation outputs across conventional and unconventional EV-A types were also with low efficiency. However, replicons of conventional types bearing exchanged 5′-untranslated region (UTR) or non-structural regions from the unconventional types were replication-competent. Furthermore, we created a viable recombinant EVA71 (conventional type) with its P3 region replaced by that from EVA89 (unconventional type). Thus, our data for the first time reveal the potential for fertile genetic exchanges between conventional and unconventional EV-A types. It also discloses that the mysterious recombination barriers may lie in uncoordinated capsid expression and particle assembly by different EV-A clusters.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain–Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood–brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml−1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

In this recommendation, we update our 2016 table of reference sequences of subtypes of hepatitis E virus (HEV; species Orthohepevirus A, family Hepeviridae) for which complete genome sequences are available (Smith et al., 2016). This takes into account subsequent publications describing novel viruses and additional proposals for subtype names; there are now eight genotypes and 36 subtypes. Although it remains difficult to define strict criteria for distinguishing between virus subtypes, and is not within the remit of the International Committee on Taxonomy of Viruses (ICTV), the use of agreed reference sequences will bring clarity and stability to researchers, epidemiologists and clinicians working with HEV.

Rotavirus A (RVA), bovine torovirus (BToV), bovine enterovirus (BEV) and bovine coronavirus (BCV) at a bovine farm in Ibaraki prefecture were monitored by one-step multiplex reverse transcription polymerase chain reaction (RT-PCR), with the aim of confirming the reduction of “viral pathogen indicators”. A total of 960 bovine fecal samples were collected from calves less than 2 month-old within the period from October 2016 to October 2018 every 2 months at the bovine farm. In each sampling, 40 samples were taken from calves 3 week-old or less, and 40 samples from calves over 3 week-old, in principle. At the end of September 2017, the farm introduced improvement of hygiene protocols on boots by exchanging boots and appropriate usage of a footbath at the entrance of calf sheds. In the comparison of the virus detection by RT-PCR, prevalence of all 4 viruses was significantly reduced (P

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host’s brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Free radical release due to oxidative stress is gaining importance in the field of viral pathogenesis. Recent studies suggest the involvement of oxidative stress and ROS levels in regulating disease virulence during RNA virus infection. Most of the RNA virus infections lead to vascular dysfunction and disease severity. However, the biology of free radicals in maintaining vascular endothelium integrity is not completely understood. In the present review, we discuss some of the common features in positive-strand RNA virus infections such as dengue and SARS-CoV-2 and suggest that anti-oxidant therapy could pave the way to develop therapeutic strategies in combating emerging and re-emerging RNA viruses.

Life cycle processes of positive-strand (+)RNA viruses are broadly conserved across families, yet they employ different strategies to grow in the cell. Using a generalized dynamical model for intracellular (+)RNA virus growth, we decipher these life cycle determinants and their dependencies for several viruses and parse the effects of viral mutations, drugs and host cell permissivity. We show that poliovirus employs rapid replication and virus assembly, whereas the Japanese encephalitis virus leverages its higher rate of translation and efficient cellular reorganization compared to the hepatitis C virus. Stochastic simulations demonstrate infection extinction if all seeding (inoculating) viral RNA degrade before establishing robust replication critical for infection. The probability of this productive cellular infection, ‘cellular infectivity’, is affected by virus–host processes and defined by early life cycle events and viral seeding. An increase in cytoplasmic RNA degradation and delay in vesicular compartment formation reduces infectivity, more so when combined. Synergy among these parameters in limiting (+)RNA virus infection as predicted by our model suggests new avenues for inhibiting infections by targeting the early life cycle bottlenecks.

Remdesivir (RDV) is a direct antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, e.g., SARS-CoV-2 and hepatitis C virus (HCV) and non-segmented negative-sense RNA viruses, e.g., Nipah virus (NiV), while several segmented negative-sense RNA viruses such as influenza (Flu) virus or Crimean-Congo hemorrhagic fever virus (CCHFV) are not sensitive to the drug. The reasons for this apparent pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases (RdRp) and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. The results of this study revealed a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Delayed chain-termination is heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP is seen with all polymerases. Molecular modeling suggests a steric conflict between the 1’-cyano group of RDV-MP and conserved residues of RdRp motif F. We conclude that future efforts in the development of nucleotide analogues with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1’-modification.

International audience; Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and the frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.

International audience; Coronavirus (CoV) genomes consist of positive-sense single-stranded RNA and are among the largest viral RNAs known todate (30 kb). As a result, CoVs deploy sophisticated mecha-nisms to replicate these extraordinarily large genomes as wellas to transcribe subgenomic messenger RNAs. Since 2003, withthe emergence of three highly pathogenic CoVs (SARS-CoV,MERS-CoV, and SARS-CoV-2), significant progress has beenmade in the molecular characterization of the viral proteinsand key mechanisms involved in CoV RNA genome replication.For example, to allow for the maintenance and integrity of theirlarge RNA genomes, CoVs have acquired RNA proofreading 30-50 exoribonuclease activity (in nonstructural protein nsp14). Inorder to replicate the large genome, the viral-RNA–dependentRNA polymerase (RdRp; in nsp12) is supplemented by aprocessivity factor (made of the viral complex nsp7/nsp8),making it the fastest known RdRp. Lastly, a viral structuralprotein, the nucleocapsid (N) protein, which is primarilyinvolved in genome encapsidation, is required for efficient viralreplication and transcription. Therefore, CoVs are a paradoxamong positive-strand RNA viruses in the sense that they useboth a processivity factor and have proofreading activityreminiscent of DNA organisms in addition to structural pro-teins that mediate efficient RNA synthesis, commonly used bynegative-strand RNA viruses. In this review, we present a his-torical perspective of these unsuspected discoveries and detailthe current knowledge on the core replicative machinerydeployed by CoVs.

Graphical abstract, The ongoing COVID-19 pandemic, periodic recurrence of viral infections, and the emergence of challenging variants has created an urgent need of alternative therapeutic approaches to combat the spread of viral infections, failing to which may pose a greater risk to mankind in future. Resilience against antiviral drugs or fast evolutionary rate of viruses is stressing the scientific community to identify new therapeutic approaches for timely control of disease. Host metabolic pathways are exquisite reservoir of energy to viruses and contribute a diverse array of functions for successful replication and pathogenesis of virus. Targeting the host factors rather than viral enzymes to cease viral infection, has emerged as an alternative antiviral strategy. This approach offers advantage in terms of increased threshold to viral resistance and can provide broad-spectrum antiviral action against different viruses. The article here provides substantial review of literature illuminating the host factors and molecular mechanisms involved in innate/adaptive responses to viral infection, hijacking of signalling pathways by viruses and the intracellular metabolic pathways required for viral replication. Host-targeted drugs acting on the pathways usurped by viruses are also addressed in this study. Host-directed antiviral therapeutics might prove to be a rewarding approach in controlling the unprecedented spread of viral infection, however the probability of cellular side effects or cytotoxicity on host cell should not be ignored at the time of clinical investigations.