A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

The minus strand of positive-sense RNA viruses encodes small proteins.

It is widely accepted that the minus strands of positive single-strand RNA (+ssRNA) viruses function as replication templates only. Gong et al. revealed that the minus strand of two unrelated +ssRNA viruses encodes proteins. This textbook-changing discovery calls for the reconsideration of the molecular mechanisms underlying the infection cycle of +ssRNA viruses.

Molecular characterization of a novel deltaflexivirus infecting the edible fungus Pleurotus ostreatus.

A novel positive single-stranded RNA virus, Pleurotus ostreatus deltaflexivirus 1 (PoDFV1), was isolated from the edible fungus Pleurotus ostreatus strain ZP6. The complete genome of PoDFV1 is 7706 nucleotides (nt) long and contains a short poly(A) tail. PoDFV1 was predicted to contain one large open reading frame (ORF1) and three small downstream ORFs (ORFs 2-4). ORF1 encodes a putative replication-associated polyprotein of 1979 amino acids (aa) containing three conserved domains - viral RNA methyltransferase (Mtr), viral RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) - which are common to all deltaflexiviruses. ORFs 2-4 encode three small hypothetical proteins (15-20 kDa) without conserved domains or known biological functions. Sequence alignments and phylogenetic analysis suggested that PoDFV1 is a member of a new species in the genus Deltaflexivirus (family Deltaflexiviridae, order Tymovirales). To our knowledge, this is the first report of a deltaflexivirus infecting P. ostreatus.

Replication of the coronavirus genome: A paradox among positive-strand RNA viruses.

Coronavirus (CoV) genomes consist of positive-sense single-stranded RNA and are among the largest viral RNAs known to date (∼30 kb). As a result, CoVs deploy sophisticated mechanisms to replicate these extraordinarily large genomes as well as to transcribe subgenomic messenger RNAs. Since 2003, with the emergence of three highly pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2), significant progress has been made in the molecular characterization of the viral proteins and key mechanisms involved in CoV RNA genome replication. For example, to allow for the maintenance and integrity of their large RNA genomes, CoVs have acquired RNA proofreading 3'-5' exoribonuclease activity (in nonstructural protein nsp14). In order to replicate the large genome, the viral-RNA-dependent RNA polymerase (RdRp; in nsp12) is supplemented by a processivity factor (made of the viral complex nsp7/nsp8), making it the fastest known RdRp. Lastly, a viral structural protein, the nucleocapsid (N) protein, which is primarily involved in genome encapsidation, is required for efficient viral replication and transcription. Therefore, CoVs are a paradox among positive-strand RNA viruses in the sense that they use both a processivity factor and have proofreading activity reminiscent of DNA organisms in addition to structural proteins that mediate efficient RNA synthesis, commonly used by negative-strand RNA viruses. In this review, we present a historical perspective of these unsuspected discoveries and detail the current knowledge on the core replicative machinery deployed by CoVs.

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

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.

Protein Nucleotidylylation in +ssRNA Viruses.

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5' end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

Identification and characterization of a novel virus associated with an eriophyid mite in extracts of fruit trees leaves.

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.

A second capsidless hadakavirus strain with 10 positive-sense single-stranded RNA genomic segments from Fusarium nygamai.

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.

An expanded class of histidine-accepting viral tRNA-like structures.

Structured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some viral RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLSHis) are examples found at the 3' termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate processes important for infection, to include genome replication. As only five TLSHis examples had been reported, we explored the possible larger phylogenetic distribution and diversity of this TLS class using bioinformatic approaches. We identified many new examples of TLSHis, yielding a rigorous consensus sequence and secondary structure model that we validated by chemical probing of representative TLSHis RNAs. We confirmed new examples as authentic TLSHis by demonstrating their ability to be histidylated in vitro, then used mutational analyses to imply a tertiary interaction that is likely analogous to the D- and T-loop interaction found in canonical tRNAs. These results expand our understanding of how diverse RNA sequences achieve tRNA-like structure and function in the context of viral RNA genomes and lay the groundwork for high-resolution structural studies of tRNA mimicry by histidine-accepting TLSs.

Genetic and structural analyses of ssRNA viruses pave the way for the discovery of novel antiviral pharmacological targets.

In the era of big data and artificial intelligence, a lot of new discoveries have influenced the fields of antiviral drug design and pharmacophore identification. Viruses have always been a threat to society in terms of public health and economic stability. Viruses not only affect humans but also livestock and agriculture with a direct impact on food safety, economy and environmental imprint. Most recently, with the pandemic of COVID-19, it was made clear that a single virus can have a devastating impact on global well-being and economy. In this direction, there is an emerging need for the identification of promising pharmacological targets in viruses. Herein, an effort has been made to discuss the current knowledge, state-of-the-art applications and future implications for the main pharmacological targets of single-stranded RNA viruses.

Silicon nitride: a potent solid-state bioceramic inactivator of ssRNA viruses.

Surface inactivation of human microbial pathogens has a long history. The Smith Papyrus (2600 ~ 2200 B.C.) described the use of copper surfaces to sterilize chest wounds and drinking water. Brass and bronze on doorknobs can discourage microbial spread in hospitals, and metal-base surface coatings are used in hygiene-sensitive environments, both as inactivators and modulators of cellular immunity. A limitation of these approaches is that the reactive oxygen radicals (ROS) generated at metal surfaces also damage human cells by oxidizing their proteins and lipids. Silicon nitride (Si3N4) is a non-oxide ceramic compound with known surface bacterial resistance. We show here that off-stoichiometric reactions at Si3N4 surfaces are also capable of inactivating different types of single-stranded RNA (ssRNA) viruses independent of whether their structure presents an envelop or not. The antiviral property of Si3N4 derives from a hydrolysis reaction at its surface and the subsequent formation of reactive nitrogen species (RNS) in doses that could be metabolized by mammalian cells but are lethal to pathogens. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of viral RNA and in situ Raman spectroscopy suggested that the products of Si3N4 hydrolysis directly react with viral proteins and RNA. Si3N4 may have a role in controlling human epidemics related to ssRNA mutant viruses.

The Halastavi árva Virus Intergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism.

Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRESs) do not require initiator tRNA, an AUG codon, or initiation factors and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A sites to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By using in vitro reconstitution and cryoelectron microscopy (cryo-EM), we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

A Novel RNA Virus in the Parasitoid Wasp Lysiphlebus fabarum: Genomic Structure, Prevalence, and Transmission.

We report on a novel RNA virus infecting the wasp Lysiphlebus fabarum, a parasitoid of aphids. This virus, tentatively named "Lysiphlebus fabarum virus" (LysV), was discovered in transcriptome sequences of wasps from an experimental evolution study in which the parasitoids were allowed to adapt to aphid hosts (Aphis fabae) with or without resistance-conferring endosymbionts. Based on phylogenetic analyses of the viral RNA-dependent RNA polymerase (RdRp), LysV belongs to the Iflaviridae family in the order of the Picornavirales, with the closest known relatives all being parasitoid wasp-infecting viruses. We developed an endpoint PCR and a more sensitive qPCR assay to screen for LysV in field samples and laboratory lines. These screens verified the occurrence of LysV in wild parasitoids and identified the likely wild-source population for lab infections in Western Switzerland. Three viral haplotypes could be distinguished in wild populations, of which two were found in the laboratory. Both vertical and horizontal transmission of LysV were demonstrated experimentally, and repeated sampling of laboratory populations suggests that the virus can form persistent infections without obvious symptoms in infected wasps.