摘要：Chikungunya fever (CHIKF) is caused by chikungunya virus (CHIKV) infection which is a re-emerging mosquito-borne zoonosis. At present, there are no approved therapeutics for CHIKF. Herein, we have investigated candidate compounds which can inhibit CHIKV infection. Screening of chemical compound libraries were performed and one candidate, a benzimidazole-related compound designated Compound-A was found to inhibit infection by several CHIKV strains and a Sindbis virus strain at nanomolar concentrations. To investigate the inhibitory mechanism of action, a Compound-A resistant CHIKV (res-CHIKV) was isolated and a key mutation associated with resistance was identified by reverse-genetic recombinant CHIKVs containing amino acid substitutions present in res-CHIKV. These results demonstrated that the target site of Compound-A was the M2295 residue in the nonstructural protein 4 (nsP4), which is located in one of the functional domains of RNA-dependent RNA-polymerase (RdRp). We also confirmed that Compound-A inhibits RdRp function of CHIKV by using CHIKV replicons.

摘要：Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae, is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro. The recombinant CDV could stably express the NanoLuc^® luciferase (NLuc), a novel enzyme that was smaller and "brighter" than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro. Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.

摘要：Caliciviruses have a positive-strand RNA genome with a length of about 7.5 kb that contains 2, 3, or 4 functional open reading frames (ORFs). A subgenomic mRNA (sg-RNA) is transcribed in the infected cell, and both major capsid protein viral protein 1 (VP1) and minor capsid protein VP2 are translated from the sg-RNA. Translation of proteins from the genomic RNA (g-RNA) and from the sg-RNA is mediated by the RNA-linked viral protein VPg (virus protein, genome linked). Most of the calicivirus genera have translation mechanisms leading to VP1 expression from the g-RNA. VP1 is part of the polyprotein for sapoviruses, lagoviruses, and neboviruses, and a termination/reinitiation mechanism was described for noroviruses. Vesiviruses have no known mechanism for the expression of VP1 from the g-RNA, and the Vesivirus genus is the only genus of the Caliciviridae that generates VP1 via a precursor capsid leader protein (LC-VP1). Analyses of feline calicivirus (FCV) g-RNA translation showed a low level of VP1 expression with an initiation downstream of the original start codon of LC-VP1, leading to a smaller, truncated LC-VP1 (tLC-VP1) protein. Deletion and substitution analyses of the region surrounding the LC-VP1 start codon allowed the identification of sequences within the leader protein coding region of FCV that have an impact on VP1 translation frequency from the g-RNA. Introduction of such mutations into the virus showed an impact of strongly reduced tLC-VP1 levels translated from the g-RNA on viral replication.IMPORTANCE Caliciviruses are a cause of important diseases in humans and animals. It is crucial to understand the prerequisites of efficient replication of these viruses in order to develop strategies for prevention and treatment of these diseases. It was shown before that all caliciviruses except vesiviruses have established mechanisms to achieve major capsid protein (VP1) translation from the genomic RNA. Here, we show for the first time that a member of the genus Vesivirus also has a translation initiation mechanism by which a precursor protein of the VP1 protein is expressed from the genomic RNA. This finding clearly points at a functional role of the calicivirus VP1 capsid protein in early replication, and we provide experimental data supporting this hypothesis.

摘要：Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

摘要：RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins.

摘要：H9N2 avian influenza viruses (AIVs) continuously cross the species barrier to infect mammalians and are repeatedly transmitted to humans, posing a significant threat to public health. Importantly, some H9N2 AIVs were found to cause lethal infection in mice, but little is known about the viral infection dynamics in vivo. To analyze the real-time infection dynamics, we described the generation of a mouse-lethal recombinant H9N2 AIV, an influenza reporter virus (VK627-NanoLuc virus) carrying a NanoLuc gene in the non-structural (NS) segment, which was available for in vivo imaging. Although attenuated for replication in MDCK cells, VK627-NanoLuc virus showed similar pathogenicity and replicative capacity in mice to its parental virus. Bioluminescent imaging of the VK627-NanoLuc virus permitted successive observations of viral infection and replication in infected mice, even following the viral clearance of a sublethal infection. Moreover, VK627-NanoLuc virus was severely restricted by the K627E mutation in PB2, as infected mice showed little weight loss and a low level of bioluminescence. In summary, we have preliminarily established a visualized tool that enables real-time observation of the infection and replication dynamics of H9N2 AIV in mice, which contributes to further understanding the mechanisms underlying the pathogenic enhancement of H9N2 AIV to mice.

摘要：Infection with Junín virus (JUNV) is currently being effectively managed in the endemic region using a combination of targeted vaccination and plasma therapy. However, the long-term sustainability of plasma therapy is unclear and similar resources are not available for other New World arenaviruses. As a result, there has been renewed interest regarding the potential of drug-based therapies. To facilitate work on this issue, we present the establishment and subsequent optimization of a JUNV minigenome system to a degree suitable for high-throughput miniaturization, thereby providing a screening platform focused solely on factors affecting RNA synthesis. Using this tool, we conducted a limited drug library screen and identified AVN-944, a non-competitive inosine monophosphate dehydrogenase (IMPDH) inhibitor, as an inhibitor of arenavirus RNA synthesis. We further developed a transcription and replication competent virus-like particle (trVLP) system based on these minigenomes and used it to screen siRNAs against IMPDH, verifying its role in supporting arenavirus RNA synthesis. The antiviral effect of AVN-944, as well as siRNA inhibition, on JUNV RNA synthesis supports that, despite playing only a minor role in the activity of ribavirin, exclusive IMPDH inhibitors may indeed have significant therapeutic potential for use against New World arenaviruses. Finally, we confirmed that AVN-944 is also active against arenavirus infection in cell culture, supporting the suitability of arenavirus lifecycle modelling systems as tools for the screening and identification, as well as the mechanistic characterization, of novel antiviral compounds.

摘要：Adenoviruses (Ads) are large human-pathogenic double-stranded DNA (dsDNA) viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR) and linear-circular homologous recombination (LCHR). We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS). We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS) of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource.

摘要：Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV.

摘要：The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.