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.

摘要：A serum neutralization test (SNT) is an essential method for the serological diagnosis of pestivirus infections, including classical swine fever, because of the cross reactivity of antibodies against pestiviruses and the non-quantitative properties of antibodies in an enzyme-linked immunosorbent assay. In conventional SNTs, an immunoperoxidase assay or observation of cytopathic effect after incubation for 3 to 7 days is needed to determine the SNT titer, which requires labor-intensive or time-consuming procedures. Therefore, a new SNT, based on the luciferase system and using classical swine fever virus, bovine viral diarrhea virus, and border disease virus possessing the 11-amino-acid subunit derived from NanoLuc luciferase was developed and evaluated; this approach enabled the rapid and easy determination of the SNT titer using a luminometer. In the new method, SNT titers can be determined tentatively at 2 days post-infection (dpi) and are comparable to those obtained by conventional SNTs at 3 or 4 dpi. In conclusion, the luciferase-based SNT can replace conventional SNTs as a high-throughput antibody test for pestivirus infections.

摘要：Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for the global pork industry, characterized for its genetic variation and unsatisfied heterological protection from vaccines. A high-throughput screening platform for developing anti-PRRSV therapies is urgently needed. Here, an 11-amino-acid subunit HiBiT derived from NanoLuc luciferase was inserted into the PRRSV genome at four loci of the Nsp2 coding region or as an additional TRS2 driven open reading frame (ORF) between the ORF7 and 3'-untranscribed region (3'-UTR), respectively, and five recombinant viruses with luciferase activity were successfully rescued. The virological characteristics of the representative virus RvJX-Nsp2325-HiBiT were investigated. In vitro, it displayed similar growth kinetics as the parental virus and keeps the luciferase activity and genetic stability after eight rounds of serial passages. The concept-proof test confirmed that RvJX-Nsp2325-HiBiT can be easily used to evaluate the efficacy of antiviral reagents by detecting the reduction of luciferase activity, showing a consistent trend with infectious titers, as well as to set a novel convenient virus neutralization assay based on the intensity of luciferase activity. Last, the viral proliferation, virulence, validity, and HiBiT stability were further investigated in pig inoculation study, showing that the luciferase activity can be directly detected in the tissue samples or indirectly from the MARC-145 cells inoculated with sera from RvJX-Nsp2325-HiBiT-inoculated pigs. Taken together, the results indicate that the HiBiT-tagged virus is a convenient and stable tool for evaluating viral propagation both in vitro and in vivo, which can provide a high-efficient platform for screening and evaluating anti-PRRSV therapies. IMPORTANCE Luciferase reporter tagged virus is crucial to viral quantification in the study of viral replication, pathogenesis and exploring antiviral reagents. It is urgently needed for PRRSV academia to construct a stable, fast, and high-throughput reporting system, which can be used both in vitro and in vivo. Here, an 11-amino-acid luciferase subunit was successfully inserted into the PRRSV genome; the feasibility, genetic stability, and efficiency for viral quantification both in vitro and in vivo were characterized; and the results demonstrated it has greatly improved the convenience and efficiency for screening the anti-PRRSV reagents. Furthermore, a novel luciferase-based virus neutralization assay was successfully set, which can eliminate the step of sample gradient dilution and greatly improve the convenience and throughput of neutralizing antibody testing. Predictably, it will greatly facilitate the screening and evaluating anti-PRRSV therapies, as well as the mechanistic study of its replication and pathogenesis in the future.

摘要：Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

摘要：Coronaviruses (CoVs) are enveloped (+) ssRNA viruses of veterinary and medical importance. Because recombinant CoVs with reporter proteins fused with viral proteins are usually non-viable or unstable, a small and quantifiable epitope tag would be beneficial to CoV research. In this study, we integrated the NanoLuc Binary Technology to the reverse genetics of infectious bronchitis virus (IBV), a prototypic gammacoronavirus. The 11-amino-acid HiBiT tag was inserted to the spike (S) or membrane (M) protein, and the recombinant IBVs (rS-HiBiT and rM-HiBiT) were characterized. Compared with the rIBV-p65 control, rS-HiBiT exhibited comparable growth kinetics, whereas rM-HiBiT replicated slightly slower. The levels of HiBiT-tagged S and M proteins in the infected cells or the culture supernatant could be both rapidly (~15 min) and efficiently (30 μL sample volume) determined using the HiBiT luminescence assay. Notably, replication of the HiBiT-tagged IBV could be monitored continuously in an infected chicken embryo, and rS-HiBiT was genetically stable for at least 20 passages. By integrating the HiBiT tagging system with CoV reverse genetics, this new reporter system may facilitate future study of CoV replication and pathogenesis.

摘要：In studies of HIV-1, virus production is normally monitored by either a reverse transcriptase assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. For example, sample dilution is always required in the ELISA procedure to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. This peptide is a fragment of NanoLuc luciferase and generates a strong luminescent signal when complemented with the remaining subunit. To employ this technology, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the resultant virus production, infectivity, or susceptibility to an integrase inhibitor. EM revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1/lentivirus production. This system can be widely applied to a variety of virological studies, along with screening for candidates of future antiviral drugs.

摘要：For the development of antiviral agents to eliminate hepatitis B virus (HBV), it is essential to establish an HBV cell culture system that can easily monitor HBV infection. Here, we created a novel HBV infection monitoring system using a luminescent 11-amino acid reporter, the high-affinity subunit of nano-luciferase binary technology (HiBiT). The HiBiT-coding sequence was inserted at the N-terminus of preS1 in a 1.2-fold plasmid encoding a genotype C HBV genome. After transfection of HepG2 cells with this HiBiT-containing plasmid, the supernatant was used to prepare a recombinant cell culture-derived virus (HiBiT-HBVcc). Primary human hepatocytes (PXB) were inoculated with HiBiT-HBVcc. Following inoculation, intracellular and extracellular HiBiT activity and the levels of various HBV markers were determined. Reinfection of naive PXB cells with HiBiT-HBVcc prepared from HiBiT-HBVcc-infected PXB cells was analyzed. When PXB cells were infected with HiBiT-HBVcc at several titers, extracellular HiBiT activity was detected in a viral titer-dependent manner and was correlated with intracellular HiBiT activity. Inhibitors of HBV entry or replication suppressed extracellular HiBiT activity. Viral DNA, RNA, and proteins were detectable, including covalently closed circular DNA, by Southern blot analysis. The synthesis of relaxed-circular DNA from single-stranded DNA in HiBiT-HBV decreased to one third of that of wild-type HBV, and the infectivity of HiBiT-HBVcc decreased to one tenth of that of wild-type HBVcc. HiBiT-HBVcc prepared from PXB cells harboring HiBiT-HBV was able to infect naive PXB cells. Conclusions: Recombinant HiBiT-HBV can undergo the entire viral life cycle, thus facilitating high-throughput screening for HBV infection in vitro using supernatants. This system will be a powerful tool for developing antiviral agents.