SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

Production of infectious reporter murine norovirus by VP2 trans-complementation.

Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics.IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans, indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.

A reporter Oropouche virus expressing ZsGreen from the M segment enables pathogenesis studies in mice.

Oropouche fever caused by Oropouche virus (OROV) is a significant zoonosis in Central and South America. Despite its public health significance, we lack high-throughput diagnostics, therapeutics, and a comprehensive knowledge of OROV biology. Reporter viruses are valuable tools to rapidly study virus dynamics and develop neutralization and antiviral screening assays. OROV is a tri-segmented bunyavirus, which makes generating a reporter virus challenging, as introducing foreign elements into the viral genome typically affects fitness. We previously demonstrated that the non-structural gene NSm on the OROV medium (M) segment is non-essential for replication in vitro. Taking advantage of this, we have now generated a recombinant OROV expressing fluorescent protein ZsGreen in place of NSm. This reporter OROV is both stable and pathogenic in IFNAR-/- mice and provides a powerful tool for OROV pathogenesis studies and assay development.IMPORTANCEEmerging and reemerging infectious agents such as zoonotic bunyaviruses are of global health concern. Oropouche virus (OROV) causes recurring outbreaks of acute febrile illness in the Central and South American human populations. Biting midges are the primary transmission vectors, whereas sloths and non-human primates are their reservoir hosts. As global temperatures increase, we will likely see an expansion in arthropod-borne pathogens such as OROV. Therefore, developing reagents to study pathogen biology to aid in identifying druggable targets is essential. Here, we demonstrate the feasibility and use of a fluorescent OROV reporter in mice to study viral dynamics and pathogenesis. We show that this reporter OROV maintains characteristics such as growth and pathogenicity similar to the wild-type virus. Using this reporter virus, we can now develop methods to assist OROV studies and establish various high-throughput assays.

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.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Development of a HiBiT-tagged reporter hepatitis E virus and its utility as an antiviral drug screening platform.

Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/ΔORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/ΔORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for in vitro assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents.

A reporter virus particle seroneutralization assay for tick-borne encephalitis virus overcomes ELISA limitations.

Tick-borne encephalitis (TBE) virus is the most prevalent tick-transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)-based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. The RVP-based SNT for TBEV exhibited a highly significant correlation with the traditional virus-based SNT (R2 = 0.8637, p < 0.0001). The RVP-based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%-97.4%) and specificity of 100% (95% CI: 81.6%-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV-positive by ELISA but negative by RVP-based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP-based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.

Novel antiviral discoveries for Japanese encephalitis virus infections through reporter virus-based high-throughput screening.

Japanese encephalitis (JE) caused by JE virus (JEV), remains a global public health concern. Currently, there is no specific antiviral drug approved for the treatment of JE. While vaccines are available for prevention, they may not cover all at-risk populations. This underscores the urgent need for prophylaxis and potent anti-JEV drugs. In this context, a high-content JEV reporter system expressing Nanoluciferase (Nluc) was developed and utilized for a high-throughput screening (HTS) of a commercial antiviral library to identify potential JEV drug candidates. Remarkably, this screening process led to the discovery of five drugs with outstanding antiviral activity. Further mechanism of action analysis revealed that cepharanthine, an old clinically approved drug, directly inhibited virus replication by blocking GTP binding to the JEV RNA-dependent RNA polymerase. Additionally, treatment with cepharanthine in mice models alleviated JEV infection. These findings warrant further investigation into the potential anti-JEV activity of cepharanthine as a new therapeutic approach for the treatment of JEV infection. The HTS method employed here proves to be an accurate and convenient approach that facilitates the rapid development of antiviral drugs.

Advances and Challenges in Molecular Imaging of Viral Infections.

Molecular imaging of viral infection, using a variety of advanced imaging techniques such as optical and nuclear imaging, can and has been used for direct visualization of the virus as well as assessment of virus-host interactions. Unlike imaging of other pathogens such as bacteria and fungi, challenging aspects of imaging viral infections include the small size of viruses, the complexity of viral infection animal models (eg, species dependence), and the high-level containment needs for many high-consequence pathogens, among others. In this review, using representative viral infections, we discuss how molecular imaging can reveal real-time infection dynamics, improve our understanding of disease pathogenesis, and guide optimization of treatment and prevention strategies. Key findings from human and animal studies are highlighted.

In Vivo Profiling of Individual Multiciliated Cells during Acute Influenza A Virus Infection.

Influenza virus infections are thought to be initiated in a small number of cells; however, the heterogeneity across the cellular responses of the epithelial cells during establishment of disease is incompletely understood. Here, we used an H1N1 influenza virus encoding a fluorescent reporter gene, a cell lineage-labeling transgenic mouse line, and single-cell RNA sequencing to explore the range of responses in a susceptible epithelial cell population during an acute influenza A virus (IAV) infection. Focusing on multiciliated cells, we identified a subpopulation that basally expresses interferon-stimulated genes (ISGs), which we hypothesize may be important for the early response to infection. We subsequently found that a population of infected ciliated cells produce most of the ciliated cell-derived inflammatory cytokines, and nearly all bystander ciliated cells induce a broadly antiviral state. From these data together, we propose that variable preexisting gene expression patterns in the initial cells targeted by the virus may ultimately affect the establishment of viral disease. IMPORTANCE Influenza A virus poses a significant threat to public health, and each year, millions of people in the United States alone are exposed to the virus. We do not currently, however, fully understand why some individuals clear the infection asymptomatically and others become severely ill. Understanding how these divergent phenotypes arise could eventually be leveraged to design therapeutics that prevent severe disease. As a first step toward understanding these different infection states, we used a technology that allowed us to determine how thousands of individual murine lung epithelial cells behaved before and during IAV infection. We found that small subsets of epithelial cells exhibited an antiviral state prior to infection, and similarly, some cells made high levels of inflammatory cytokines during infection. We propose that different ratios of these individual cellular responses may contribute to the broader antiviral state of the lung and may ultimately affect disease severity.

Development of Recombinant Infectious Hepatitis E Virus Harboring the nanoKAZ Gene and Its Application in Drug Screening.

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. IMPORTANCE Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.

Development of an HiBiT-tagged reporter H3N2 influenza A virus and its utility as an antiviral screening platform.

The balance of the segmented genome derived from naturally occurring influenza A viruses (IAVs) is delicate and vulnerable to foreign insertions, thus most reporter IAVs up to date are generated using the backbone of the laboratory-adapted strains. In this study, we constructed a reporter influenza A/H3N2 virus (A/NY-HiBiT) which was derived from a clinical isolate, by placing a minimized HiBiT tag to the N-terminus of the viral nuclear-export protein (NEP). Here, we show that this 11-amino acid HiBiT tag did not adversely impact the viral genome balance, and the recombinant A/NY-HiBiT virus maintains its relative stability. Moreover, the replication profile of the HiBiT-tagged virus can be measured by a simple Nano-Glo assay, providing a robust high-throughput screening (THS) platform. We used this platform to evaluate a collection of the pre-purified fractions which were derived from rare Chinese medicinal materials, and we identified three fractions, including wild Trametes robiniophila (50% methanol fraction), Ganoderma (water fraction), and wild Phellinus igniarius (ethyl acetate fraction), as potent anti-IAV actives. Our results demonstrate that this IAV reporter can be used as a powerful HTS platform for antiviral development.

A Recombinant Genotype I Japanese Encephalitis Virus Expressing a Gaussia Luciferase Gene for Antiviral Drug Screening Assay and Neutralizing Antibodies Detection.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in humans throughout Asia. In the past twenty years, the emergence of the genotype I (GI) JEV as the dominant genotype in Asian countries has raised a significant threat to public health security. However, no clinically approved drug is available for the specific treatment of JEV infection, and the commercial vaccines derived from the genotype III JEV strains merely provided partial protection against the GI JEV. Thus, an easy-to-perform platform in high-throughput is urgently needed for the antiviral drug screening and assessment of neutralizing antibodies specific against the GI JEV. In this study, we established a reverse genetics system for the GI JEV strain (YZ-1) using a homologous recombination strategy. Using this reverse genetic system, a gaussia luciferase (Gluc) expression cassette was inserted into the JEV genome to generate a reporter virus (rGI-Gluc). The reporter virus exhibited similar growth kinetics to the parental virus and remained genetically stable for at least ten passages in vitro. Of note, the bioluminescence signal strength of Gluc in the culture supernatants was well correlated with the viral progenies determined by viral titration. Taking advantage of this reporter virus, we established Gluc readout-based assays for antiviral drug screening and neutralizing antibody detection against the GI JEV. These Gluc readout-based assays exhibited comparable performance to the assays using an actual virus and are less time consuming and are applicable for a high-throughput format. Taken together, we generated a GI JEV reporter virus expressing a Gluc gene that could be a valuable tool for an antiviral drug screening assay and neutralization assay.

Development of Reverse Genetics for the Prototype New World Mammarenavirus Tacaribe Virus.

The New World mammarenavirus Tacaribe virus (TCRV) has been isolated from fruit bats, mosquitoes, and ticks, whereas all other known New World mammarenaviruses are maintained in rodents. TCRV has not been linked to human disease, but it has been shown to protect against Argentine hemorrhagic fever-like disease in marmosets infected with the New World mammarenavirus Junín virus (JUNV), indicating the potential of TCRV as a live-attenuated vaccine for the treatment of Argentine hemorrhagic fever. Implementation of TCRV as a live-attenuated vaccine or a vaccine vector would be facilitated by the establishment of reverse genetics systems for the genetic manipulation of the TCRV genome. In this study, we developed, for the first time, reverse genetics approaches for the generation of recombinant TCRV (rTCRV). We successfully rescued a wild-type (WT) rTCRV (a trisegmented form of TCRV expressing two reporter genes [r3TCRV]) and a bisegmented TCRV expressing a single reporter gene from a bicistronic viral mRNA (rTCRV/GFP). These reverse genetics approaches represent an excellent tool to investigate the biology of TCRV and to explore its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of other viral infections. Notably, we identified a 39-nucleotide (nt) deletion (Δ39) in the noncoding intergenic region (IGR) of the viral large (L) segment that is required for optimal virus multiplication. Accordingly, an rTCRV containing this 39-nt deletion in the L-IGR (rTCRV/Δ39) exhibited decreased viral fitness in cultured cells, suggesting the feasibility of using this deletion in the L-IGR as an approach to attenuate TCRV, and potentially other mammarenaviruses, for their implementation as live-attenuated vaccines or vaccine vectors.IMPORTANCE To date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever caused by mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin, which is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is nonpathogenic in humans but able to provide protection against Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever, demonstrating the feasibility of using TCRV as a live-attenuated vaccine vector for the treatment of JUNV and potentially other viral infections. Here, we describe for the first time the feasibility of generating recombinant TCRV (rTCRV) using reverse genetics approaches, which paves the way to study the biology of TCRV and also its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of mammarenavirus and/or other viral infections in humans.

Infectious recombinant Senecavirus A expressing novel reporter proteins.

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. The construction of SVA virus carrying foreign reporter gene provides a powerful tool in virus research. However, it is often fraught with rescuing a recombinant picornavirus harboring a foreign gene or maintaining the stability of foreign gene in the virus genome. Here, we successfully generated recombinant SVA GD05/2017 viruses (V-GD05-clone) expressing the green fluorescent protein (iLOV), red fluorescent protein (RFP), or NanoLuc luciferase (Nluc). These recombinant viruses have comparable growth kinetics to the parental virus. Genetic stability analysis indicated that V-GD05-iLOV was highly stable in retaining iLOV gene for more than 10 passages, while V-GD05-RFP and V-GD05-Nluc lost the foreign genes in five passages. In addition, high-intensity fluorescent signals were found in the V-GD05-RFP- and V-GD05-iLOV-infected cells by fluorescence observation and flow cytometry analysis, and the luciferase activity assay could quantitatively monitor the replication of V-GD05-Nluc. In order to identify the porcine cell receptor for SVA, anthrax toxin receptor 1 (ANTXR1) was knocked out or overexpressed in the ST-R cells. The ANTXR1 knock-out cells lost the ability for SVA infection, while overexpression of ANTXR1 significantly increased the cell permissivity. These results confirmed that ANTXR1 was the receptor for SVA to invade porcine cells as reported in the human cells. Overall, this study suggests that these SVA reporter viruses will be useful tools in elucidating virus pathogenesis and developing control measures. KEY POINTS: • We successfully generated SVA viruses expressing the iLOV, RFP, or Nluc. • The iLOV was genetically stable in the V-GD05-iLOV genome over ten passages. • ANTXR1 was the receptor for SVA to invade porcine cells.

Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination.

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.

Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release.

BACKGROUND: The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. RESULTS: The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. CONCLUSION: These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small molecules that promote or inhibit HIV-1 particle production.

Development of Stable Rotavirus Reporter Expression Systems.

Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5'- and 3'-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens).IMPORTANCE Development of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5'- and 3'-terminal sequences in terms of genome replication, assembly, and packaging.

In Vivo Dynamics of Reporter Flaviviridae Viruses.

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.