Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication.

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

Characterization of virus‒host recombinant variants of the hepatitis E virus.

Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity. IMPORTANCE: Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.

A novel BSL-2 Lassa virus reverse genetics system modelling the complete viral life cycle.

Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we developed a novel LASV reverse genetics system which, to our knowledge, is the first to study the complete LASV life cycle under BSL-2 conditions. Viral particles can be produced efficiently when LASV minigenomic RNA harbouring minimal viral cis-elements and reporter genes is transfected into a helper cell line stably expressing viral NP, GP, Z and L proteins. The resulting defective virions, named LASVmg, can propagate only in the helper cell line, providing a BSL-2 model to study the complete LASV life cycle. Using this model, we found that a previously reported cellular receptor α-dystroglycan is dispensable for LASVmg infection. Furthermore, we showed that ribavirin can inhibit LASVmg infection by inducing viral mutations. This new BSL-2 system should facilitate studying the LASV life cycle and screening antivirals.

Favipiravir Treatment Prolongs Survival in a Lethal BALB/c Mouse Model of Ebinur Lake Virus Infection.

Orthobunyavirus is the largest and most diverse genus in the family Peribunyaviridae. Orthobunyaviruses are widely distributed globally and pose threats to human and animal health. Ebinur Lake virus (EBIV) is a newly classified Orthobunyavirus detected in China, Russia, and Kenya. This study explored the antiviral effects of two broad-spectrum antiviral drugs, favipiravir and ribavirin, in a BALB/c mouse model. Favipiravir significantly improved the clinical symptoms of infected mice, reduced viral titer and RNA copies in serum, and extended overall survival. The median survival times of mice in the vehicle- and favipiravir-treated groups were 5 and 7 days, respectively. Favipiravir significantly reduced virus titers 10- to 100-fold in sera at all three time points compared to vehicle-treated mice. And favipiravir treatment effectively reduced the virus copies by approximately 10-fold across the three time points, relative to vehicle-treated mice. The findings expand the antiviral spectrum of favipiravir for orthobunyaviruses in vivo.

Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.

A nanobody-mediated drug system against largemouth bass virus delivered by bacterial nanocellulose in Micropterus salmoides.

Diseases caused by pathogens severely hampered the development of aquaculture, especially largemouth bass virus (LMBV) has caused massive mortality and severe economic losses to the culture of largemouth bass (Micropterus salmoides). Considering the environmental hazards and human health, effective and environmentally friendly therapy strategy against LMBV is of vital importance and in pressing need. In the present study, a novel nanobody (NbE4) specific for LMBV was selected from a phage display nanobody library. Immunofluorescence and indirect ELISA showed that NbE4 could recognize LMBV virions and had strong binding capacity, but RT-qPCR evidenced that NBE4 did not render the virus uninfectious. Besides, antiviral drug ribavirin was used to construct a targeted drug system delivered by bacterial nanocellulose (BNC). RT-qPCR revealed that NbE4 could significantly enhance the antiviral activity of ribavirin in vitro and in vivo. The targeted drug delivery system (BNC-Ribavirin-NbE4, BRN) reduced the inflammatory response caused by LMBV infection and improved survival rate (BRN-L, 33.3 %; BRN-M, 46.7 %; BRN-H, 56.7 %)compared with control group (13.3 %), ribavirin group (RBV, 26.7 %) and BNC-ribavirin (BNC-R, 40.0 %), respectively. This research provided an effective antiviral strategy that improved the drug therapeutic effect and thus reduced the dosage.

Identification of Novel Bisamide-Decorated Benzotriazole Derivatives as Anti-Phytopathogenic Virus Agents: Bioactivity Evaluation and Computational Simulation.

As a notorious phytopathogenic virus, the tobacco mosaic virus (TMV) severely reduced the quality of crops worldwide and caused critical constraints on agricultural production. The development of novel virucides is a persuasive strategy to address this predicament. Herein, a series of novel bisamide-decorated benzotriazole derivatives were elaborately prepared and screened. Biological tests implied that the optimized compound 7d possessed the most brilliant antiviral inactive profile (EC50 = 157.6 µg/mL) and apparently surpassed that of commercial ribavirin (EC50 = 442.1 µg/mL) 2.8-fold. The preliminary antiviral mechanism was elaborately investigated via transmission electron microscopy, microscale thermophoresis (MST) determination, RT-qPCR, and Western blot analysis. The results showed that compound 7d blocked the assembly of TMV by binding with coat protein (Kd = 0.7 µM) and suppressed TMV coat protein gene expression and biosynthesis process. Computational simulations indicated that 7d displayed strong H-bonds and pi interactions with TMV coat protein, affording a lower binding energy (ΔGbind = -17.8 kcal/mol) compared with Ribavirin (ΔGbind = -10.7 kcal/mol). Overall, current results present a valuable perception of bisamide decorated benzotriazole derivatives with appreciably virustatic competence and should be profoundly developed as virucidal candidates in agrochemical.

Effects on viral suppression and the early-immune expression of ribavirin against spring viremia of carp virus in vitro.

Spring viremia of carp virus (SVCV) is a globally distributed virus that causes severe clinical symptoms and high mortality in fish belonging to the families Cyprinidae and Siluridae. To protect the host against viral infection, understanding the relatedness between viral susceptibility and antiviral mechanisms must be crucial. Thus, we evaluated the viral suppression efficacy of ribavirin by measuring the transcription levels of viral and immune genes in vitro. The results showed that following ribavirin treatment after SVCV infection (MOI 0.1), ribavirin inhibited SVCV replication in epithelioma papulosum cyprini (EPC) cells and completely inhibited viral gene (G and N) expression at concentrations above 10 µg/mL at 48 h post-infection. Ribavirin does not directly damage SVCV particles but inhibits early viral replication. In the absence of SVCV infection, the immunological dynamics triggered by ribavirin resulted in upregulated pattern recognition receptors and proinflammatory cytokine-related genes (i.e., PI3K, MYD88, IRAK1, RIG-І, MAVS, Mx1, TNF-α, and NF-κB). Furthermore, EPC cells treated with ribavirin following SVCV infection showed upregulation of PI3K, MYD88, IRAK1, RIG-І, TNF-α, and NF-κB genes within 24 h post-SVCV infection, suggesting that ribavirin positively inhibits the SVCV infection in vitro.

Drug repurposing screen identifies vidofludimus calcium and pyrazofurin as novel chemical entities for the development of hepatitis E interventions.

Hepatitis E virus (HEV) infection can cause severe complications and high mortality, particularly in pregnant women, organ transplant recipients, individuals with pre-existing liver disease and immunosuppressed patients. However, there are still unmet needs for treating chronic HEV infections. Herein, we screened a best-in-class drug repurposing library consisting of 262 drugs/compounds. Upon screening, we identified vidofludimus calcium and pyrazofurin as novel anti-HEV entities. Vidofludimus calcium is the next-generation dihydroorotate dehydrogenase (DHODH) inhibitor in the phase 3 pipeline to treat autoimmune diseases or SARS-CoV-2 infection. Pyrazofurin selectively targets uridine monophosphate synthetase (UMPS). Their anti-HEV effects were further investigated in a range of cell culture models and human liver organoids models with wild type HEV strains and ribavirin treatment failure-associated HEV strains. Encouragingly, both drugs exhibited a sizeable therapeutic window against HEV. For instance, the IC50 value of vidofludimus calcium is 4.6-7.6-fold lower than the current therapeutic doses in patients. Mechanistically, their anti-HEV mode of action depends on the blockage of pyrimidine synthesis. Notably, two drugs robustly inhibited ribavirin treatment failure-associated HEV mutants (Y1320H, G1634R). Their combination with IFN-α resulted in synergistic antiviral activity. In conclusion, we identified vidofludimus calcium and pyrazofurin as potent candidates for the treatment of HEV infections. Based on their antiviral potency, and also the favorable safety profile identified in clinical studies, our study supports the initiation of clinical studies to repurpose these drugs for treating chronic hepatitis E.

Discovery of tryptanthrin analogues bearing F and piperazine moieties as novel phytopathogenic antibacterial and antiviral agents.

BACKGROUND: Plant bacterial infections and plant viruses seriously affect the yield and quality of crops. Based on the various activities of tryptanthrin, a series of tryptanthrin analogues bearing F and piperazine moieties were designed, synthesized, and evaluated for their biological activities against three plant bacteria and tobacco mosaic virus (TMV). RESULTS: Bioassay results indicated that compounds 6a-6l displayed excellent antibacterial activities in vitro and 6a-6c and 6g exhibited better antiviral activities against TMV than commercial ribavirin. In particular, 6b showed the most effect on Xanthomonas oryzae pv. oryzae (Xoo) with a half-maximal effective concentration (EC50 ) of 1.26 µg mL-1 , compared with the commercial pesticide bismerthiazol (BT; EC50 = 34.3 µg mL-1 ) and thiodiazole copper (TC; EC50 = 73.3 µg mL-1 ). Meanwhile, 6a also had the best antiviral activity at 500 µg mL-1 for curative, protection, and inactivation purposes, compared with ribavirin in vivo. CONCLUSION: Compound 6b could cause changes in bacterial morphology, induce the accumulation of reactive oxygen species, promote apoptosis of bacterial cells, inhibit the formation of biofilm, and block the growth of Xoo cells. Proteomic analysis revealed major differences in the bacterial secretory system pathways T2SS and T6SS, which inhibited membrane transport. Molecular docking revealed that 6a and 6g could interact with TMV coat protein preventing virus assembly. These results suggest that tryptanthrin analogues bearing F and piperazine moieties could be promising candidate agents for antibacterial and antiviral use in agricultural production. © 2023 Society of Chemical Industry.

Ribavirin attenuates carcinogenesis by downregulating IL-6 and IL-8 in vitro in human lung adenocarcinoma.

Lung cancer is one of the leading causes of death worldwide, whereby the major contributing factors are cigarette smoking and exposure to environmental pollutants. Despite the availability of numerous treatment options, including chemotherapy, the five-year survival rate is still extremely low, highlighting the urgent need to develop novel, more effective therapeutic strategies. In this context, the repurposing of previously approved drugs is an advantage in terms of time and resources invested. Ribavirin is an antiviral drug approved for the treatment of hepatitis C, which shows potential for repurposing as an anticancer agent. Among the many signaling molecules promoting carcinogenesis, the interleukins (ILs) IL-6 and IL-8 are interesting therapeutic targets as they promote a variety of cancer hallmarks such as cell proliferation, migration, metastasis, and angiogenesis. In the present study, we show that ribavirin significantly downregulates the expression of IL-6 and IL-8 in vitro in A549 human lung adenocarcinoma cells. The results of this study shed light on the anticancer mechanisms of ribavirin, providing further proof of its potential as a repurposed drug for the treatment of lung cancer.

Effects of glycine 64 substitutions in RNA-dependent RNA polymerase on ribavirin sensitivity and pathogenicity of coxsackievirus A6.

Hand, foot, and mouth disease (HFMD) caused by a group of enteroviruses is a global public health problem. In recent years, coxsackievirus A6 (CVA6) has emerged as an important HFMD agent. Previous studies have shown that mutations of glycine 64 in RNA-dependent RNA polymerase (3D polymerase), which is central to viral replication, cause phenotypic changes such as ribavirin resistance, increased replication fidelity, and virulence attenuation in poliovirus and enterovirus A71. In this study, we constructed CVA6 mutants with G64R, G64S, and G64T substitutions by site-directed mutagenesis in full-length cDNA of an infectious CVA6 strain cloned in pcDNA3.1. Viral RNA was obtained by in vitro transcription, and the rescued virus strains were propagated in RD cells. Sequencing after six passages revealed that G64S and G64T mutations were stably inherited, whereas G64R was genetically unstable and reversed to the wild type. Comparison of the biological characteristics of the wild-type and mutant CVA6 strains in an in vivo model (one-day-old ICR mice) revealed that the pathogenicity of CVA6-G64S and CVA6-G64T was significantly reduced compared to wild-type CVA6. In vitro experiments indicated the mutant CVA6-G64S and CVA6-G64T strains had increased resistance to 0.8 mM ribavirin and a decreased replication rate in the presence of 0.8 mM guanidine hydrochloride. Our results show that mutation of residue 64 reduces CVA6 susceptibility to ribavirin and increases CVA6 susceptibility to guanidine hydrochloride, together with increased replication fidelity and attenuated viral pathogenicity, thus laying a foundation for the development of safe and effective live attenuated CVA6 vaccine.

In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments.

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible.

Development and Mechanism Investigation of Novel Thioacetalized Indoles as Antiphytoviral Agents.

Potato virus Y (PVY) is a highly destructive pathogen that infects Solanum tuberosumvL., commonly known as potato, a crop that produces one of the most crucial food staples of the world. The PVY viral infection can considerably reduce the yield and quality of potatoes, thereby causing significant economic ramifications. Given the unsatisfactory performance of commercially available antiviral agents against PVY, we synthesized a series of novel indole-derived compounds followed by their bioevaluation and investigation of the mechanisms governing their anti-PVY activity. These indole-based derivatives contain dithioacetal as a key chemical moiety, and most of them exhibit promising anti-PVY activities. In particular, compound B2 displays remarkable in vivo protective and inactivating properties, with half-maximal effective concentration (EC50) values of 209.3 and 113.0 µg/mL, respectively, in stark contrast to commercial agents such as ningnanmycin (EC50 = 281.4 and 136.3 µg/mL, respectively) and ribavirin (EC50 = 744.8 and 655.4 µg/mL, respectively). The mechanism using which B2 enhances plant immune response to protect plants from PVY is elucidated using enzyme activity tests, real-time quantitative polymerase chain reaction (RT-qPCR), and proteomics techniques. This study aims to pave the way for developing candidate pesticides and related molecules using antiphytoviral activity.

Cytokine polymorphisms and genotypic susceptibility of HCV infection in ribavirin response to peg interferon.

Immune responses are largely regulated by cytokines. Genetic polymorphisms of the regulatory coding regions are recognized to impact the expression of cytokines. The abnormal cytokine levels in hepatitis C virus (HCV) infection seems to be involved in disease progression, viral survival, and therapeutic response. The current study assesses the polymorphisms associated with IL-6, IL-10, IL28B, IFN-γ, TGF-ß, and TNF-α on the genotypic susceptibility to HCV infection and Ribavirin response to Peg interferon. Droplet digital polymerase chain reaction (PCR) was used to assess the gene polymorphisms associated with IL-6 A/G (rs2069837), IL-10-1082 G/A (rs1800896)], IL28B C/T (rs12979860), IFN-γ +874 A/T (rs2430561), TGF-ß 1-509 C/T (rs1800469) and TNF-α-308 G/A promoter (rs1800629) from stored samples of 200 healthy individuals and 300 HCV infected patients. There was a significant association of AG and AA genotypes of IL28B, IFN-γ, TGF-ß1, and TNF-α over HCV susceptibility and treatment outcome. However, no association between IL-6 and IL-10 gene polymorphism to HCV susceptibility response to the treatment. The observations indicate IL28B CT, TGF-ß1 CT, TT and TNF- AG with AA genotypes influence the cytokine expression, which is related to susceptibility and resistance to HCV infection and combined antiviral therapy.

In-cell Western assay to quantify infection with pathogenic orthohantavirus Puumala virus in replication kinetics and antiviral drug testing.

Hemorrhagic fever with renal syndrome (HFRS) represents a serious zoonotic disease caused by orthohantaviruses in Eurasia. A specific antiviral therapy is not available. HFRS is characterized by acute kidney injury (AKI) with often massive proteinuria. Infection of kidney cells may contribute to the clinical picture. However, orthohantaviral replication in kidney cells is not well characterized. Therefore, we aimed to perform a reliable high-throughput assay that allows the quantification of infection rates and testing of antiviral compounds in different cell types. We quantified relative infection rates of Eurasian pathogenic Puumala virus (PUUV) by staining of nucleocapsid protein (N protein) in an in-cell Western (ICW) assay. Vero E6 cells, derived from the African green monkey and commonly used in viral cell culture studies, and the human podocyte cell line CIHP (conditionally immortalized human podocytes) were used to test the ICW assay for replication kinetics and antiviral drug testing. Quantification of infection by ICW revealed reliable results for both cell types, as shown by their correlation with immunofluorescence quantification results by counting infected cells. Evaluation of antiviral efficacy of ribavirin by ICW assay revealed differences in the toxicity (TC) and inhibitory concentrations (IC) between Vero E6 cells and podocytes. IC5O of ribavirin in podocytes is about 12-fold lower than in Vero E6 cells. In summary, ICW assay together with relevant human target cells represents an important tool for the study of hantaviral replication and drug testing.

Whole genome sequencing reveals insights into hepatitis E virus genome diversity, and virus compartmentalization in chronic hepatitis E.

BACKGROUND: Hepatitis E virus (HEV) is a leading cause of acute hepatitis and can cause chronic infections in immunocompromised patients. Although HEV infections can be treated with ribavirin, antiviral efficacy is hampered by resistance mutations, normally detected by virus sequencing. OBJECTIVES: High-throughput sequencing (HTS) allows for cost-effective complete viral genome sequencing. This enables the discovery and delineation of new subtypes, and revised the recognition of quasispecies and putative resistance mutations. However, HTS is challenged by factors including low viral load, sample degradation, high host background, and high viral diversity. STUDY DESIGN: We apply complete genome sequencing strategies for HEV, including a targeted enrichment approach. These approaches were used to investigate sequence diversity in HEV RNA-positive animal and human samples and intra-host diversity in a chronically infected patient. RESULTS: Here, we describe the identification of potential novel subtypes in a blood donation (genotype 3) and in an ancient livestock sample (genotype 7). In a chronically infected patient, we successfully investigated intra-host virus diversity, including the presence of ribavirin resistance mutations. Furthermore, we found convincing evidence for HEV compartmentalization, including the central nervous system, in this patient. CONCLUSIONS: Targeted enrichment of viral sequences enables the generation of complete genome sequences from a variety of difficult sample materials. Moreover, it enables the generation of greater sequence coverage allowing more advanced analyses. This is key for a better understanding of virus diversity. Investigation of existing ribavirin resistance, in the context of minorities or compartmentalization, may be critical in treatment strategies of HEV patients.

Elimination of Solanum nigrum ilarvirus 1 and Apple Hammerhead Viroid from Apple Cultivars Using Antivirals Ribavirin, Rimantadine, and Zidovudine.

Apple hammerhead viroid (AHVd) was detected in the apple cultivar 'Sampion' and in mixed infection with Solanum nigrum ilarvirus 1 (SnIV-1) in the cultivars 'Selena' and 'Jonagored Supra', using a high-throughput sequencing method. Experiments were conducted to eliminate both pathogens in apples using meristem tip cultures in combination with the antivirotics ribavirin, rimantadine, and zidovudine. Elimination of both pathogens was verified by repeated RT-PCR and qRT-PCR assays after 7-11 months. Elimination of SnIV-1 from all cultivars was successful with each of the three antivirotics at concentrations of 20, 40, and 80 mg L-1. Elimination of AHVd was also achieved, although less effectively and only with ribavirin in the concentration range of 20-160 mg L-1.

Favipiravir and Ribavirin protect immunocompetent mice from lethal CCHFV infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever (CCHF) in humans with high morbidity and mortality. Currently, there is neither an approved antiviral drug nor a vaccine against CCHFV. In this study, we describe a lethal model of CCHFV infection using a mouse-adapted strain of CCHFV (MA-CCHFV) in adult wild-type male mice. Infected mice developed high viral loads, tissue pathology, and inflammatory immune responses before ultimately succumbing to the infection. We used the model to evaluate the protective efficacy of nucleoside analogs monulpiravir, favipiravir, ribavirin, the antibiotic tigecycline and the corticosteroids dexamethasone and methylprednisolone against lethal CCHFV infection. Tigecycline, monulpiravir and the corticosteroids failed to protect mice from lethal MA-CCHFV infection. In contrast, favipiravir and ribavirin protected animals from clinical disease and death even when treatment was delayed. Despite demonstrating uniform protection, CCHFV RNA persisted in survivors treated with favipiravir and ribavirin. Nevertheless, the study demonstrated the anti-CCHFV efficacy of favipiravir and ribavirin in a model with intact innate immunity and establishes this model for continued development of CCHFV countermeasures.

Establishment of a robust rat hepatitis E virus fecal-oral infection model and validation for antiviral studies.

The hepatitis E virus (HEV) is a major cause of hepatitis, with an estimated 3.3 million symptomatic cases annually. There is no HEV-specific treatment besides the off-label use of ribavirin and a vaccine is only available in China and Pakistan. To aid the development of therapeutic and preventive strategies, there is a need for convenient HEV infection models in small laboratory animals. To this end, we make use of the rat hepatitis E virus. Human infections with this virus have been reported in recent years, making it a relevant pathogen for the establishment of a small animal infection model. We here report that oral gavage of a feces suspension, containing a pre-defined viral RNA load, results in a reproducible synchronized infection in athymic nude rats. This route of administration mimics fecal-oral transmission in a standardized fashion. The suitability of the model to study the effect of antiviral drugs was assessed by using ribavirin, which significantly reduced viral loads in the feces, liver, and other tissues.