当前期刊: Antiviral Research Go to current issue    加入关注   
显示样式:        排序: 导出
  • Identification and structural characterization of small molecule fragments targeting Zika virus NS2B-NS3 protease
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-15
    Jun Ping Quek; Shuang Liu; Zhenzhen Zhang; Yan Li; Elizabeth Yihui Ng; Ying Ru Loh; Alvin W. Hung; Dahai Luo; CongBao Kang

    Zika virus (ZIKV) NS2B-NS3 protease is a validated antiviral target as it is essential for maturation of viral proteins. However, its negatively charged active site hinders the development of orthosteric small-molecule inhibitors. Fragment-based drug discovery (FBDD) is a powerful tool to generate novel chemical starting points against difficult drug targets. In this study, we screened a fragment compound library against the Zika protease using a primary thermal shift assay and identified twenty-two fragments which (bind to and) stabilize the protease. We then determined the X-ray crystal structures of two hits from different classes, all of which bind to the S1 pocket next to the protease active site. We confirmed that these two fragments bind to the protease without inducing significant conformational changes using solution NMR spectroscopy. These fragment scaffolds serve as the starting point for subsequent lead compound development.

  • Advances in the genotypic diagnosis of cytomegalovirus antiviral drug resistance
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-12
    Sunwen Chou

    Cytomegalovirus (CMV) drug resistance mutation maps are updated with recent information for polymerase inhibitors, the terminase inhibitor letermovir and the UL97 kinase inhibitor maribavir. Newly mapped mutations and their phenotypes provide more detail on cross-resistance properties and suggest the need to expand the CMV gene regions covered in diagnostic testing. Next-generation deep sequencing technology offers a more sensitive, higher resolution view of emerging antiviral resistance and is recommended for use in clinical trials. Issues of standardization and diagnostic utility in comparison with traditional Sanger sequencing remain unresolved. Quality control is important for the accurate and reproducible detection of mutant viral populations in clinical specimens.

  • The discovery and development of filociclovir for the prevention and treatment of human cytomegalovirus-related disease
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-12
    Islam T.M. Hussein; Jennifer Brooks; Terry L. Bowlin

    Human cytomegalovirus (HCMV) infections are widespread among the human population. Infection is persistent and mostly asymptomatic, except in immunocompromised individuals, particularly transplant patients, where significant morbidity and mortality can occur. Currently approved drugs for treating HCMV-related disease [including ganciclovir (GCV), valganciclovir (VGCV), cidofovir (CDV) and foscarnet (FOS)] all target the viral DNA polymerase and suffer from dose-limiting toxicity and resistance issues. The most recently approved drug, letermovir (LMV), was approved only for prophylaxis in adult HCMV-seropositive stem cell transplant recipients. Although LMV is highly potent, high-grade resistance mutations in the terminase gene were shown to readily emerge in vitro and in treated patients. Therefore, there is a need for new drugs that can be used for combinatorial therapeutic and/or prophylactic regimens to counteract the emergence of resistant mutants. Filociclovir (FCV), also known as cyclopropavir or MBX-400, is a methylenecyclopropane nucleoside analog, which has successfully completed Phase I safety studies, and is now entering Phase II clinical efficacy studies for the treatment of HCMV-related disease in transplant patients. FCV is 10-fold more active than GCV against HCMV in vitro, and has activity against all human herpesviruses except HSV-1 and HSV-2. Recently, FCV was also shown to be highly potent against human adenoviruses. This activity spectrum suggests that FCV could be used to treat/prevent infection with several viruses that pose significant risk to transplant patients. The active triphosphate form of FCV (FCV-TP) reaches higher peak levels than GCV-TP in HCMV-infected cells, and exhibits about 10-fold higher affinity to HCMV DNA polymerase UL54. Furthermore, FCV was shown to retain activity against a panel of GCV-resistant HCMV isolates, suggesting that it could be a useful alternative therapy for treating patients infected with some GCV-resistant HCMV strains. This review summarizes the early discovery work of FCV and highlights the recent advances in the continued development of this clinical candidate.

  • Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-12
    Eunji Jo; Dong-Kyun Ryu; Alexander König; Soonju Park; Yoojin Cho; Sang-Hyun Park; Tae-Hee Kim; Seung Kew Yoon; Wang-Shick Ryu; Jonathan Cechetto; Marc P. Windisch

    Currently, therapies to treat chronic hepatitis B (CHB) infection are based on the use of interferon-α or nucleos(t)ide analogs (NAs) to prevent viral DNA synthesis by inhibiting the reverse transcriptase activity of the hepatitis B virus (HBV) polymerase (Pol). However, these therapies are not curative; thus, the development of novel anti-HBV agents is needed. In accordance with this unmet medical need, we devised a new target- and cell-based, high-throughput screening assay to identify novel small molecules that block the initial interaction of the HBV Pol with its replication template the viral pregenomic RNA (pgRNA). We screened approximately 110,000 small molecules for the ability to prevent HBV Pol recognition of the pgRNA 5′ epsilon (ε) stem-loop structure, identifying (Z)-2-(allylamino)-4-amino-N′-cyanothiazole-5-carboximidamide (AACC). Viral nucleocapsid-captured quantitative RT-PCR and Western blot results revealed that AACC significantly decreased encapsidated pgRNA levels and blocked capsid assembly without affecting core protein expression in stable HBV-replicating cells. As a result, both intra- and extracellular accumulation of viral DNA was strongly reduced. AACC treatment of HepG2-sodium taurocholate transporting polypeptide (NTCP) cells and primary human hepatocytes infected with cell culture- or patient-derived HBV isolates showed both time- and dose-dependent inhibition of infectious viral progeny and rcDNA production. Furthermore, AACC showed cross-genotypic activity against genotypes B, C, and D. Of note, AACC inhibited the viral replication of lamivudine and a capsid inhibitor-resistant HBV, and showed synergistic effects with NAs and a capsid inhibitor. In conclusion, we identified a novel class of compounds specifically targeting the ε-Pol interaction and thereby preventing the encapsidation of pgRNAs into viral capsids. This promising new HBV inhibitor class potently inhibits HBV amplification with distinct characteristics from existing NAs and other drugs currently under development, promising to add value to existing therapies for CHB.

  • Potent in vitro activity of β-D-4ʹ-chloromethyl-2ʹ-deoxy-2ʹ-fluorocytidine against Nipah virus
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-11
    Michael K. Lo; Franck Amblard; Mike Flint; Payel Chatterjee; Mahesh Kasthuri; Chengwei Li; Olivia Russell; Kiran Verma; Leda Bassit; Raymond F. Schinazi; Stuart T. Nichol; Christina F. Spiropoulou

    Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that continues to cause outbreaks in humans characterized by high mortality and significant clinical sequelae in survivors. Currently, no therapeutics are approved for use in humans against NiV infection. Here, we report that 4ʹ-chloromethyl-2ʹ-deoxy-2ʹ-fluorocytidine (ALS-8112) inhibits NiV. ALS-8112 is the parent nucleoside of lumicitabine, which has been evaluated in phase I and II clinical trials to treat pediatric and adult respiratory syncytial virus infection. In this study, we tested ALS-8112 against NiV and other major human respiratory pneumo- and paramyxoviruses in 2 human lung epithelial cell lines, and demonstrated the ability of ALS-8112 to reduce infectious wild-type NiV yield by over 6 orders of magnitude with no apparent cytotoxicity. However, further cytotoxicity testing in primary cells and bone marrow progenitor cells indicated cytotoxicity at higher concentrations of ALS-8112. Our results warrant the evaluation of lumicitabine against NiV infection in relevant animal models.

  • Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-10
    Filippo Dragoni; Adele Boccuto; Francesca Picarazzi; Alessia Giannini; Federica Giammarino; Francesco Saladini; Mattia Mori; Eloise Mastrangelo; Maurizio Zazzi; Ilaria Vicenti

    Sofosbuvir, a licensed nucleotide analog targeting hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), has been recently evaluated as a broad anti-Flavivirus lead candidate revealing activity against Zika and Dengue viruses both in vitro and in animal models. In this study, the in vitro antiviral activity of sofosbuvir against West Nile virus (WNV) was determined by plaque assay (PA) and Immunodetection Assay (IA) in human cell lines and by enzymatic RdRp assay. By PA, the sofosbuvir half-maximal inhibitory concentration (IC50) was 1.2 ± 0.3 μM in Huh-7, 5.3 ± 0.9 μM in U87, 7.8 ± 2.5 μM in LN-18 and 63.4 ± 14.1 μM in A549 cells. By IA, anti-WNV activity was confirmed in both hepatic (Huh-7, 1.7 ± 0.5 μM) and neuronal (U87, 7.3 ± 2.0 μM) cell types. Sofosbuvir was confirmed to inhibit the purified WNV RdRp (IC50 11.1 ± 4.6 μM). In vitro resistance selection experiments were performed by propagating WNV in the Huh-7 cell line with two-fold increasing concentrations of sofosbuvir. At 80 μM, a significantly longer time for viral breakthrough was observed compared with lower concentrations (18 vs. 7–9 days post infection; p = 0.029), along with the detection of the S604T mutation, corresponding to the well-known S282T substitution in the motif B of HCV NS5B, which confers resistance to sofosbuvir. Molecular docking experiments confirmed that the S604T mutation within the catalytic site of RdRp affected the binding mode of sofosbuvir. To our knowledge, this is the first report of the antiviral activity of sofosbuvir against WNV as well as of selection of mutants in vitro.

  • Comparison of broad-spectrum antiviral activities of the synthetic rocaglate CR-31-B (−) and the eIF4A-inhibitor Silvestrol
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-10
    Christin Müller; Wiebke Obermann; Falk W. Schulte; Kerstin Lange-Grünweller; Lisa Oestereich; Fabian Elgner; Mirco Glitscher; Eberhard Hildt; Kamini Singh; Wendel Hans-Guido; Roland K. Hartmann; John Ziebuhr; Arnold Grünweller
  • The use of mice lacking type I or both type I and type II interferon responses in research on hemorrhagic fever viruses. Part 1: Potential effects on adaptive immunity and response to vaccination
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-10
    Elizabeth C. Clarke; Steven B. Bradfute

    For more than 20 years, researchers have used laboratory mice lacking type I or both type I and type II interferon (IFN) responses to study viruses that cause hemorrhagic fever (HF) in humans. Whereas immunocompetent mice do not become ill when infected with Ebola, Lassa, dengue and other HF viruses, IFN-deficient mice typically develop severe or fatal disease when inoculated with these pathogens. The ease of employment of these “mouse models” has led to their extensive use in biocontainment laboratories to assess the efficacy of novel vaccines, often without consideration of whether adaptive immune responses in IFN-deficient mice accurately mirror those in humans. Failure to consider these questions may lead to inappropriate expectations of the predictive value of mouse experiments. In two invited articles, we investigate this question. This paper examines how the lack of type I or both type I and type II IFN signaling may affect the development of adaptive immune responses in mice and the outcome of vaccine studies. A second article reviews the published literature on the use of IFN-deficient mice for the assessment of novel vaccines against HF viruses.

  • MCPIP1 inhibits Hepatitis B virus replication by destabilizing viral RNA and negatively regulates the virus-induced innate inflammatory responses
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-09
    Min Li; Jie Yang; Yinxia Zhao; Yahui Song; Shengxia Yin; Jing Guo; Hongkai Zhang; Kezhen Wang; Lin Wei; Shuijun Li; Wei Xu

    Monocyte chemotactic protein-induced protein 1 (MCPIP1) is an inflammatory regulator in immune response. Recently, MCPIP1 has also been identified as a host antiviral factor against certain virus infection including human immunodeficiency virus, dengue virus and hepatitis C virus. However, whether MCPIP1 could restrict the replication of hepatitis B virus (HBV), a DNA pararetrovirus belonging to Hepadnaviridae family, has not been investigated. In this study, we found that MCPIP1 expression was up-regulated in mouse livers upon acute HBV replication and in HBV-replicated hepatoma cells or HBV-stimulated macrophages. Enforced MCPIP1 expression by hydrodynamic DNA injection in vivo significantly inhibited HBV replication in the mouse livers. Then in vitro studies by overexpression or knockdown assays in cell-lines identified the direct antiviral effect of MCPIP1 on HBV replication. RNA immunoprecipitation and decay assay further suggested that MCPIP1 potently restricted HBV replication through directly binding viral RNA and degrading RNA via its RNase activity, but not deubiquitinase activity. Moreover, we further verified that MCPIP1 negatively regulated HBV-induced proinflammatory cytokines, such as IL-1β, TNF-α and IL-6 in macrophages. Taken together, our data expand MCPIP1's range of viral targets to DNA virus and also demonstrate the negative regulatory role of MCPIP1 in suppressing virus-induced inflammatory response, suggesting MCPIP1 as a potential therapeutic target for treating HBV-related diseases via inducing a host defense against HBV and reducing inflammatory injury meanwhile.

  • Deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells
    Antivir. Res. (IF 4.130) Pub Date : 2020-01-07
    Weibo Liao; Xin Liu; Quanlue Yang; Huifang Liu; Bingyu Liang; Junjun Jiang; Jiegang Huang; Chuanyi Ning; Ning Zang; Bo Zhou; Yanyan Liao; Jingzhao Chen; Li Tian; Wenzhe Ho; Abu S. Abdullah; Lingbao Kong; Hao Liang; Hui Chen; Li Ye

    Aims Deguelin, a natural compound derived from Mundulea sericea (Leguminosae) and some other plants exhibits an activity to inhibit autophagy, a cellular machinery required for hepatitis C virus (HCV) replication. This study aimed to illuminate the impact of deguelin on HCV replication and mechanism(s) involved. Methods HCV JFH-1-Huh7 infectious system was used for the investigation. Real time RT-PCR, Western blot, fluorescent microscopy assay were used to measure the expression levels of viral or cellular factors. Overexpression and silencing expression techniques were used to determine the role of key cellular factors. Results Deguelin treatment of Huh7 cells significantly inhibited HCV JFH-1 replication in a dose- and time-dependent manner. Deguelin treatment suppressed autophagy in Huh7 cells, evidenced by the decrease of LC3B-II levels, the conversion of LC3B–I to LC3B-II, and the formation of GFP-LC3 puncta as well as the increase of p62 level in deguelin-treated cells compared with control cells. HCV infection could induce autophagy which was also suppressed by deguelin treatment. Mechanism research reveals that deguelin inhibited expression of Beclin1, which is a key cellular factor for the initiation of the autophagosome formation in autophagy. Overexpression or silencing expression of Beclin1 in deguelin-treated Huh7 cells could weaken or enhance the inhibitory effect on autophagy by deguelin, respectively, and thus partially recover or further inhibit HCV replication correspondingly. Conclusions: Deguelin may serve as a novel anti-HCV compound via its inhibitory effect on autophagy, which warrants further investigation as a potential therapeutic agent for HCV infection.

  • N-Acetyl cysteine effectively alleviates Coxsackievirus B-Induced myocarditis through suppressing viral replication and inflammatory response
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-26
    Yao Wang; Shuoxuan Zhao; Yang Chen; Ying Wang; Tianying Wang; Xiaoman Wo; Yanyan Dong; Jian Zhang; Weizhen Xu; Cong Qu; Xiaofeng Feng; Xiaoyu Wu; Yan Wang; Zhaohua Zhong; Wenran Zhao

    Viral myocarditis caused by Coxsackievirus B (CVB) infection is a severe inflammatory disease of the myocardium, which may develop to cardiomyopathy and heart failure. No effective specific treatment is available presently. Our previous study demonstrated that suppression of proinflammatory caspase-1 activation effectively inhibited CVB replication. N-acetyl cysteine (NAC) is a widely used antioxidant. In this study, we found that NAC significantly alleviated the myocardial injury caused by CVB type 3 (CVB3) under in vivo condition. Importantly, NAC treatment simultaneously suppressed viral replication and inflammatory response in both myocardium and cell culture. The antiviral and anti-inflammation mechanism of NAC, while independent of its antioxidant property, relies on its inhibition on caspase-1 activation. Moreover, NAC promotes procaspase-1 degradation via ubiquitin proteasome system, which further contributes to caspase-1 down-regulation. NAC also inhibits the activity of viral proteases. Taken together, this study shows that NAC exerts potent anti-CVB and anti-inflammation effect through targeting caspase-1. Given that NAC is a clinically approved medicine, we recommend NAC as a valuable therapeutic agent for viral myocarditis caused by CVB.

  • The antifungal isavuconazole inhibits the entry of lassa virus by targeting the stable signal peptide-GP2 subunit interface of lassa virus glycoprotein
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-23
    Xiaoyu Zhang; Ke Tang; Ying Guo

    Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever in humans, and the limited therapeutic treatment for Lassa fever poses significant threat to public health in West Africa. Using an HIV based pseudovirus platform, we identified isavuconazole, a triazole antifungal for systemic use, as a LASV entry inhibitor with an EC50 of 1.2 μM. Isavuconazole inhibits Lassa virus entry by blocking the pH dependent viral fusion mediated by the Lassa virus surface glycoprotein. Fragment replacement mutational study indicated that isavuconazole targets the stable signal peptide (SSP)-membrane fusion subunit (GP2) interface of Lassa glycoprotein. Further mutational study of the SSP-GP2 region of LASV glycoprotein revealed that S27 in the N-terminal transmembrane region of SSP and V431, F434 and V435 in the transmembrane domain of GP2 affect anti-LASV activity of isavuconazole. Isavuconazole also displays antiviral activity to five New World (NW) mammarenaviruses that cause hemorrhagic fever. This study facilitates the potential repurposing of isavuconazole for therapeutic intervention against human-pathogenic arenaviruses, and provides the basis for further structural optimization of arenavirus fusion inhibitors based on the predicted structural characteristics of the unique SSP-GP2 interface.

  • Mechanisms of inter-epidemic maintenance of Rift Valley fever phlebovirus
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-21
    Melanie Rissmann; Franziska Stoek; Matthew J. Pickin; Martin H. Groschup

    Rift Valley fever phlebovirus (RVFV) is an arthropod-borne virus that has caused substantial epidemics throughout Africa and in the Arabian Peninsula. The virus can cause severe disease in livestock and humans and therefore the control and prevention of viral outbreaks is of utmost importance. The epidemiology of RVFV has some particular characteristics. Unexpected and significant epidemics have been observed in spatially and temporally divergent patterns across the African continent. Sudden epidemics in previously unaffected areas are followed by periods of long-term apparent absence of virus and sudden, unpredictable reoccurrence in disparate regions. Therefore, the elucidation of underlying mechanisms of viral maintenance is one of the largest gaps in the knowledge of RVFV ecology. It remains unknown whether the virus needs to be reintroduced before RVFV outbreaks can occur, or if unperceived viral circulation in local vertebrates or mosquitoes is sufficient for maintenance of the virus. To gain insight into these knowledge gaps, we here review existing data that describe potential mechanisms of RVFV maintenance, as well as molecular and serological studies in endemic and non-endemic areas that provide evidence of an inter- or pre-epidemic virus presence. Basic and country-specific mechanisms of RVFV introduction into non-endemic countries are summarized and an overview of studies using mathematical modeling of RVFV persistence is given.

  • In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-18
    Sirin Theerawatanasirikul; Chih Jung Kuo; Nanthawan Phetcharat; Porntippa Lekcharoensuk

    The computational search of chemical libraries has been used as a powerful tool for the rapid discovery of candidate compounds. To find small molecules with anti-feline infectious peritonitis virus (FIPV) properties, we utilized a virtual screening technique to identify the active site on the viral protease for the binding of the available natural compounds. The protease 3CL (3CLpro) plays an important role in the replication cycle of FIPV and other viruses within the family Coronaviridae. The 15 best-ranked candidate consensus compounds, based on three docking tools, were evaluated for further assays. The protease inhibitor assay on recombinant FIPV 3CLpro was performed to screen the inhibitory effect of the candidate compounds with IC50 ranging from 6.36 ± 2.15 to 78.40 ± 2.60 μM. As determined by the cell-based assay, the compounds NSC345647, NSC87511, and NSC343256 showed better EC50 values than the broad-spectrum antiviral drug ribavirin and the protease inhibitor lopinavir, under all the test conditions including pre-viral entry, post-viral entry, and prophylactic activity. The NSC87511 particularly yielded the best selective index (>4; range of SI = 13.80–22.90). These results indicated that the natural small-molecular compounds specifically targeted the 3CLpro of FIPV and inhibited its replication. Structural modification of these compounds may generate a higher anti-viral potency for the further development of a novel therapy against FIP.

  • Generation of pRSAD2 gene knock-in pig via CRISPR/Cas9 technology
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-17
    Zicong Xie; Huping Jiao; Haonan Xiao; Yuan Jiang; Zhenying Liu; Chunyun Qi; Dehua Zhao; Shuyu Jiao; Tingting Yu; Xiaochun Tang; Daxin Pang; Hongsheng Ouyang

    A wide range of endemic and epidemic viruses, including classic swine fever virus (CSFV), pseudorabies virus (PRV) and others, are among the most economically important pathogens in pigs and have severely affected the national economy, human health and animal welfare and productivity. The RSAD2 exhibits antiviral activity against various DNA and RNA viruses. In this study, we successfully accomplished site-specific insertion of the porcine RSAD2 gene (pRSAD2) at the porcine ROSA26 (pROSA26) locus, generating pRSAD2 gene knock-in (pRSAD2-KI) PK-15 cells and porcine foetal fibroblasts (PFFs) via CRISPR/Cas9 technology. Gene expression analysis confirmed that pRSAD2-KI cells stably and efficiently overexpressed the pRSAD2 gene. Furthermore, viral challenge studies in vitro indicated that site-specific integration of the pRSAD2 gene not only effectively reduced CSFV infection but also PRV infection. More importantly, we ultimately successfully produced a pRSAD2-KI pig that constitutively overexpressed the pRSAD2, viral challenge results indicated that fibroblasts isolated from the pRSAD2-KI pig reduced CSFV infection. Taken together, these results suggest that CRISPR/Cas9-mediated knock-in strategy can be used for producing pRSAD2-KI pigs.

  • Effectiveness of Elbasvir/Grazoprevir in patients with hepatitis C virus genotype 1 infection and chronic kidney disease in the United States veterans population
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-17
    Debra T. Choi; Amy Puenpatom; Xian Yu; Kevin F. Erickson; Fasiha Kanwal; Hashem B. El-Serag; Jennifer R. Kramer

    Background & aims Randomized controlled trials of EBR/GZR have reported high treatment efficacy, safety and tolerability in patients undergoing dialysis. However, real world effectiveness data for EBR/GZR in this population is lacking. We evaluated the effectiveness of EBR/GZR in an HCV-infected population with all stages of CKD including dialysis compared with control patients with estimated glomerular filtration rate (eGFR) ≥60 in the US Department of Veterans Affairs (VA). Methods We conducted a retrospective cohort study of patients with chronic HCV genotype 1 infection with EBR/GZR prescriptions dispensed during February 1, 2016–August 31, 2017 in 128 VA Medical Centers. We collected patient information regarding history of dialysis, end stage renal disease (ESRD), and/or eGFR values. We measured SVR based on undetectable HCV RNA at least 4 weeks after the completion of treatment. We examined SVR rates by CKD stage compared to control patients and within patient subgroups using logistic regression models. Results We identified 5,961 patients (42.5% genotype 1a, 55.0% genotype 1b) who met eligibility criteria and completed a EBR/GZR treatment course (≥11 weeks). Approximately 73.2% (n = 4,361) had eGFR ≥60 who served as control patients, 14.4% (n = 860) had Stage 3 CKD, and 12.4% (n = 740) had Stage 4–5 CKD or ESRD. Of patients with Stage 4–5 CKD/ESRD, 76.1% underwent dialysis (n = 563). The overall SVR was 96.7% in all patients, 96.4% for eGFR≥60, 98.3% in Stage 3 CKD, and 96.5% in Stage 4–5 CKD/ESRD. No statistically significant differences were found in the SVR rates in patients with or without dialysis in the Stage 4–5 CKD/ESRD patients (adjusted OR 0.91; 95% CI 0.56–1.47 and OR 1.74; 95% CI 0.63–4.81) compared with those with eGFR≥60. Conclusion We found EBR/GZR was effective in patients with HCV GT1 infection regardless of CKD severity or receipt of dialysis in the US VA population.

  • Structure-activity relationships of fluorene compounds inhibiting HCV variants
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-17
    Hee Sun Kim; Youngsu You; Jae Gon Mun; Changdev G. Gadhe; Heejo Moon; Jae Seung Lee; Ae Nim Pae; Michinori Kohara; Gyochang Keum; Byeong Moon Kim; Sung Key Jang
  • Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-16
    Qian Chen; Celia Perales; María Eugenia Soria; Damir García-Cehic; Josep Gregori; Francisco Rodríguez-Frías; María Buti; Javier Crespo; José Luis Calleja; David Tabernero; Marta Vila; Fernando Lázaro; Ariadna Rando; Leonardo Nieto-Aponte; Meritxell Llorens-Revull; Maria Francesca Cortese; Irati Fernandez-Alonso; José Castellote; José Antonio del-Campo

    A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) α-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions.

  • The Delta-4 fibrosis score (D4FS): A novel fibrosis score in chronic hepatitis D
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-16
    Ben L. Da; Pallavi Surana; David E. Kleiner; Theo Heller; Christopher Koh

    Background Chronic Hepatitis D virus (HDV) infection results in the most severe form of viral hepatitis with a rapid progression to cirrhosis. However, non-invasive fibrosis tests that can accurately predict cirrhosis have not been adequately validated. We aimed to develop a clinically useful non-invasive score that can accurately detect cirrhosis. Material and methods Patients with chronic HDV diagnosed by liver histology or serum PCR were evaluated. Data regarding demographics, laboratory, imaging, vibration-controlled transient elastography (VCTE), and liver biopsy were collected. The total cohort was randomized into a training and validation cohort. The training cohort was used to develop a novel score, the Delta-4 fibrosis score (D4FS) which was then compared to other non-invasive tests in the validation cohort by area under receiver operating characteristics (AUROC). Results 77 patients with chronic HDV were evaluated: mean age 42.6 (SD:11.1) years, 59.7% male, and 57.1% Asian. The total cohort was then separated into a training (n = 45) and validation (n = 32) cohort with no significant differences in terms of clinical characteristics between the two. From the training cohort, the D4FS was derived from variables of statistical and clinical interest (gamma-glutamyl transpeptidase (GGT), platelet count, alanine aminotransferase (ALT), and liver stiffness measurement (LSM)). The D4FS demonstrated the best AUROC in the validation cohort (0.94) followed by VCTE (0.90), FIB-4 (0.86), APRI (0.81), and AAR (0.71). Discussion The D4FS is a clinically useful non-invasive fibrosis score that can accurately detect cirrhosis in patients with chronic HDV infection. Further studies should be performed to further validate clinical utility.

  • Recombination of B- and T-cell epitope-rich loci from Aedes- and Culex-borne flaviviruses shapes Zika virus epidemiology
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-16
    Michael W. Gaunt; Duane J. Gubler; John H.-O. Pettersson; Goro Kuno; Annelies Wilder-Smith; Xavier de Lamballerie; Ernest A. Gould; Andrew K. Falconar

    Sporadic human Zika virus (ZIKV) infections have been recorded in Africa and Asia since the 1950s. Major epidemics occurred only after ZIKV emerged in the Pacific islands and spread to the Americas. Specific biological determinants of the explosive epidemic nature of ZIKV have not been identified. Phylogenetic studies revealed incongruence in ZIKV placement in relation to Aedes-borne dengue viruses (DENV) and Culex-borne flaviviruses. We hypothesized that this incongruence reflects interspecies recombination resulting in ZIKV evasion of cross-protective T-cell immunity. We investigated ZIKV phylogenetic incongruence In relation to: 1) DENV T-cell maps experimentally identified ex vivo, 2) published B-cell epitope loci, and 3) CD8+ epitopes predicted in silico for mosquito-borne flaviviruses. Our findings demonstrate that the ZIKV proteome is a hybrid of Aedes-borne DENV proteins interspersed amongst Culex-borne flavivirus proteins derived through independent interspecies recombination events. These analyses infer that DENV-associated proteins in the ZIKV hybrid proteome generated immunodominant human B-cell responses, whereas ZIKV recombinant derived Culex-borne flavivirus-associated proteins generated immunodominant CD8+ and/or CD4+ T-cell responses. In silico CD8+ epitope ZIKV cross-reactive prediction analyses verified this observation. We propose that by acquiring T-cell epitope-rich regions from Culex-borne flaviviruses, ZIKV evaded DENV-generated T-cell immune cross-protection. Thus, Culex-borne flaviviruses, including West Nile virus and Japanese encephalitis virus, might induce cross-protective T-cell responses against ZIKV. This would explain why explosive ZIKV epidemics occurred in DENV-endemic regions of Micronesia, Polynesia and the Americas where Culex-borne flavivirus outbreaks are infrequent and why ZIKV did not cause major epidemics in Asia where Culex-borne flaviviruses are widespread.

  • Abacavir induces the transcriptional activity of YY1 and other oncogenic transcription factors in gastric cancer cells
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-14
    Ponmathi Panneerpandian; Helen Jemimah Devanandan; Anantharaj Marimuthu; Chandrabose Karthikeyan; Kumaresan Ganesan

    Yin Yang 1 (YY1) is a ubiquitous transcription factor with both transcriptional activating and repressing functions. Targeting YY1 is considered as a potential therapeutic strategy for several malignancies. Telomerase Reverse Transcriptase (TERT) is also considered as a potential target for cancer therapeutics. To enable the large-scale screening and identification of potential YY1 targeting drugs, a gastric cancer cell line-based drug screening assay was developed. In a YY1 targeted drug repurpose screen, abacavir sulfate, a nucleoside analog reverse transcriptase inhibitor and known to target TERT was identified to show the feature of activating YY1 mediated transcription. We further explored i) the molecular targets of abacavir, ii) activation pattern of pathways regulated by abacavir in gastric tumors, and iii) therapeutic potential of abacavir for gastric cancer cells. Oncogenic signaling pathways like MYC, HIF1-α, ERK, WNT, E2F, NFκB and NRF1/2 were also found to be highly activated by abacavir. Abacavir was found to have less impact on the viability of gastric cancer cells. Across gastric tumors, we observed the co-activation of TERT, alternative lengthening of telomere (ALT), DNA repair, and the oncogenic pathways MYC, E2F/DP1, ERK, YY1, HIF1α, and NFκB specific gene-sets, in a subset of gastric tumors. The observed connectivity among TERT, DNA repair, and multiple oncogenic pathways indicate the need for the development of combinatorial therapeutics for the gastric tumors with the activated TERT.

  • Safety, efficacy, and pharmacokinetics of pradefovir for the treatment of chronic hepatitis B infection
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-13
    Hong Zhang; Jingrui Liu; Xiaoxue Zhu; Xiaojiao Li; Weili Jin; Hong Chen; Min Wu; Cuiyun Li; Chengjiao Liu; JunqiNiu; Yanhua Ding

    Background & aims Pradefovir is a liver targeted novel prodrug of adefovir (PMEA) developed to provide higher antiviral activity with reduced systemic toxicities. This study evaluated the tolerability, pharmacokinetics, and antiviral activity of pradefovir in patients with chronic hepatitis B (CHB) virus infection. Methods Non-cirrhotic, treatment-naïve subjects with CHB were divided into five groups (10 patients each) and randomized within each group in a ratio of 6:2:2 to receive an ascending dose of 30, 60, 75, 90, or 120 mg pradefovir, 10 mg adefovir dipivoxil (ADV), or 300 mg tenofovir disoproxil fumarate (TDF) once a day for 28 days. Results A total of 51 subjects were randomized and 49 subjects completed the study. The groups were well matched and included 39 males, of whom 71% were hepatitis B e-antigen-negative with a mean hepatitis B virus (HBV) DNA level of 6.4–7.16 log10 IU/mL. No subject experienced a serious adverse event or nephrotoxicity. The most frequently reported adverse event was asymptomatic reduction in blood cholinesterase levels in the pradefovir group which recovered without any treatment about 13 ± 7 days after drug discontinuation. This adverse event was not observed in the ADV and TDF groups. The mean changes in serum HBV DNA were −2.78, −2.77, −3.08, −3.18, −3.44, −2.34, and −3.07 log10 IU/mL at 30, 60, 75, 90, and 120 mg pradefovir, 10 mg ADV and 300 mg TDF, respectively, with plateau levels reached with 60 mg pradefovir. Pradefovir and its metabolite PMEA showed linear pharmacokinetics proportional to the dose. The half-life of PMEA in the pradefovir group was 11.47–17.63 h. Conclusions Short-term use of pradefovir was well tolerated. A decline in HBV DNA levels was superior to TDF at higher doses of pradefovir. 30–60 mg pradefovir is recommended for CHB treatment. Clinical trial number CTR20150224.

  • Wedelolactone inhibits human cytomegalovirus replication by targeting distinct steps of the viral replication cycle
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-10
    Adriana Svrlanska, Anna Ruhland, Manfred Marschall, Nina Reuter, Thomas Stamminger

    Wedelolactone (WDL) is a coumestan present in the plants Eclipta prostrata and Wedelia calendulacea which are used for treatment of a multitude of health problems in traditional medicine. It has previously been shown that WDL exerts antiviral activity against human immunodeficiency virus and hepatitis C virus. In this study, we investigated the effect of WDL on lytic human cytomegalovirus (HCMV) infection. We demonstrate a strong interference with HCMV replication as analyzed in multi-round replication settings. A more detailed analysis of the underlying mechanisms revealed that WDL acts at two distinct steps of the viral replication cycle. During immediate early (IE) times, we observe an inhibition of IE1/IE2 expression. Although WDL was reported to interfere with NF-κB signaling our results suggest the existence of additional mechanisms that impede viral IE expression. During later time points of infection, WDL induced a disruption of the interaction between EZH2 and EED, components of the virus-supportive polycomb repressive complex 2 (PRC2). Thereby, the stability of the PRC2 complex as well as the related complex PRC1 was disturbed leading to diminished viral DNA synthesis. Taken together, we identify WDL as a potent agent against HCMV which interferes at two distinct steps of viral replication.

  • Protection of swine by potent neutralizing anti-Japanese encephalitis virus monoclonal antibodies derived from vaccination
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-09
    Christian L. Young, Amy C. Lyons, Wei-Wen Hsu, Dana L. Vanlandingham, So Lee Park, Ashley N. Doerfler, Victoria B. Ayers, Susan M. Hettenbach, Ashley M. Zelenka, Konner R. Cool, Gregory J. Peterson, Stephen Higgs, Yan-Jang S. Huang

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus endemic in the Asia Pacific region. Despite use of several highly effective vaccines, it is estimated that up to 44,000 new cases of Japanese encephalitis (JE) occur every year including 14,000 deaths and 24,000 survivors with permanent sequelae. Humoral immunity induced by vaccination is critical for effective protection. Potently neutralizing antibodies reactive with the JEV envelope (E) protein are important since protective immune responses induced by both live-attenuated and inactivated JE vaccines target the E protein. Our understanding of how vaccine-induced humoral immunity protects vaccinees from morbidity and mortality is, however, limited and largely obtained from in vitro studies. With the exception of neurovirulence mouse models, very few platforms are available for evaluating the protective efficacy of neutralizing antibodies against JEV in vivo. Swine are a major amplifying host in the natural JEV transmission cycle and develop multiple pathological outcomes similar to humans infected with JEV. In this study, prophylactic passive immunization was performed in a miniature swine model, using two vaccination-induced monoclonal antibodies (mAb), JEV-31 and JEV-169. These were selected as representatives for antibodies reactive with the major antigenic structures in the E protein of JEV and related flaviviruses. JEV-31 recognizes the lateral ridge of E protein domain III (EDIII) whilst JEV-169 has a broad footprint of binding involving residues throughout domains I (EDI) and II (EDII) of the E protein. Detection of neutralizing antibodies in the serum of immunized animals mimics the presence of neutralizing antibodies in vaccinated individuals. Passive immunization with both mAbs significantly reduced the severity of diseases that resemble the symptoms of human JE including fever, viremia, viral shedding, systemic infection, and neuroinvasion. In contrast to the uniformed decrease of viral loads in lymphoid and central nervous systems, distinct kinetics in the onset of fever and viremia between animals receiving JEV-31 and JEV-169 suggest potential differences in immune protection mechanisms between anti-EDI and anti-EDIII neutralizing antibodies elicited by vaccination. Our data demonstrate the feasibility of using swine models in characterizing the protective humoral immunity against JEV and increase our understanding of how clonal populations of anti-E mAbs derived from JE vaccination protect against infection in vivo.

  • Inhibition of a novel coumarin on an aquatic rhabdovirus by targeting the early stage of viral infection demonstrates potential application in aquaculture
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-09
    Lei Liu, Tian-Xiu Qiu, Da-Wei Song, Li-Peng Shan, Jiong Chen

    Spring viremia of carp virus (SVCV) is one of the most serious pathogens in aquaculture, resulting in devastating damage in cyprinid. In this study, we designed and synthesized a novel coumarin derivative (C3007) for evaluating its in vitro and in vivo anti-SVCV effects. Here, we determined that up to 25 mg/L C3007 significantly decreased SVCV protein gene expression levels in EPC cells by a maximum inhibitory rate of >95%. When C3007 was preincubated with SVCV, infectivity was significantly inhibited in vitro in a time-dependent manner, with complete inhibition at 25 mg/L. For in vivo studies, C3007 exhibited an anti-SVCV effect by substantially enhancing the survival rate of virus-infected fish via intraperitoneal injection. Although the horizontal transmission of SVCV was hindered by C3007 in a static cohabitation challenge model, it was not completely blocked, showing that the viral loads in recipient fish were obviously reduced. Thus, C3007 could potentially be used as a therapeutic agent with great potential in aquatic systems and may also be suitable for applications in pond aquaculture settings against viral transmission. Additionally, the C3007-preincubated virus induced an antiviral immune response with high levels of IFN expression, suggesting that C3007 pre-treatment could be used in vaccine development.

  • Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-06
    Andrew M. Skidmore, Robert S. Adcock, Colleen B. Jonsson, Jennifer E. Golden, Dong-Hoon Chung

    Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2–4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 μM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.

  • Discovery of dihydroxyindole-2-carboxylic acid derivatives as dual allosteric HIV-1 Integrase and Reverse Transcriptase associated Ribonuclease H inhibitors
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-05
    Francesca Esposito, Mario Sechi, Nicolino Pala, Adele Sanna, Pratibha Chowdary Koneru, Mamuka Kvaratskhelia, Lieve Naesens, Angela Corona, Nicole Grandi, Roberto di Santo, Vincenzo Maria D'Amore, Francesco Saverio Di Leva, Ettore Novellino, Sandro Cosconati, Enzo Tramontano

    The management of Human Immunodeficiency Virus type 1 (HIV-1) infection requires life-long treatment that is associated with chronic toxicity and possible selection of drug-resistant strains. A new opportunity for drug intervention is offered by antivirals that act as allosteric inhibitors targeting two viral functions (dual inhibitors). In this work, we investigated the effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derivatives on both HIV-1 Integrase (IN) and Reverse Transcriptase associated Ribonuclease H (RNase H) activities. Among the tested compounds, the dihydroxyindole-carboxamide 5 was able to inhibit in the low micromolar range (1–18 μM) multiple functions of IN, including functional IN-IN interactions, IN-LEDGF/p75 binding and IN catalytic activity. Docking and site-directed mutagenesis studies have suggested that compound 5 binds to a previously described HIV-1 IN allosteric pocket. These observations indicate that 5 is structurally and mechanistically distinct from the published allosteric HIV-1 IN inhibitors. Moreover, compound 5 also inhibited HIV-1 RNase H function, classifying this molecule as a dual HIV-1 IN and RNase H inhibitor able to impair the HIV-1 virus replication in cell culture. Overall, we identified a new scaffold as a suitable platform for the development of novel dual HIV-1 inhibitors.

  • Antiviral activity of phage display-selected peptides against Japanese encephalitis virus infection in vitro and in vivo
    Antivir. Res. (IF 4.130) Pub Date : 2019-12-05
    Jianchao Wei, Muddassar Hameed, Xin Wang, Junjie Zhang, Shuang Guo, Muhammad Naveed Anwar, Linlin Pang, Ke Liu, Beibei Li, Donghua Shao, Yafeng Qiu, Dengke Zhong, Bin Zhou, Zhiyong Ma

    Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that is the most significant etiological agent of childhood viral neurological infections. However, no specific antiviral drug is currently available to treat JEV infections. The JEV envelope (E) protein is a class II viral fusion protein that mediates host cell entry, making interference with the interaction between the E protein of JEV and its cognate receptors an attractive strategy for anti-JEV drug development. In this study, we identified a peptide derived from a phage display peptide library against the E protein of JEV, designated P1, that potentially inhibits in vitro and in vivo JEV infections. P1 inhibits JEV infection in BHK-21 cells with 50% inhibitory capacity at a concentration of 35.9 μM. The time-of-addition assay indicates that JEV replication is significantly inhibited during pre-infection and co-infection of P1 with JEV while post-infection treatments with P1 have very little impact on JEV proliferation, showing that P1 inhibits JEV infection at early stages and indicating the potential prophylactic effect of P1. We adapted an in vitro BiFC assay system and demonstrated that P1 interacts with JEV E proteins and blocks their entry into cells. We also evaluated the therapeutic efficacy of P1 in a lethal JEV mouse model exhibiting systemic and brain infections. Interestingly, P1 treatment protected C57BL/6 mice against mortality, markedly reduced the viral loads in blood and brain, and diminished the histopathological lesions in the brain cells. In addition to controlling systemic infection, P1 has a very low level of cytotoxicity and acts in a sequence-specific manner, as scrambled peptide sP1 does not show any antiviral activity. In conclusion, our in vitro and in vivo experimental findings show that P1 possesses antiviral activity against JEV infections, is safe to use, and has potential for further development as an antiviral treatment against JEV infections.

  • Influenza B viruses from different genetic backgrounds are variably impaired by neuraminidase inhibitor resistance–associated substitutions
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-29
    Philippe Noriel Q. Pascua, Bindumadhav M. Marathe, Shivantika Bisen, Richard J. Webby, Elena A. Govorkova

    Identifying evolutionary routes to antiviral resistance among influenza viruses informs molecular-based resistance surveillance and clinical decisions. To improve antiviral management and understand whether clinically identified neuraminidase (NA) inhibitor (NAI) resistance–associated markers affect influenza B viruses of the Victoria- or Yamagata-lineages differentially, we generated a panel of NAI-resistant viruses (carrying E105K, G145E, R150K, D197N, I221 L/N/T/V, H273Y, N294S, or G407S substitutions; B numbering) in B/Brisbane/60/2008 (BR/08) and B/Phuket/3073/2013 (PH/13). In both backgrounds, I221 L/N/T/V resulted in reduced or highly reduced inhibition (HRI) by one to three currently available NAIs. D197N reduced inhibition by all NAIs in BR/08 but only by oseltamivir and peramivir in PH/13; R150K caused HRI by all NAIs in PH/13. Although PH/13 generally retained or enhanced NA activity in the presence of the substitutions, enzymatic activity in BR/08 was detrimentally affected. Similarly, substrate affinity and catalysis were relatively stable in PH/13, but not in the BR/08 variants. E105K, R150K, and D197N attenuated replication efficiency of BR/08 in vitro and in mice; only E105K had this effect in PH/13. Notably, the I221 L/N/T/V substitutions did not severely impair replication, particularly in PH/13. Overall, our data show differential effects of NA substitutions in representative Victoria- and Yamagata-lineage viruses, suggesting distinct evolution of these viruses caused variable fitness and NAI susceptibility profiles when similar key NA substitutions arise. Because the viruses harboring the I221 NA substitutions displayed undiminished fitness and are commonly reported, this position is likely to be the most clinically relevant marker for NAI resistance among contemporary influenza B viruses.

  • A cell-based, infectious-free, platform to identify inhibitors of lassa virus ribonucleoprotein (vRNP) activity
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-28
    Beatrice Cubitt, Emilio Ortiz-Riano, Benson YH. Cheng, Yu-Jin Kim, Charles D. Yeh, Catherine Z. Chen, N.O.E. Southall, Wei Zheng, Luis Martinez-Sobrido, Juan C. de la Torre

    The mammarenavirus Lassa (LASV) is highly prevalent in West Africa where it infects several hundred thousand individuals annually resulting in a high number of Lassa fever (LF) cases, a febrile disease associated with high morbidity and significant mortality. Mounting evidence indicates that the worldwide-distributed prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. There are not Food and Drug Administration (FDA) licensed vaccines and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective and can cause significant side effects. Therefore, there is an unmet need for novel antiviral drugs to combat LASV. This task would be facilitated by the implementation of high throughput screens (HTS) to identify inhibitors of the activity of the virus ribonucleoprotein (vRNP) responsible for directing virus RNA genome replication and gene transcription. The use of live LASV for this purpose is jeopardized by the requirement of biosafety level 4 (BSL4) containment. We have developed a virus-free cell platform, where expression levels of reporter genes serve as accurate surrogates of vRNP activity, to develop cell-based assays compatible with HTS to identify inhibitors of LASV and LCMV mammarenavirus vRNP activities.

  • In silico drug repurposing for the identification of potential candidate molecules against arboviruses infection
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-28
    Diana Montes-Grajales, Henry Puerta-Guardo, Diego A. Espinosa, Eva Harris, William Caicedo-Torres, Jesus Olivero-Verbel, Esperanza Martínez-Romero
  • Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-23
    Jozlyn R. Clasman, Renata K. Everett, Karthik Srinivasan, Andrew D. Mesecar

    Coronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts.

  • Development of horse neutralizing immunoglobulin and immunoglobulin fragments against Junín virus
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-21
    Xiaoyan Pan, Yan Wu, Wei Wang, Leike Zhang, Gengfu Xiao

    Argentine haemorrhagic fever (AHF) is a rodent-borne disease with a lethality as high as ∼30%, which is caused by the New World arenavirus, Junín virus (JUNV). It was once a major epidemic in South America and puts millions of people in Argentina at risk. Here, we aimed to develop horse antibodies or antibody fragments against JUNV. Before preparing the horse antibodies, a strategy to efficiently generate horse antisera was established based on comparisons among immunogens and immunization methods in both mice and horses. Antisera against JUNV were finally obtained by vaccinating horses with vesicular stomatitis virus pseudotypes bearing JUNV GP. The horse antibodies IgG and F(ab’)2 were subsequently demonstrated to effectively neutralize vesicular stomatitis virus pseudotypes bearing JUNV GP and to show some cross-neutralization against pathogenic New World arenaviruses. Further research revealed that Asp123 on GP1 is an important site for the binding of antibodies targeting mainly JUNV GP1 for neutralization. Collectively, this study presents an efficient strategy to develop horse antisera against JUNV and provides GP1-specific horse antibodies as potential therapeutics for AHF.

  • Receptor tyrosine kinase inhibitors block proliferation of TGEV mainly through p38 mitogen-activated protein kinase pathways
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-18
    Wanyu Dong, Wenting Xie, Yunbo Liu, Baokun Sui, Hao Zhang, Liran Liu, Yubei Tan, Xiaohan Tong, Zhen F. Fu, Ping Yin, Liurong Fang, Guiqing Peng

    Emerging coronaviruses (CoVs) primarily cause severe gastroenteric or respiratory diseases in humans and animals, and no approved therapeutics are currently available. Here, A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, is identified as a robust inhibitor of transmissible gastroenteritis virus (TGEV) infection in cell-based assays. Moreover, A9 exhibited potent antiviral activity against the replication of various CoVs, including murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV). We further performed a comparative phosphoproteomic analysis to investigate the mechanism of action of A9 against TGEV infection in vitro. We specifically identified p38 and JNK1, which are the downstream molecules of receptor tyrosine kinases (RTKs) required for efficient TGEV replication, as A9 targets through plaque assays, qRT-PCR and Western blotting assays. p38 and JNK1 inhibitors and RNA interference further showed that the inhibitory activity of A9 against TGEV infection was mainly mediated by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. All these findings indicated that the RTKI A9 directly inhibits TGEV replication and that its inhibitory activity against TGEV replication mainly occurs by targeting p38, which provides vital clues to the design of novel drugs against CoVs.

  • Recombinant pseudorabies virus expressing E2 of classical swine fever virus (CSFV) protects against both virulent pseudorabies virus and CSFV
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-18
    Wu Tong, Hao Zheng, Guo-xin Li, Fei Gao, Tong-ling Shan, Yan-jun Zhou, Hai Yu, Yi-feng Jiang, Ling-xue Yu, Li-wei Li, Ning Kong, Guang-zhi Tong, Ji-chang Li

    Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV.

  • Emetine protects mice from enterovirus infection by inhibiting viral translation
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-14
    Qi Tang, Siliang Li, Liaoqi Du, Shihua Chen, Jingyi Gao, Yang Cai, Zhichao Xu, Zixuan Zhao, Ke Lan, Shuwen Wu

    IRES-driven translation plays an essential role in picornavirus infection. However, there are seldom reports of compounds targeting this pathway with effective protection in animal models. Here, we identified emetine, an antiprotozoal drug, which inhibits EV-A71 with an EC50 value of 0.04 μM and a CC50 value of 10 μM in RD cell culture. Interestingly, emetine exhibits activities against a series of human enteroviruses, including CV-A16, CV-B1, EV-D68, Echov-6, etc., at the nanomolar level. When orally administered at 0.20 mg/kg twice a day in an EV-A71 mouse model, emetine reduced viral loads in various organs and completely prevented diseases and death. A mechanistic study demonstrated that emetine suppressed EV-A71 by inhibiting viral IRES-driven translation. Taken together, these data indicate emetine as a promising candidate to treat picornavirus infection.

  • A novel isothiocyanate derivative inhibits HIV-1 gene expression and replication by modulating the nuclear matrix associated protein SMAR1
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-09
    Jay Trivedi, Aftab Alam, Shruti Joshi, Pavan Kumar, Venkatraju Chippala, Prathama S. Mainkar, Srivari Chandrasekhar, Samit Chattopadhyay, Debashis Mitra

    The essential role of SMAR1 in HIV-1 transcription and LTR driven gene expression suggests SMAR1 as an HIV dependency factor (HDF) and a potential anti-HIV therapeutic target. Here, we report for the first time, anti-HIV activity of 8 novel isothiocyanate (ITC) derivatives that differentially stabilise SMAR1. Out of 8 novel ITC derivatives, SCS-OCL-381 was observed to inhibit HIV-1 replication most significantly at the noncytotoxic concentration in reporter T-cell line, CEM-GFP. Further, the highly conserved anti-HIV activity of SCS-OCL-381 is a cell type, virus isolate and viral load independent phenomena and is approximately 3 fold more effective than the representative ITC, Sulforaphane (SFN). Further, SCS-OCL-381 does not hamper the activity of viral enzymes reverse transcriptase, integrase and protease. Mechanistically, SCS-OCL-381 stabilises SMAR1 which, otherwise undergoes proteasomal degradation upon HIV-1 infection in T-cells. This stabilisation results in the recruitment of repressor complex on HIV-1 LTR resulting in repression of LTR mediated transcription and gene expression. These inhibitory consequences were further confirmed by reporter based LTR activity assays in different cell lines. Taken together, these findings highlight the anti-HIV potential of novel ITC derivatives by the stabilisation of SMAR1 and strongly support further in vivo characterisation and potential translational applications of SCS-OCL-381.

  • Small molecule screening identifies inhibitors of the Epstein-Barr virus deubiquitinating enzyme, BPLF1
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-08
    Sage L. Atkins, Safiyyah Motaib, Laura C. Wiser, Sharon E. Hopcraft, Paul B. Hardy, Julia Shackelford, Peter Foote, Ashley H. Wade, Blossom Damania, Joseph S. Pagano, Kenneth H. Pearce, Christopher B. Whitehurst

    Herpesviral deubiquitinating enzymes (DUBs) were discovered in 2005, are highly conserved across the family, and are proving to be increasingly important players in herpesviral infection. EBV's DUB, BPLF1, is known to regulate both cellular and viral target activities, yet remains largely unstudied. Our work has implicated BPLF1 in a wide range of processes including infectivity, viral DNA replication, and DNA repair. Additionally, knockout of BPLF1 delays and reduces human B-cell immortalization and lymphoma formation in humanized mice. These findings underscore the importance of BPLF1 in viral infectivity and pathogenesis and suggest that inhibition of EBV's DUB activity may offer a new approach to specific therapy for EBV infections. We set out to discover and characterize small molecule inhibitors of BPLF1 deubiquitinating activity, through high-throughput screening. An initial small pilot screen resulted in discovery of 10 compounds yielding >80% decrease in BPLF1 DUB activity at a 10 μM concentration. Follow-up dose response curves of top hits identified several compounds with an IC50 in the low micromolar range. Four of these hits were tested for their ability to cleave ubiquitin chains as well as their effects on viral infectivity and cell viability. Further characterization of the top hit, commonly known as suramin was found to not be selective yet decreased viral infectivity by approximately 90% with no apparent effects on cell viability. Due to the conserved nature of Herpesviral deubiquitinating enzymes, identification of an inhibitor of BPLF1 may prove to be an effective and promising new avenue of therapy for EBV and other herpesviral family members.

  • Characterization of the dynamics of human cytomegalovirus resistance to antiviral drugs by ultra-deep sequencing
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-07
    Hélène Guermouche, Sonia Burrel, Mélanie Mercier-Darty, Thomas Kofman, Olivier Rogier, Jean-Michel Pawlotsky, David Boutolleau, Christophe Rodriguez

    Prophylactic or preemptive treatment strategies are required to prevent human cytomegalovirus (CMV) infections in transplant recipients. However, treatment failure occurs when CMV resistant-associated variants (RAVs) are selected. Although the diversity of CMV is lower than that of RNA viruses, CMV appears to show some genetic instability, with possible minor emerging resistance that may be undetectable by Sanger sequencing. We aimed to examine CMV-resistance mutations over time by ultra-deep sequencing (UDS) and Sanger sequencing in a kidney transplant recipient experiencing CMV infection. This patient showed a transient response to three different antiviral drugs (valganciclovir, foscarnet, and maribavir) and four episodes of CMV resistance over two years. The full-length UL97 (2.3kpb) and partial UL54 (2.4kpb) CMV genes were studied by UDS and Sanger sequencing and linkage mutations calculated to determine RAVs. We detected four major and five minor resistance mutations. Minor resistant variants (2–20%) were detected by UDS, whereas major resistance substitutions (>20%) were identified by both UDS and Sanger method. We detected cross-resistance to three drugs, despite high CMV loads, suggesting that the fitness of the viral mutants was not impaired. In conclusion, CMV showed complex dynamic of resistance under antiviral drug pressure, as described for highly variable viruses. The emergence of successive RAVs constitutes a clinically challenging complication and contributes to the difficulty of therapeutic management of patients.

  • Non-invasive bioluminescence imaging of HCoV-OC43 infection and therapy in the central nervous system of live mice
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-06
    Junwei Niu, Liang Shen, Baoying Huang, Fei Ye, Li Zhao, Huijuan Wang, Yao Deng, Wenjie Tan

    Human coronaviruses (HCoVs) are important pathogens that cause upper respiratory tract infections and have neuroinvasive abilities; however, little is known about the dynamic infection process of CoVs in vivo, and there are currently no specific antiviral drugs to prevent or treat HCoV infection. Here, we verified the replication ability and pathogenicity of a reporter HCoV-OC43 strain expressing Renilla luciferase (Rluc; rOC43-ns2DelRluc) in mice with different genetic backgrounds (C57BL/6 and BALB/c). Additionally, we monitored the spatial and temporal progression of HCoV-OC43 through the central newrvous system (CNS) of live BALB/c mice after intranasal or intracerebral inoculation with rOC43-ns2DelRluc. We found that rOC43-ns2DelRluc was fatal to suckling mice after intranasal inoculation, and that viral titers and Rluc expression were detected in the brains and spinal cords of mice infected with rOC43-ns2DelRluc. Moreover, viral replication was initially observed in the brain by non-invasive bioluminescence imaging before the infection spread to the spinal cord of BALB/c mice, consistent with its tropism in the CNS. Furthermore, the Rluc readout correlated with the HCoV replication ability and protein expression, which allowed quantification of antiviral activity in live mice. Additionally, we validated that chloroquine strongly inhibited rOC43-ns2DelRluc replication in vivo. These results provide new insights into the temporal and spatial dissemination of HCoV-OC43 in the CNS, and our methods provide an extremely sensitive platform for evaluating the efficacy of antiviral therapies to treat neuroinvasive HCoVs in live mice.

  • 2019 meeting of the global virus network
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-04
    Ramesh Akkina, Robert Garry, Christian Bréchot, Heinz Ellerbrok, Hideki Hasegawa, Luis Menéndez-Arias, Natalia Mercer, Johan Neyts, Victor Romanowski, Joaquim Segalés, Anders Vahlne

    The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 52 GVN Centers of Excellence and 9 Affiliate laboratories in 32 countries. The 11th International GVN meeting was held from June 9–11, 2019 in Barcelona, Spain and was jointly organized with the Spanish Society of Virology. A common theme throughout the meeting was globalization and climate change. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including severe virus epidemics, anticipation and preparedness for changing disease dynamics, host-pathogen interactions, zoonotic virus infections, ethical preparedness for epidemics and pandemics, one health and antivirals.

  • A synthetic derivative of houttuynoid B prevents cell entry of Zika virus
    Antivir. Res. (IF 4.130) Pub Date : 2019-11-04
    Michael Basic, Fabian Elgner, Daniela Bender, Catarina Sabino, Marie-Luise Herrlein, Hanna Roth, Mirco Glitscher, Alicia Fath, Thomas Kerl, Hans-Günther Schmalz, Eberhard Hildt

    Zika virus (ZIKV) is a re-emerging virus belonging to the family of Flaviviridae, which contains several human pathogens. A great deal of attention came through the association of ZIKV infection with an increasing number of microcephaly cases in newborns during the 2016 outbreak in Brazil. Currently, no anti-viral drug or vaccine is available. Houttuynoids are a group of structurally related flavonoid glycosides that can be isolated from Houttuynia cordata belonging to the family of Sauraceae. Moreover, H. cordata was described to have an antiviral effect on herpes simplex virus type 1 (HSV-1), human immunodeficiency virus type 1 (HIV-1) and influenza A virus (Hayashi et al., 1995). In light of this, this study aimed to investigate a potential antiviral effect of the synthetic houttuynoids TK1023 and TK1024 (i.e. houttuynoid B) on two ZIKV isolates (Uganda and French Polynesia). A significant decrease in the amount of intra- and extracellular viral genomes as well as infectious viral particles was observed after treatment with the tetra-O-acetylated houttuynoid TK1023 independent from the analyzed virus isolate. In contrast, TK1024 (houttuynoid B) had no effect on ZIKV. Treatment with TK1023 significantly decreases the number of infected cells 24 h and 48 h after infection, as compared to the control. Analysis of the mode of action revealed that TK1023 neither affects the viral genome replication nor the production of viral proteins nor morphogenesis or release. Binding and entry assays showed that TK1023 interferes with the entry of the virus in the cell. Thereby, the spread of ZIKV infection is impaired as the infection of the individual cell is inhibited. These data indicate that for both analyzed virus isolates the spread of ZIKV infection can be impaired by the synthetic houttuynoid TK1023 due to an inhibition of the viral entry.

  • Identification of inhibitors of dengue viral replication using replicon cells expressing secretory luciferase
    Antivir. Res. (IF 4.130) Pub Date : 2019-10-31
    Fumihiro Kato, Yasunori Nio, Kazumi Yagasaki, Rieko Suzuki, Makoto Hijikata, Tomoyuki Miura, Isao Miyazaki, Shigeru Tajima, Chang-Kwang Lim, Masayuki Saijo, Tomohiko Takasaki, Takayuki Hishiki

    Dengue virus (DENV) is the causative agent of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) and continues to be a public health problem in the tropical and subtropical areas. However, there is currently no antiviral treatment for DENV infection. In this study, our aim was to develop a stable reporter replicon cell system that supports constant viral RNA replication in cultured cells. The isolated replicon cells exhibited high levels of luciferase activity in the culture supernatant concomitant with expression of virus-encoded NS1, NS3 and NS5 proteins in the cells. The NS1, NS3 proteins and dsRNA were detected in the replicon cells by immunofluorescence analysis. Furthermore, the anti-DENV inhibitors ribavirin and bromocriptine significantly reduced the luciferase activity in a dose-dependent manner. High-throughput screening with a compound library using the stably-transfected replicon cells showed a Z′ factor value of 0.57. Our screening yielded several candidates including one compound that has already shown anti-DENV activity. Taken together, our results demonstrate that this DENV subgenomic replicon cell system expressing a secretory luciferase gene can be useful for the high-throughput screening of anti-DENV compounds and the analysis of the replication mechanism of the DENV RNA.

  • Fatty acid synthase and stearoyl-CoA desaturase-1 are conserved druggable cofactors of Old World Alphavirus genome replication
    Antivir. Res. (IF 4.130) Pub Date : 2019-10-31
    William Bakhache, Aymeric Neyret, Joe McKellar, Camille Clop, Eric Bernard, James Weger-Lucarelli, Laurence Briant
  • APOBEC and iNOS are not the main intracellular effectors of IFN-gamma-mediated inactivation of Hepatitis B virus replication.
    Antivir. Res. (IF 4.130) Pub Date : 2008-03-04
    Sandra Proto,John A Taylor,Shilpa Chokshi,Naveenan Navaratnam,Nikolai V Naoumov

    BACKGROUND/AIM Interferon-gamma (IFN-gamma) produced by activated T-cells is the principle mediator of non-cytolytic Hepatitis B virus (HBV) inactivation; however the intracellular pathways responsible are poorly defined. We investigated the role of IFN-gamma-inducible nitric oxide synthase (iNOS) and APOBEC3 (A3) enzyme family in the inhibition of HBV replication by IFN-gamma. METHODS Hepatoma-cell lines transfected with HBV DNA were treated with IFN-gamma. Viral replication, iNOS and A3 mRNAs were quantitated by TaqManPCR and the direct nitric oxide (NO) effect on HBV replication was investigated using an NO-donor. A3G antiviral activity was verified by co-transfection with its inhibitor, human immunodeficiency virus (HIV)-associated virion infectivity factor (Vif). RESULTS IFN-gamma caused a dose-dependent reduction (>50%) of HBV DNA in the absence of cytotoxicity. Although iNOS mRNA increased 45-fold in IFN-gamma treated cells, NO2- was not detectable in supernatants and the use of an NO-donor did not inhibit HBV replication. A3 enzyme mRNAs varied between cells and were >10-fold higher in lymphocytes than in liver tissue. IFN-gamma up-regulated A3G mRNA by three-fold, associated with significant HBV DNA decrease. However, A3G degradation by Vif did not abolish the antiviral effect of IFN-gamma against HBV. CONCLUSIONS IFN-gamma inhibits HBV replication and up-regulates both iNOS and A3G. However, other pathways appear to have a greater role in IFN-gamma-induced HBV inactivation in the liver.

  • GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 2: Epidemiological distribution of o'nyong-nyong virus.
    Antivir. Res. (IF 4.130) Pub Date : 2019-09-24
    L Pezzi,A D LaBeaud,C B Reusken,J F Drexler,N Vasilakis,M Diallo,F Simon,T Jaenisch,P Gallian,A Sall,A B Failloux,S C Weaver,X de Lamballerie,

    The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to identify gaps of knowledge about the natural history, epidemiology and medical management of infection by these viruses, and to provide adapted recommendations for future investigations. Here, we present a report dedicated to ONNV epidemiological distribution. Two large-scale ONNV outbreaks have been identified in Africa in the last 60 years, interspersed with sporadic serosurveys and case reports of returning travelers. The assessment of the real scale of ONNV circulation in Africa remains a difficult task and surveillance studies are necessary to fill this gap. The identification of ONNV etiology is made complicated by the absence of multiplex tools in co-circulation areas and that of reference standards, as well as the high cross-reactivity with related pathogens observed in serological tests, in particular with CHIKV. This is a specific obstacle for seroprevalence studies, that necessitate an improvement of serological tools to provide robust results. The scarcity of existent genetic data currently limits molecular epidemiology studies. ONNV epidemiology would also benefit from reinforced entomological and environmental surveillance. Finally, the natural history of the disease deserves to be further investigated, with a specific attention paid to long-term complications. Considering our incomplete knowledge on ONNV distribution, GloPID-R CHIKV, ONNV and MAYV experts recommend that a major effort should be done to fill existing gaps.

  • GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 3: Epidemiological distribution of Mayaro virus.
    Antivir. Res. (IF 4.130) Pub Date : 2019-09-24
    L Pezzi,A J Rodriguez-Morales,C B Reusken,G S Ribeiro,A D LaBeaud,R Lourenço-de-Oliveira,P Brasil,M Lecuit,A B Failloux,P Gallian,T Jaenisch,F Simon,A M Siqueira,M G Rosa-Freitas,A Vega Rua,S C Weaver,J F Drexler,N Vasilakis,de Lamballerie X,

  • Activities of JNJ63623872 and oseltamivir against influenza A H1N1pdm and H3N2 virus infections in mice.
    Antivir. Res. (IF 4.130) Pub Date : 2016-10-25
    Donald F Smee,Dale L Barnard,Steven M Jones

    JNJ63623872 (formerly known as VX-787) is an inhibitor of influenza A virus polymerases through interaction with the viral PB2 subunit. This interaction blocks the cap-snatching activity of the virus that is essential for virus replication. Previously published work has documented antiviral activity of JNJ63623872 in cell culture and mouse infection studies. In this report, we extend the in vivo observations by comparing the efficacies of JNJ63623872 and oseltamivir in mice infected with influenza A/California/04/2009 (H1N1pdm) and A/Victoria/3/75 (H3N2) viruses. Animals received JNJ63623872 or oseltamivir orally twice daily for 10 days starting 2 h pre-infection. JNJ63623872 (2, 6, and 20 mg/kg/day) and oseltamivir (20 mg/kg/day) completely prevented death in the H1N1pdm virus infection. Weight loss at nadir was only 12% in mice receiving 2 mg/kg/day of JNJ63623872 compared to 23% and 32%, respectively, in oseltamivir-treated (20 mg/kg/day) and placebo groups. Lung hemorrhage scores, lung weights, and lung virus titers on day 6 were reduced in a dose-responsive manner by JNJ63623872 treatments, whereas oseltamivir treatments were not as effective. JNJ63623872 was less active against H3N2 virus infection, with more body weight loss occurring and only 30% survival at the 2-mg/kg/day dose. Lung scores, lung weights, and H3N2 viral titers in lungs of mice were reduced less by JNJ63623872 treatments compared to the H1N1pdm infection. Nevertheless, the 20-mg/kg/day dose of JNJ63623872 was more effective than oseltamivir (20 mg/kg/day) in improving body weight and reducing the severity of lung infection. JNJ63623872 appears to be an important new drug candidate to treat influenza A H1N1pdm and H3N2 virus infections.

  • Pharmacodynamics of folic acid receptor targeted antiretroviral nanotherapy in HIV-1-infected humanized mice.
    Antivir. Res. (IF 4.130) Pub Date : 2015-06-01
    Pavan Puligujja,Mariluz Araínga,Prasanta Dash,Diana Palandri,R Lee Mosley,Santhi Gorantla,Larisa Poluektova,JoEllyn McMillan,Howard E Gendelman

    Long-acting nanoformulated antiretroviral therapy (nanoART) can sustain plasma drug levels and improve its biodistribution. Cell targeted-nanoART can achieve this and bring drug efficiently to viral reservoirs. However, whether such improvements affect antiretroviral responses remains unknown. To these ends, we tested folic acid (FA)-linked poloxamer407-coated ritonavir-boosted atazanavir (FA-nanoATV/r) nanoparticles for their ability to affect chronic HIV-1 infection in humanized mice. Following three, 100mg/kg FA-nanoATV/r intramuscular injections administered every other week to infected animals, viral RNA was at or below the detection limit, cell-associated HIV-1p24 reduced and CD4+ T cell counts protected. The dosing regimen improved treatment outcomes more than two fold from untargeted nanoATV/r. We posit that these nanoformulations have potential for translation to human use.

  • Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc.
    Antivir. Res. (IF 4.130) Pub Date : 2014-12-03
    Olga Latinovic,Kate Schneider,Henryk Szmacinski,Joseph R Lakowicz,Alonso Heredia,Robert R Redfield

    The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies inhibit HIV-1 infection, suggesting a need for more potent reagents. Here we evaluated full length single chain (FLSC) IgG1, a novel IgG-CD4-gp120(BAL) fusion protein with several characteristics that make it an attractive candidate for treatment of HIV-1 infections, including bivalency and a potentially increased serum half-life over FLSC, the parental molecule. FLSC IgG1 binds two domains on CCR5, the N-terminus and the second extracellular loop, lowering the levels of available CCR5 viral attachment sites. Furthermore, FLSC IgG1 synergizes with Maraviroc (MVC), the only licensed CCR5 antagonist. In this study, we used both microscopy and functional assays to address the mechanistic aspects of the interactions of FLSC IgG1 and MVC in the context of CCR5 conformational changes and viral infection. We used a novel stochastic optical reconstruction microscopy (STORM), based on high resolution localization of photoswitchable dyes to visualize direct contacts between FLSC IgG1 and CCR5. We compared viral entry inhibition by FLSC IgG1 with that of other CCR5 blockers and showed FLSC IgG1 to be the most potent. We also showed that lower CCR5 surface densities in HIV-1 infected primary cells result in lower FLSC IgG1 EC50 values. In addition, CCR5 binding by FLSC IgG1, but not CCR5 Ab 2D7, was significantly increased when cells were treated with MVC, suggesting MVC allosterically increases exposure of the FLSC IgG1 binding site. These data have implications for future antiviral therapy development.

  • Spectrum of activity testing for therapeutics against all four dengue virus serotypes in AG129 mouse models: Proof-of-concept studies with the adenosine nucleoside inhibitor NITD-008.
    Antivir. Res. (IF 4.130) Pub Date : 2018-04-18
    Gregg N Milligan,Mellodee White,Diana Zavala,Richard B Pyles,Vanessa V Sarathy,Alan D T Barrett,Nigel Bourne

    Dengue is a mosquito-borne disease of global public health importance caused by four genetically and serologically related viruses (DENV-1 to DENV-4). Efforts to develop effective vaccines and therapeutics for dengue have been slowed by the paucity of preclinical models that mimic human disease. DENV-2 models in interferon receptor deficient AG129 mice were an important advance but only allowed testing against a single DENV serotype. We have developed complementary AG129 mouse models of severe disseminated dengue infection using strains of the other three DENV serotypes. Here we used the adenosine nucleoside inhibitor NITD-008 to show that these models provide the ability to perform comparative preclinical efficacy testing of candidate antivirals in vivo against the full-spectrum of DENV serotypes. Although NITD-008 was effective in modulating disease caused by all DENV serotypes, the variability in protection among DENV serotypes was greater than expected from differences in activity in in vitro testing studies emphasizing the need to undertake spectrum of activity testing to help in prioritization of candidate compounds for further development.

  • 4'-Azidocytidine (R1479) inhibits henipaviruses and other paramyxoviruses with high potency.
    Antivir. Res. (IF 4.130) Pub Date : 2017-06-21
    Anne L Hotard,Biao He,Stuart T Nichol,Christina F Spiropoulou,Michael K Lo

    The henipaviruses Nipah virus and Hendra virus are highly pathogenic zoonotic paramyxoviruses which have caused fatal outbreaks of encephalitis and respiratory disease in humans. Despite the availability of a licensed equine Hendra virus vaccine and a neutralizing monoclonal antibody shown to be efficacious against henipavirus infections in non-human primates, there remains no approved therapeutics or vaccines for human use. To explore the possibility of developing small-molecule nucleoside inhibitors against henipaviruses, we evaluated the antiviral activity of 4'-azidocytidine (R1479), a drug previously identified to inhibit flaviviruses, against henipaviruses along with other representative members of the family Paramyxoviridae. We observed similar levels of R1479 antiviral activity across the family, regardless of virus genus. Our brief study expands the documented range of viruses susceptible to R1479, and provides the basis for future investigation and development of 4'-modified nucleoside analogs as potential broad-spectrum antiviral therapeutics across both positive and negative-sense RNA virus families.

  • Successful strategies implemented towards the elimination of canine rabies in the Western Hemisphere.
    Antivir. Res. (IF 4.130) Pub Date : 2017-04-08
    Andres Velasco-Villa,Luis E Escobar,Anthony Sanchez,Mang Shi,Daniel G Streicker,Nadia F Gallardo-Romero,Fernando Vargas-Pino,Veronica Gutierrez-Cedillo,Inger Damon,Ginny Emerson

    Almost all cases of human rabies result from dog bites, making the elimination of canine rabies a global priority. During recent decades, many countries in the Western Hemisphere have carried out large-scale dog vaccination campaigns, controlled their free-ranging dog populations and enforced legislation for responsible pet ownership. This article reviews progress in eliminating canine rabies from the Western Hemisphere. After briefly summarizing the history of control efforts and describing the approaches listed above, we note that programs in some countries have been hindered by societal attitudes and severe economic disparities, which underlines the need to discuss measures that will be required to complete the elimination of canine rabies throughout the region. We also note that there is a constant threat for dog-maintained epizootics to re-occur, so as long as dog-maintained rabies "hot spots" are still present, free-roaming dog populations remain large, herd immunity becomes low and dog-derived rabies lyssavirus (RABLV) variants continue to circulate in close proximity to rabies-naïve dog populations. The elimination of dog-maintained rabies will be only feasible if both dog-maintained and dog-derived RABLV lineages and variants are permanently eliminated. This may be possible by keeping dog herd immunity above 70% at all times, fostering sustained laboratory-based surveillance through reliable rabies diagnosis and RABLV genetic typing in dogs, domestic animals and wildlife, as well as continuing to educate the population on the risk of rabies transmission, prevention and responsible pet ownership. Complete elimination of canine rabies requires permanent funding, with governments and people committed to make it a reality. An accompanying article reviews the history and epidemiology of canine rabies in the Western Hemisphere, beginning with its introduction during the period of European colonization, and discusses how spillovers of viruses between dogs and various wild carnivores will affect future eradication efforts (Velasco-Villa et al., 2017).

  • Antiviral lectins: Selective inhibitors of viral entry.
    Antivir. Res. (IF 4.130) Pub Date : 2017-03-23
    Carter A Mitchell,Koreen Ramessar,Barry R O'Keefe

    Many natural lectins have been reported to have antiviral activity. As some of these have been put forward as potential development candidates for preventing or treating viral infections, we have set out in this review to survey the literature on antiviral lectins. The review groups lectins by structural class and class of source organism we also detail their carbohydrate specificity and their reported antiviral activities. The review concludes with a brief discussion of several of the pertinent hurdles that heterologous proteins must clear to be useful clinical candidates and cites examples where such studies have been reported for antiviral lectins. Though the clearest path currently being followed is the use of antiviral lectins as anti-HIV microbicides via topical mucosal administration, some investigators have also found systemic efficacy against acute infections following subcutaneous administration.

  • Meeting report: Third Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS).
    Antivir. Res. (IF 4.130) Pub Date : 2016-12-13
    Stuart F J Le Grice,Joanna Sztuba-Solinska,Elias Maccioni,Katarzyna J Purzycka,Cristina Parolin,Enzo Tramontano

    The third Summer School on Innovative Approaches for Identification of Antiviral Agents (IAAASS) was held from September 28th to October 2nd, 2016 at the Sardegna Ricerche Research Park in Santa Margherita di Pula, Sardinia, Italy. The school brought together graduate students and postdoctoral fellows early in their careers with a faculty of internationally recognized experts, to encourage the sharing of knowledge and experience in virology research and drug development in an informal and interactive environment. The first IAAASS was held in Sardinia in 2012 and the second in 2014. The meetings provide a unique combination of plenary lectures on topics in virology, biochemistry, molecular modeling, crystallography and medicinal chemistry with small group sessions, in which students have the opportunity to ask questions and put forward their own ideas, and senior researchers offer advice, based on their own experience. This report summarizes presentations and presentations at the 3rd IAAASS.

  • Synergistic drug combination effectively blocks Ebola virus infection.
    Antivir. Res. (IF 4.130) Pub Date : 2016-11-29
    Wei Sun,Shihua He,Carles Martínez-Romero,Jennifer Kouznetsova,Gregory Tawa,Miao Xu,Paul Shinn,Ethan Fisher,Yan Long,Omid Motabar,Shu Yang,Philip E Sanderson,Peter R Williamson,Adolfo García-Sastre,Xiangguo Qiu,Wei Zheng

    Although a group of FDA-approved drugs were previously identified with activity against Ebola virus (EBOV), most of them are not clinically useful because their human blood concentrations are not high enough to inhibit EBOV infection. We screened 795 unique three-drug combinations in an EBOV entry assay. Two sets of three-drug combinations, toremifene-mefloquine-posaconazole and toremifene-clarithromycin-posaconazole, were identified that effectively blocked EBOV entry and were further validated for inhibition of live EBOV infection. The individual drug concentrations in the combinations were reduced to clinically relevant levels. We identified mechanisms of action of these drugs: functional inhibitions of Niemann-Pick C1, acid sphingomyelinase, and lysosomal calcium release. Our findings identify the drug combinations with potential to treat EBOV infection.

  • Amending Koch's postulates for viral disease: When "growth in pure culture" leads to a loss of virulence.
    Antivir. Res. (IF 4.130) Pub Date : 2016-11-12
    Joseph Prescott,Heinz Feldmann,David Safronetz

    It is a common laboratory practice to propagate viruses in cell culture. While convenient, these methodologies often result in unintentional genetic alterations, which have led to adaptation and even attenuation in animal models of disease. An example is the attenuation of hantaviruses (family: Bunyaviridae, genus: Hantavirus) when cultured in vitro. In this case, viruses propagated in the natural reservoir species cause disease in nonhuman primates that closely mimics the human disease, but passaging in cell culture attenuates these viruses to the extent that do not cause any measurable disease in nonhuman primates. As efforts to develop animal models progress, it will be important to take into account the influences that culture in vitro may have on the virulence of viruses. In this review we discuss this phenomenon in the context of past and recent examples in the published literature.

  • An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody with prophylactic and therapeutic activity in vivo.
    Antivir. Res. (IF 4.130) Pub Date : 2016-10-28
    Jason R Wilson,Zhu Guo,Adrian Reber,Ram P Kamal,Nedzad Music,Shane Gansebom,Yaohui Bai,Min Levine,Paul Carney,Wen-Pin Tzeng,James Stevens,Ian A York

    Zoonotic A(H7N9) avian influenza viruses emerged in China in 2013 and continue to be a threat to human public health, having infected over 800 individuals with a mortality rate approaching 40%. Treatment options for people infected with A(H7N9) include the use of neuraminidase (NA) inhibitors. However, like other influenza viruses, A(H7N9) can become resistant to these drugs. The use of monoclonal antibodies is a rapidly developing strategy for controlling influenza virus infection. Here we generated a murine monoclonal antibody (3c10-3) directed against the NA of A(H7N9) and show that prophylactic systemic administration of 3c10-3 fully protected mice from lethal challenge with wild-type A/Anhui/1/2013 (H7N9). Further, post-infection treatment with a single systemic dose of 3c10-3 at either 24, 48 or 72 h post A(H7N9) challenge resulted in both dose- and time-dependent protection of up to 100% of mice, demonstrating therapeutic potential for 3c10-3. Epitope mapping revealed that 3c10-3 binds near the enzyme active site of NA, and functional characterization showed that 3c10-3 inhibits the enzyme activity of NA and restricts the cell-to-cell spread of the virus in cultured cells. Affinity analysis also revealed that 3c10-3 binds equally well to recombinant NA of wild-type A/Anhui/1/2013 and to a variant NA carrying a R289K mutation known to infer NAI resistance. These results suggest that 3c10-3 has the potential to be used as a therapeutic to treat A(H7N9) infections either as an alternative to, or in combination with, current NA antiviral inhibitors.

  • A chronological review of experimental infection studies of the role of wild animals and livestock in the maintenance and transmission of Crimean-Congo hemorrhagic fever virus.
    Antivir. Res. (IF 4.130) Pub Date : 2016-10-28
    Jessica R Spengler,Agustín Estrada-Peña,Aura R Garrison,Connie Schmaljohn,Christina F Spiropoulou,Éric Bergeron,Dennis A Bente

    This article provides a definitive review of experimental studies of the role of wild animals and livestock in the maintenance and transmission of Crimean-Congo hemorrhagic fever virus (CCHFV), the etiologic agent of Crimean-Congo hemorrhagic fever (CCHF), beginning with the first recognized outbreak of the human disease in Crimea in 1944. Published reports by researchers in the former Soviet Union, Bulgaria, South Africa, and other countries where CCHF has been observed show that CCHFV is maintained in nature in a tick-vertebrate-tick enzootic cycle. Human disease most commonly results from the bite of an infected tick, but may also follow crushing of infected ticks or exposure to the blood and tissues of infected animals during slaughter. Wild and domestic animals are susceptible to infection with CCHFV, but do not develop clinical illness. Vertebrates are important in CCHF epidemiology, as they provide blood meals to support tick populations, transport ticks across wide geographic areas, and transmit CCHFV to ticks and humans during the period of viremia. Many aspects of vertebrate involvement in the maintenance and spread of CCHFV are still poorly understood. Experimental investigations in wild animals and livestock provide important data to aid our understanding of CCHFV ecology. This article is the second in a series of reviews of more than 70 years of research on CCHF, summarizing important findings, identifying gaps in knowledge, and suggesting directions for future research.

  • Signaling via pattern recognition receptors NOD2 and TLR2 contributes to immunomodulatory control of lethal pneumovirus infection.
    Antivir. Res. (IF 4.130) Pub Date : 2016-06-18
    Tyler A Rice,Todd A Brenner,Caroline M Percopo,Michelle Ma,Jesse D Keicher,Joseph B Domachowske,Helene F Rosenberg

    Pattern recognition receptors (PRRs) engage microbial components in the lung, although their role in providing primary host defense against respiratory virus infection is not fully understood. We have previously shown that Gram-positive Lactobacillus plantarum (Lp) administered to the respiratory tract promotes full and sustained protection in response to an otherwise lethal mouse pneumovirus (PVM) infection, a robust example of heterologous immunity. While Lp engages PRRs TLR2 and NOD2 in ex vivo signaling assays, we found that Lp-mediated protection was unimpaired in single gene-deleted TLR2(-/-) and NOD2(-/-) mice. Here we demonstrate substantial loss of Lp-mediated protection in a double gene-deleted NOD2(-/-)TLR2(-/-) strain. Furthermore, we demonstrate protection against PVM infection by administration of the bi-functional NOD2-TLR2 agonist, CL-429. The bi-functional NOD2-TLR2 ligand CL-429 not only suppresses virus-induced inflammation, it is significantly more effective at preventing lethal infection than equivalent amounts of mono-molecular TLR2 and NOD2 agonists. Interestingly, and in contrast to biochemical NOD2 and/or TLR2 agonists, Lp remained capable of eliciting primary proinflammatory responses from NOD2(-/-)TLR2(-/-) mice in vivo and from alveolar macrophages challenged ex vivo. Taken together, we conclude that coordinate engagement of NOD2 and TLR2 constitutes a key step in the genesis of Lp-mediated protection from a lethal respiratory virus infection, and represents a critical target for modulation of virus-induced inflammatory pathology.

Contents have been reproduced by permission of the publishers.