Joana Rocha-Pereira

Favipiravir (T-705) inhibits in vitro norovirus replication

Human noroviruses are the primary cause of foodborne gastroenteritis. Potent and safe inhibitors are needed for the treatment/prophylaxis of norovirus infections. We demonstrate that Favipiravir [T-705, a drug in advanced clinical development for the treatment of infections with the influenza virus] inhibits in vitro murine norovirus replication. Time-of-drug addition studies reveal that T-705 exerts its activity at a time-point that coincides with onset of viral RNA synthesis, which is in line with the viral polymerase as the presumed target.

The Viral Polymerase Inhibitor 2′-C-Methylcytidine Inhibits Norwalk Virus Replication and Protects against Norovirus-Induced Diarrhea and Mortality in a Mouse Model

ABSTRACT Human noroviruses are a major cause of food-borne illness, accountable for 50% of all-etiologies outbreaks of acute gastroenteritis (in both developing and developed countries). There is no vaccine or antiviral drug for the prophylaxis or treatment of norovirus-induced gastroenteritis. We recently reported the inhibitory effect of 2′-C-methylcytidine (2CMC), a hepatitis C virus polymerase inhibitor, on the in vitro replication of murine norovirus (MNV). Here we evaluated the inhibitory effect of 2CMC on in vitro human norovirus replication through a Norwalk virus replicon model and in a mouse model by using AG129 mice orally infected with MNV. Survival, weight, and fecal consistency were monitored, and viral loads in stool samples and organs were quantified. Intestines were examined histologically. 2CMC reduced Norwalk virus replicon replication in a dose-dependent manner and was able to clear cells of the replicon. Treatment of MNV-infected AG129 mice with 2CMC (i) prevented norovirus-induced diarrhea; (ii) markedly delayed the appearance of viral RNA and reduced viral RNA titers in the intestine, mesenteric lymph nodes, spleen, lungs, and stool; (iii) completely prevented virus-induced mortality; and (iv) resulted in protective immunity against a rechallenge. We demonstrate for the first time that a small-molecule inhibitor of norovirus replication protects from virus-induced disease and mortality in a relevant animal model. These findings pave the way for the development of potent and safe antivirals as prophylaxis and therapy of norovirus infection.

Norovirus: Targets and tools in antiviral drug discovery

Abstract The development of antiviral strategies to treat or prevent norovirus infections is a pressing matter. Noroviruses are the number 1 cause of acute gastroenteritis, of foodborne illness, of sporadic gastroenteritis in all age groups and of severe acute gastroenteritis in children less than 5 years old seeking medical assistance [USA/CDC]. In developing countries, noroviruses are linked to significant mortality (∼200000 children <5 years old). Noroviruses are a major culprit for the closure of hospital wards, and associated with increased hospitalization and mortality among the elderly. Transplant patients have significant risk of acquiring persistent norovirus gastroenteritis. Control and prevention strategies are limited to the use of disinfectants and hand sanitizers, whose efficacy is frequently insufficient. Hence, there is an ample need for antiviral treatment and prophylaxis of norovirus infections. The fact that only a handful of inhibitors of norovirus replication have been reported can largely be attributable to the hampering inability to cultivate human noroviruses in cell culture. The Norwalk replicon-bearing cells and the murine norovirus-infected cell lines are the available models to assess in vitro antiviral activity of compounds. Human noroviruses have been shown to replicate (to some extent) in mice, calves, gnotobiotic pigs, and chimpanzees. Infection of interferon-deficient mice with the murine norovirus results in virus-induced diarrhea. Here we review recent developments in understanding which norovirus proteins or host cell factors may serve as targets for inhibition of viral replication. Given the recent advances, significant progress in the search for antiviral strategies against norovirus infections is expected in the upcoming years.

Molecular Chaperone Hsp90 Is a Therapeutic Target for Noroviruses

ABSTRACT Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5′ and 3′ extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients.

The Enterovirus Protease Inhibitor Rupintrivir Exerts Cross-Genotypic Anti-Norovirus Activity and Clears Cells from the Norovirus Replicon

ABSTRACT Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2′-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

Outbreak of acute gastroenteritis caused by adenovirus type 41 in a kindergarten

In response to an alert due to epidemic gastroenteritis in children in a kindergarten, an outbreak investigation was carried out in a Portuguese municipality. The objectives were to establish an aetiological diagnosis, assess vaccine efficacy if possible, and to take corrective measures if necessary. The warden at the kindergarten was interviewed, and we visited the premises. The overall attack rate was 11·4% and most cases were mild. Stool samples from three symptomatic children were collected and screened for the presence of noroviruses, rotaviruses and adenoviruses. High vaccination coverage against rotaviruses was recorded in children aged <2 years. We initially thought that noroviruses and rotaviruses were more likely to have been the aetiological cause of the disease, but the outbreak was caused by infection with adenovirus 41. These viruses should not be overlooked in the laboratory protocol in the study of acute gastroenteritis outbreaks.

Norovirus genetic diversity and evolution: implications for antiviral therapy.

Human noroviruses are the leading cause of foodborne illness causing both acute and chronic gastroenteritis. In recent years, a number of vaccine candidates entered (pre-) clinical development and the first efforts to develop antiviral therapy have been made. We here discuss aspects of norovirus genetic evolution, persistence in immunocompromised patients as well as the risk and potential consequences of resistance development toward future antiviral drugs.

Interferon lambda (IFN-λ) efficiently blocks norovirus transmission in a mouse model

ABSTRACT Human noroviruses are highly efficient in person to person transmission thus associated with explosive outbreaks of acute gastroenteritis. Outbreak control is limited to disinfection and isolation measures. Strategies to control the spread of noroviruses should be developed and models to study norovirus transmission will greatly facilitate this. Here, a mouse‐to‐mouse transmission model, in which mice develop acute murine norovirus (MNV)‐induced diarrhea, was used to explore the role of interferon lambda (IFN‐&lgr;) in the control of a norovirus infection. Sentinel AG129 mice [deficient in IFN‐&agr;/&bgr; and IFN‐&ggr; receptors] that were co‐housed with MNV‐infected mice shedding high amounts of virus in their stool, developed a MNV‐infection with associated diarrhea. Inoculation of such sentinel mice with an IFN‐&lgr; expression plasmid resulted in the production of circulating IFN‐&lgr; and upregulation of the expression of IFN‐stimulated genes (ISGs) of the gut. Injection of the IFN‐&lgr;‐expressing plasmid to sentinels prevents MNV‐induced disease upon exposure to MNV‐infected mice, as well as MNV replication in the small intestine, the associated signs of inflammation and the mounting of a specific IgG‐based immune response. This demonstrates that IFN‐&lgr; can alone mediate protection against transmission of norovirus. The development of a simple delivery method for IFN‐&lgr; could be explored as a strategy to control norovirus outbreaks and protect vulnerable populations such as the elderly and immunocompromised. HighlightsNorovirus causes large gastroenteritis outbreaks with person‐to‐person transmission.We explored the effect of IFN‐&lgr; on an acute (diarrhea‐causing) norovirus infection.IFN‐&lgr; plasmid inoculation protected sentinels against mouse‐to‐mouse transmission.IFN‐&lgr;‐prophylaxis is able to efficiently prevent infection with noroviruses.

Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-dihydropyrimidinones

Abstract A series of 4-substituted 3,4-dihydropyrimidine-2-ones (DHPM) was synthesized, characterized by IR, 1H NMR, 13C NMR and HRMS spectra. The compounds were evaluated in vitro for their antiviral activity against a broad range of DNA and RNA viruses, along with assessment for potential cytotoxicity in diverse mammalian cell lines. Compound 4m, which possesses a long lipophilic side chain, was found to be a potent and selective inhibitor of Punta Toro virus, a member of the Bunyaviridae. For Rift Valley fever virus, which is another Bunyavirus, the activity of 4m was negligible. DHPMs with a C-4 aryl moiety bearing halogen substitution (4b, 4c and 4d) were found to be cytotoxic in MT4 cells.

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs

Gastroenteritis is globally responsible for great morbidity and mortality among all ages. In the developing countries, it still represents one of the top causes of death for children <5 years of age, resulting in 1,8 million fatalities every year (Boschi-Pinto et al., 2008; Bryce et al., 2005). Viruses are responsible for the majority of cases of gastroenteritis with rotaviruses and noroviruses being the major pathogens.