Favipiravir Inhibits Zika Virus (ZIKV) Replication in HeLa Cells by Altering Viral Infectivity.

This study aims to evaluate the antiviral potential of the nucleoside analogue favipiravir (FAV) against ZIKV, an arbovirus for which there are no approved antiviral therapies, in three human-derived cell lines. HeLa (cervical), SK-N-MC (neuronal), and HUH-7 (liver) cells were infected with ZIKV and exposed to different concentrations of FAV. Viral supernatant was sampled daily, and infectious viral burden was quantified by plaque assay. Changes in ZIKV infectivity were quantified by calculating specific infectivity. FAV-related toxicities were also assessed for each cell line in both infected and uninfected cells. Our results demonstrate that FAV activity was most pronounced in HeLa cells, as substantial declines in infectious titers and viral infectivity were observed in this cell type. The decline in infectious virus occurred in an exposure-dependent manner and was more pronounced as FAV exposure times increased. Additionally, toxicity studies showed that FAV was not toxic to any of the three cell lines and, surprisingly, caused substantial improvements in the viability of infected HeLa cells. Although SK-N-MC and HUH-7 cells were susceptible to FAV's anti-ZIKV activity, similar effects on viral infectivity and improvements in cell viability with therapy were not observed. These results indicate that FAV's ability to substantially alter viral infectivity is host cell specific and suggest that the robust antiviral effect observed in HeLa cells is mediated through drug-induced losses of viral infectivity.

Favipiravir Suppresses Zika Virus (ZIKV) through Activity as a Mutagen.

In a companion paper, we demonstrated that the nucleoside analogue favipiravir (FAV) suppressed Zika virus (ZIKV) replication in three human-derived cell lines-HeLa, SK-N-MC, and HUH-7. Our results revealed that FAV's effect was most pronounced in HeLa cells. In this work, we aimed to explain variation in FAV activity, investigating its mechanism of action and characterizing host cell factors relevant to tissue-specific differences in drug effect. Using viral genome sequencing, we show that FAV therapy was associated with an increase in the number of mutations and promoted the production of defective viral particles in all three cell lines. Our findings demonstrate that defective viral particles made up a larger portion of the viral population released from HeLa cells both at increasing FAV concentrations and at increasing exposure times. Taken together, our companion papers show that FAV acts via lethal mutagenesis against ZIKV and highlight the host cell's influence on the activation and antiviral activity of nucleoside analogues. Furthermore, the information gleaned from these companion papers can be applied to gain a more comprehensive understanding of the activity of nucleoside analogues and the impact of host cell factors against other viral infections for which we currently have no approved antiviral therapies.

Combination Therapy with UV-4B and Molnupiravir Enhances SARS-CoV-2 Suppression.

The host targeting antiviral, UV-4B, and the RNA polymerase inhibitor, molnupiravir, are two orally available, broad-spectrum antivirals that have demonstrated potent activity against SARS-CoV-2 as monotherapy. In this work, we evaluated the effectiveness of UV-4B and EIDD-1931 (molnupiravir's main circulating metabolite) combination regimens against the SARS-CoV-2 beta, delta, and omicron BA.2 variants in a human lung cell line. Infected ACE2 transfected A549 (ACE2-A549) cells were treated with UV-4B and EIDD-1931 both as monotherapy and in combination. Viral supernatant was sampled on day three when viral titers peaked in the no-treatment control arm, and levels of infectious virus were measured by plaque assay. The drug-drug effect interaction between UV-4B and EIDD-1931 was also defined using the Greco Universal Response Surface Approach (URSA) model. Antiviral evaluations demonstrated that treatment with UV-4B plus EIDD-1931 enhanced antiviral activity against all three variants relative to monotherapy. These results were in accordance with those obtained from the Greco model, as these identified the interaction between UV-4B and EIDD-1931 as additive against the beta and omicron variants and synergistic against the delta variant. Our findings highlight the anti-SARS-CoV-2 potential of UV-4B and EIDD-1931 combination regimens, and present combination therapy as a promising therapeutic strategy against SARS-CoV-2.

Structure-Based Design of Potent Iminosugar Inhibitors of Endoplasmic Reticulum α-Glucosidase I with Anti-SARS-CoV-2 Activity.

Enveloped viruses depend on the host endoplasmic reticulum (ER) quality control (QC) machinery for proper glycoprotein folding. The endoplasmic reticulum quality control (ERQC) enzyme α-glucosidase I (α-GluI) is an attractive target for developing broad-spectrum antivirals. We synthesized 28 inhibitors designed to interact with all four subsites of the α-GluI active site. These inhibitors are derivatives of the iminosugars 1-deoxynojirimycin (1-DNJ) and valiolamine. Crystal structures of ER α-GluI bound to 25 1-DNJ and three valiolamine derivatives revealed the basis for inhibitory potency. We established the structure-activity relationship (SAR) and used the Site Identification by Ligand Competitive Saturation (SILCS) method to develop a model for predicting α-GluI inhibition. We screened the compounds against SARS-CoV-2 in vitro to identify those with greater antiviral activity than the benchmark α-glucosidase inhibitor UV-4. These host-targeting compounds are candidates for investigation in animal models of SARS-CoV-2 and for testing against other viruses that rely on ERQC for correct glycoprotein folding.

Why Molnupiravir Fails in Hospitalized Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has radically altered daily life. Effective antiviral therapies to combat COVID-19, especially severe disease, remain scarce. Molnupiravir is an antiviral that has shown clinical efficacy against mild-to-moderate COVID-19 but failed to provide benefit to hospitalized patients with severe disease. Here, we explained the mechanism behind the failure of molnupiravir in hospitalized patients and identified alternative dosing strategies that would improve therapeutic outcomes in all patients with COVID-19. We showed that delaying therapy initiation markedly decreased the antiviral effect of molnupiravir, and these results were directly related to intracellular drug triphosphate pools and intracellular viral burden at the start of therapy. The adverse influence of therapeutic delay could be overcome by increasing drug exposure, which increased intracellular molnupiravir triphosphate concentrations that inhibited viral replication. These findings illustrated that molnupiravir must be administered as early as possible following COVID-19 symptom onset to maximize therapeutic efficacy. Higher doses may be effective in patients hospitalized with severe disease, but the safety of high-dose molnupiravir regimens is unknown. Our findings could be extended to design effective regimens with nucleoside analogs for other RNA viruses, especially those with pandemic potential. IMPORTANCE In this study, we showed that early intervention with molnupiravir resulted in a greater antiviral effect, and we explained the mechanism behind this phenomenon. Our results predicted and explained the failure of molnupiravir in hospitalized patients and highlighted the utility of preclinical pharmacodynamic studies to design optimal antiviral regimens for the treatment of viral diseases. This contrasts with the procedure that was implemented early in the pandemic in which clinical studies were conducted in the absence of preclinical experimentation. These findings are significant and demonstrated the importance of experimental approaches in antiviral development for treatments against COVID-19 as well as other viral diseases.

UV-4B potently inhibits replication of multiple SARS-CoV-2 strains in clinically relevant human cell lines.

BACKGROUND: SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic. Although it poses a substantial public health threat, antiviral regimens against SARS-CoV-2 remain scarce. Here, we evaluated the antiviral potential of UV-4B, a host targeting antiviral, against SARS-CoV-2 in clinically relevant human cell lines. METHODS: Cells derived from human lung (A549 cells transfected with human angiotensin converting enzyme 2 receptor (ACE2; ACE2-A549)) and colon (Caco-2) were infected with either a wild type or beta variant strain of SARS-CoV-2 and exposed to various concentrations of UV-4B. Supernatant was sampled daily and viral burden was quantified by plaque assay on Vero E6 cells. RESULTS: Therapeutically feasible concentrations of UV-4B inhibited the replication of the wild type strain in ACE2-A549 and Caco-2 cells yielding EC50 values of 2.694 and 2.489 µM, respectively. UV-4B's antiviral effect was also robust against the beta variant in both cell lines (ACE2-A549 EC50: 4.369 µM; Caco-2 EC50: 6.816 µM). CONCLUSIONS: These results highlight UV-4B's antiviral potential against several strains of SARS-CoV-2.

Extracorporeal photopheresis and multimodality therapy in patients with T-cell cutaneous lymphomas: Real-life experience in Argentina.

BACKGROUND: Extracorporeal photopheresis (ECP) as a part of multimodality therapy, is one of the treatments for Sézary syndrome (SS) and advanced stage mycosis fungoides (MF). This study aims to describe cutaneous and peripheral blood responses of patients with MF and SS who received multimodality therapy. METHODS: In this cross-sectional retrospective study, patients with MF or SS who received ECP treatment in combination with at least one additional systemic treatment between 2011 and 2018 were included. ECP consisted of a two-session cycle every 2 to 4 weeks. Cutaneous and blood responses were evaluated with updated criteria. RESULTS: Twenty-eight patients (11 (39%) with MF and 17 (51%) with SS) were included. Their median age at diagnosis was 63 (57-67) years. The median number of treatments before ECP was 2 (1-3). Seven out of 11 patients with MF (63%) underwent an assessment of cutaneous response. Five patients (70%) presented a partial response; 1 (15%), stable disease, and 1 (15%) progressive disease. Thirteen of the 17 patients with SS (76%) underwent evaluation. One patient (8%) presented a complete cutaneous response; 6 (46%), a partial response; 5 (38%), stable disease; and 1 (8%), progressive disease. None of them relapsed during the study period in both groups. No ECP-related adverse effects occurred during the study. CONCLUSION: Most patients with SS and MF who underwent multimodality therapy with ECP had favorable cutaneous and blood response. It is safe to combine ECP with other treatments. Studies with large numbers of patients are necessary to assess the effects of ECP on patient survival.

Antiviral Evaluation of UV-4B and Interferon-Alpha Combination Regimens against Dengue Virus.

Dengue virus (DENV) is a flavivirus associated with clinical manifestations ranging in severity from self-limiting dengue fever, to the potentially life threatening condition, severe dengue. There are currently no approved antiviral therapies for the treatment of DENV. Here, we evaluated the antiviral potential of four broad-spectrum antivirals, UV-4B, interferon-alpha (IFN), sofosbuvir (SOF), and favipiravir (FAV) against DENV serotype 2 as mono- and combination therapy in cell lines that are physiologically relevant to human infection. Cell lines derived from human liver (HUH-7), neurons (SK-N-MC), and skin (HFF-1) were infected with DENV and treated with UV-4B, IFN, SOF, or FAV. Viral supernatant was sampled daily and infectious viral burden was quantified by plaque assay on Vero cells. Drug effect on cell proliferation in uninfected and infected cells was also assessed. UV-4B inhibited DENV in HUH-7, SK-N-MC, and HFF-1 cells yielding EC50 values of 23.75, 49.44, and 37.38 µM, respectively. Clinically achievable IFN concentrations substantially reduced viral burden in HUH-7 (EC50 = 102.7 IU/mL), SK-N-MC (EC50 = 86.59 IU/mL), and HFF-1 (EC50 = 163.1 IU/mL) cells. SOF potently inhibited DENV in HUH-7 cells but failed to produce the same effect in SK-N-MC and HFF-1 cells. Finally, FAV provided minimal suppression in HUH-7 and SK-N-MC cells, but was ineffective in HFF-1 cells. The two most potent anti-DENV agents, UV-4B and IFN, were also assessed in combination. UV-4B + IFN treatment enhanced antiviral activity in HUH-7, SK-N-MC, and HFF-1 cells relative to monotherapy. Our results demonstrate the antiviral potential of UV-4B and IFN against DENV in multiple physiologically relevant cell types.

Combination Regimens of Favipiravir Plus Interferon Alpha Inhibit Chikungunya Virus Replication in Clinically Relevant Human Cell Lines.

Chikungunya virus (CHIKV) is an alphavirus associated with a broad tissue tropism for which no antivirals or vaccines are approved. This study evaluated the antiviral potential of favipiravir (FAV), interferon-alpha (IFN), and ribavirin (RBV) against CHIKV as mono- and combination-therapy in cell lines that are clinically relevant to human infection. Cells derived from human connective tissue (HT-1080), neurons (SK-N-MC), and skin (HFF-1) were infected with CHIKV and treated with different concentrations of FAV, IFN, or RBV. Viral supernatant was sampled daily and the burden was quantified by plaque assay on Vero cells. FAV and IFN were the most effective against CHIKV on various cell lines, suppressing the viral burden at clinically achievable concentrations; although the degree of antiviral activity was heavily influenced by cell type. RBV was not effective and demonstrated substantial toxicity, indicating that it is not a feasible candidate for CHIKV. The combination of FAV and IFN was then assessed on all cell lines. Combination therapy enhanced antiviral activity in HT-1080 and SK-N-MC cells, but not in HFF-1 cells. We developed a pharmacokinetic/pharmacodynamic model that described the viral burden and inhibitory antiviral effect. Simulations from this model predicted clinically relevant concentrations of FAV plus IFN completely suppressed CHIKV replication in HT-1080 cells, and considerably slowed down the rate of viral replication in SK-N-MC cells. The model predicted substantial inhibition of viral replication by clinical IFN regimens in HFF-1 cells. Our results highlight the antiviral potential of FAV and IFN combination regimens against CHIKV in clinically relevant cell types.

Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody.

RATIONALE: The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. METHODS: Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo. RESULTS: Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. CONCLUSIONS: These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.

Use of afoxolaner for the treatment of lice (Goniodes pavonis) in different genera (Chrysolophus spp, Lophura spp, Phasianus spp, and Syrmaticus spp) and species of pheasants and West Mexican Chachalacas (Ortalis poliocephala).

Lice are ectoparasites capable of affecting birds, and can result in direct and indirect damage to their host. Afoxolaner is an isoxazoline that has been shown to be effective against these ectoparasites without known adverse effects. The objective of this research was to evaluate the effect of afoxolaner on lice in pheasants and plain chachalacas. A total of 29 pheasants of different genera and species (Chrysolophus pictus, C. amherstiae, Lophura swinghoii, L. nycthemera, Phasianus colchicus, and Syrmaticus reevesii) and 18 West Mexican Chachalacas (Ortalis poliocephala) naturally infested with Goniodes pavonis were used. The birds were allocated to one of two groups: group 1 treated with 2.50 mg/kg of afoxolaner, and group 2 given no treatment. Ectoparasites were collected using the adhesive tape technique and identified. Afoxolaner was administered later as a single dose to group 1, and the clinical assessment to detect ectoparasites was repeated 28 days post-treatment. On day 28 post-treatment, group 1 was found to be negative for the presence of lice. The body weights were compared at the beginning and end of the clinical assessment in both groups and a significant difference in weight of treated birds was found. The mean body weight decreased by 0.017 g in control group, whereas it increased by 0.016 g in treated group. Oral administration of afoxolaner is an effective option for the treatment of Goniodes pavonis infestations in zoo birds.

Primary cutaneous lymphoma in Argentina: a report of a nationwide study of 416 patients.

BACKGROUND: The aim of this study was to determine the relative frequency of primary cutaneous lymphoma (PCL) in Argentina according to the new World Health Organization (WHO)-European Organization for the Research and Treatment of Cancer (EORTC) classification system. METHODS: A total of 416 patients from 21 dermatology services were included during a 5-year period (2010-2015); these patients were classified using WHO-EORTC criteria. RESULTS: There were 231 (55.2%) males and 185 (44.8%) females; the male-to-female ratio was 1.35. The median age of the patients was 57 years (range, 0-90 years). Most patients were Caucasian (79%), and only 16% of patients were registered as Amerindian. Most patients (387/416, 93%) had cutaneous T-cell lymphoma (CTCL); 28 patients (6.7%) were diagnosed with cutaneous B-cell lymphoma (CBCL). The most frequent CTCL subtypes, in decreasing order of prevalence, were mycosis fungoides (MF), including its variants (75.7%); CD30+ primary cutaneous lymphoproliferative disorders (7.2%); and Sézary syndrome (SS) (3.1%). Cutaneous follicle center lymphoma was the most common CBCL subtype (2.9%). In the subset of patients ≤20 years of age, the most common condition was MF (57%), followed by extranodal NK-T nasal-type lymphoma (14%). CONCLUSIONS: This study revealed relatively higher rates of MF and lower rates of CBCL in Argentinean patients that have been reported in American and European countries.

Sobrecarga del cuidador informal de adultos mayores internados en el servicio de clínica médica del hospital Geriátrico "Juana Francisca Cabral". Año 2018 / Overload of the informal caregiver of older adults admitted to the medical clinic service of the "Juana Francisca Cabral" Geriatric Hospital. Year 2018

Introducción: El incremento en las tasas de supervivencia a enfermedades crónicas y discapacidades físicas o psíquicas, ha elevado el número de personas dedicadas a cuidar de familiares enfermos. Asistir a una persona envejecida dependiente es una tarea difícil de gran responsabilidad, que demanda actividades para las que no se está preparado, que no siempre son agradables. La carga es percibida por los cuidadores de diferentes maneras, en función al rol del cuidador Objetivo: Describir la existencia de sobrecarga en cuidadores informales de adultos mayores internados en el hospital geriátrico de agudos "Juana Francisca Cabral" entre agosto a octubre de 2018. Material y métodos: Estudio cuantitativo descriptivo, transversal. Se aplico instrumento de recolección de datos, durante agosto, septiembre del 2018, en servicio de internación de agudos. Resultado: En muestra conformada por 67 cuidadores informales. se observó edad promedio de 43 años, predominaba el sexo femenino con el 58%. Prevalecían hijos, parejas que son quienes proporcionan los cuidados. La mayoría de los cuidadores no presentaban capacitación para el cuidado del adulto mayor. Según el nivel de instrucción el 52% había concluido los estudios secundarios. El 43% consideraba como buena su salud. Según el nivel de sobrecarga de los cuidadores informales el 70% de los cuidadores presentaba sobrecarga. Conclusión: Se observa con frecuencia pacientes dependendientes, donde 7 de 10 cuidadores presentan sobrecarga percibiendo su salud como buena a regular y no hay relación entre el tiempo del cuidado y sobrecarga, ya que no se evidencia que a mayor tiempo de cuidado presenten sobrecarga

Abstract: The increase in the rates of survival to chronic diseases and physical or mental disabilities has increased the number of people dedicated to caring for sick family members. Assisting a dependent elderly person is a difficult task of great responsibility, which demands activities for which one is not prepared and which are not always pleasant. Objective: To describe the existence of overload in informal caregivers of elderly people interned in the "Juana Francisca Cabral" geriatric acute care hospital during the period from August to October 2018. Material and methods: This is a quantitative, descriptive, prospective, cross-sectional study. The sample consisted of 67 informal caregivers. Result: an average age of 43 years was observed, 58% of the caregivers were female. In terms of kinship, 39% belonged to the child category. It was found that 99% did not have any training towards the care of the elderly. According to the level of education, 52% had finished secondary school. 43% considered their health "good" According to the level of overload of the informal caregivers, 40% of the caregivers presented slight overload, while 30% of the overloading was intense. Conclusion: A patient with dependence is observed with great frequency, where 7 out of 10 caregivers present overload perceiving their health as good to be regular and we identify that there is no relationship between the time of care and overload, since it is not evident that a longer time of Beware of overload

The effectiveness of antiviral agents with broad-spectrum activity against chikungunya virus varies between host cell lines.

Chikungunya virus (CHIKV) is a mosquito-borne virus that has recently emerged in the Western Hemisphere. Approved antiviral therapies or vaccines for the treatment or prevention of CHIKV infections are not available. This study aims to evaluate the antiviral activity of commercially available broad-spectrum antivirals against CHIKV. Due to host cell-specific variability in uptake and intracellular processing of drug, we evaluated the antiviral effects of each agent in three cell lines. Antiviral activities of ribavirin (RBV), interferon-alfa (IFN-α) and favipiravir (FAV) were assessed in CHIKV-infected Vero, HUH-7, and A549 cells. CHIKV-infected cells were treated with increasing concentrations of each agent for three days and viral burden was quantified by plaque assay on Vero cells. Cytotoxic effects of RBV, FAV and IFN-α were also evaluated. Antiviral activity differed depending on the cell line used for evaluation. RBV had the greatest antiviral effect in HUH-7 cells (EC50 = 2.575 µg/mL); IFN-α was most effective in A549 cells (EC50 = 4.235 IU/mL); and FAV in HUH-7 cells (EC50 = 20.00 µg/mL). The results of our study show FAV and IFN-α are the most promising candidates, as their use led to substantial reductions in viral burden at clinically achievable concentrations in two human-derived cell lines. FAV is an especially attractive candidate for further investigation due to its oral bioavailability. These findings also highlight the importance of cell line selection for preclinical drug trials.