Protocol to evaluate the antiviral effect of FDA-approved drugs against dengue virus in Huh7 cells and AG129 mice.

Finding an effective therapy against diseases caused by flaviviruses remains a challenge. Here, we present a protocol to test Food and Drug Administration-approved drugs that inhibit host nuclear protein import, promoting a reduction of dengue infection. We describe steps for analyzing the drug effect on nuclear import inhibition of cellular and viral proteins by confocal microscopy or western blotting. We then describe procedures for measuring the antiviral drug effects on virus-infected cells by flow cytometry and testing drug efficacy in dengue-infected AG129 mice by survival assays. For complete details on the use and execution of this protocol, please refer to Palacios-Rápalo et al.1.

A Dual Pharmacological Strategy against COVID-19: The Therapeutic Potential of Metformin and Atorvastatin.

Metformin (MET) and atorvastatin (ATO) are promising treatments for COVID-19. This review explores the potential of MET and ATO, commonly prescribed for diabetes and dyslipidemia, respectively, as versatile medicines against SARS-CoV-2. Due to their immunomodulatory and antiviral capabilities, as well as their cost-effectiveness and ubiquitous availability, they are highly suitable options for treating the virus. MET's effect extends beyond managing blood sugar, impacting pathways that can potentially decrease the severity and fatality rates linked with COVID-19. It can partially block mitochondrial complex I and stimulate AMPK, which indicates that it can be used more widely in managing viral infections. ATO, however, impacts cholesterol metabolism, a crucial element of the viral replicative cycle, and demonstrates anti-inflammatory characteristics that could modulate intense immune reactions in individuals with COVID-19. Retrospective investigations and clinical trials show decreased hospitalizations, severity, and mortality rates in patients receiving these medications. Nevertheless, the journey from observing something to applying it in a therapeutic setting is intricate, and the inherent diversity of the data necessitates carefully executed, forward-looking clinical trials. This review highlights the requirement for efficacious, easily obtainable, and secure COVID-19 therapeutics and identifies MET and ATO as promising treatments in this worldwide health emergency.

The Role of Aspartate Aminotransferase-to-Lymphocyte Ratio Index (ALRI) in Predicting Mortality in SARS-CoV-2 Infection.

COVID-19 has a mortality rate exceeding 5.4 million worldwide. The early identification of patients at a high risk of mortality is essential to save their lives. The AST-to-lymphocyte ratio index (ALRI) is a novel biomarker of survival in patients with hepatocellular carcinoma, an organ susceptible to SARS-CoV-2 infection. For this study, the prognostic value of ALRI as a marker of COVID-19 mortality was evaluated. For this purpose, ALRI was compared with the main biomarkers for COVID-19 mortality (neutrophil-to-lymphocyte ratio [NLR], systemic immune-inflammation index [SII], platelet-to-lymphocyte ratio [PLR], lactate dehydrogenase (LDH)/lymphocyte ratio [LDH/LR]). A retrospective cohort of 225 patients with SARS-CoV-2 infection and without chronic liver disease was evaluated. In the non-survival group, the ALRI, NLR, SII, and LDH/LR were significantly higher than in the survival group (pcorrected < 0.05). ALRI had an area under the curve (AUC) of 0.81, a sensitivity of 70.37%, and a specificity of 75%, with a best cut-off value >42.42. COVID-19 patients with high ALRI levels had a mean survival time of 7.8 days. Multivariate Cox regression revealed that ALRI > 42.42 (HR = 2.32, 95% CI: 1.35-3.97; pcorrected = 0.01) was a prognostic factor of COVID-19 mortality. These findings prove that ALRI is an independent predictor of COVID-19 mortality and that it may help identify high-risk subjects with SARS-CoV-2 infection upon admission.

An ivermectin - atorvastatin combination impairs nuclear transport inhibiting dengue infection in vitro and in vivo.

Dengue virus (DENV) uses cellular nuclear transport machinery to import some proteins into the nucleus. Recently, the non-structural protein 3 (NS3) of DENV was localized in the nucleus of infected cells; however, its nuclear import mechanism is still unknown. In this study, we demonstrate that Ivermectin (IVM) inhibits the nuclear localization of NS3 through the inhibition of the Importin α/ß1 pathway. We also report that Atorvastatin (ATV) can modulate the nuclear transport of NS3 protease and NS5 polymerase of DENV-2. On the other hand, we found that IVM and ATV treatments reduce the alteration of nuclear pore complex (NPC) proteins, and an IVM+ATV combination reduced DENV infection both in vitro and in vivo. Hence, we conclude that ATV transport inhibition is an additional antiviral effect of this drug, suggesting a potential anti-DENV therapy in combination with IVM.

The ∆Pv-aCO2/∆Ca-vO2 ratio as a predictor of mortality in patients with severe acute respiratory distress syndrome related to COVID-19.

OBJECTIVE: To evaluate the central venous-to-arterial carbon dioxide difference combined with arterial-to-venous oxygen content difference (∆Pv-aCO2/∆Ca-vO2 ratio) as a predictor of mortality in patients with COVID-19-related severe acute respiratory distress syndrome (ARDS). METHODS: Patients admitted to the intensive care unit with severe ARDS secondary to SARS-CoV-2, and invasive mechanical ventilation were included in this single-center and retrospective cohort study performed between April 18, 2020, and January 18, 2022. The tissue perfusion indexes (lactate, central venous oxygen saturation [ScvO2], and venous-to-arterial carbon dioxide pressure difference [∆Pv-aCO2]), anaerobic metabolism index (∆Pv-aCO2/∆Ca-vO2 ratio), and severity index (Simplified Acute Physiology Score II [SAPSII]) were evaluated to determine its association with the mortality through Cox regression analysis, Kaplan-Meier curve and receiver operating characteristic (ROC) curve. RESULTS: One hundred fifteen patients were included in the study and classified into two groups, the survivor group (n = 54) and the non-survivor group (n = 61). The lactate, ScvO2, ∆Pv-aCO2, and ∆Pv-aCO2/∆Ca-vO2 ratio medians were 1.6 mEq/L, 75%, 5 mmHg, and 1.56 mmHg/mL, respectively. The ∆Pv-aCO2/∆Ca-vO2 ratio (Hazard Ratio (HR) = 1.17, 95% confidence interval (CI) = 1.06-1.29, p = 0.001) was identified as a mortality biomarker for patients with COVID-19-related severe ARDS. The area under the curve for ∆Pv-aCO2/∆Ca-vO2 ratio was 0.691 (95% CI 0.598-0.774, p = 0.0001). The best cut-off point for ∆Pv-aCO2/∆Ca-vO2 ratio was >2.14 mmHg/mL, with a sensitivity of 49.18%, specificity of 85.19%, a positive likelihood of 3.32, and a negative likelihood of 0.6. The Kaplan-Meier curve showed that survival rates were significantly worse in patients with values greater than this cut-off point. CONCLUSIONS: The ∆Pv-aCO2/∆Ca-vO2 ratio could be used as a predictor of mortality in patients with severe ARDS secondary to SARS-CoV-2.

Cholesterol-Lowering Drugs as Potential Antivirals: A Repurposing Approach against Flavivirus Infections.

Flaviviruses, including Dengue (DENV), Zika (ZIKV), and Yellow Fever (YFV) viruses, represent a significant global health burden. The development of effective antiviral therapies against these viruses is crucial to mitigate their impact. This study investigated the antiviral potential of the cholesterol-lowering drugs atorvastatin and ezetimibe in monotherapy and combination against DENV, ZIKV, and YFV. In vitro results demonstrated a dose-dependent reduction in the percentage of infected cells for both drugs. The combination of atorvastatin and ezetimibe showed a synergistic effect against DENV 2, an additive effect against DENV 4 and ZIKV, and an antagonistic effect against YFV. In AG129 mice infected with DENV 2, monotherapy with atorvastatin or ezetimibe significantly reduced clinical signs and increased survival. However, the combination of both drugs did not significantly affect survival. This study provides valuable insights into the potential of atorvastatin and ezetimibe as antiviral agents against flaviviruses and highlights the need for further investigations into their combined therapeutic effects.

External Validation of Two Scoring Tools to Predict the Operative Duration and Open Conversion of Elective Laparoscopic Cholecystectomy in a Mexican Population.

INTRODUCTION: This study aimed to evaluate the use of laparoscopic cholecystectomy (LC) operative time (CholeS score) and conversion to an open procedure (CLOC score) outside their validation dataset in Mexican population. METHODS: Patients >18 years who underwent elective LC were analyzed in a single-center retrospective chart review study. Association between scores (CholeS and CLOC) with operative time and conversion to open procedures was assessed with Spearman correlation. The predictive accuracy of the CholeS score and CLOC score was evaluated by receiver operator characteristic. RESULTS: 200 patients were included in the study (33 excluded for emergency case or missing data). Spearman coefficient correlations between CholeS or CLOC score and operative time were 0.456 (p < 0.0001) and 0.356 (p < 0.0001), respectively. Area under the curve (AUC) for operative prediction time (>90 min) by CholeS score was 0.786 with a 3.5-point cutoff (80% sensitivity and 63.2% specificity). AUC for open conversion (CLOC score) was 0.78 with a 5-point cutoff (60% sensitivity and 91% specificity). The CLOC score had a 0.740 AUC (64% sensitivity and 72.8% specificity) for operative time >90 min. CONCLUSIONS: The CholeS and the CLOC scores predicted LC long operative time and risk for conversion to an open procedure, respectively, outside their original validation set.

Sperm concentrations do not correlate with semen parameters and hormone profiles in males recovered from COVID-19.

Background: Although the negative impact on fertility of men recovered from coronavirus disease 2019 (COVID-19) has been suggested, there is insufficient evidence, and the data are limited and contradictory. The present prospective study aimed to evaluate the sex-related hormones, semen parameters, erectile dysfunction (ED), and lower urinary tract symptoms (LUTS) in a cohort of men who recovered from COVID-19 and age-matched control men. Methods: Semen samples were collected from twenty-two men recovered from COVID-19 with a median time of 91.5 days and thirty-six control males. The semen parameters were evaluated according to the World Health Organization (WHO) laboratory manual to examine and process human semen. The blood samples were collected to assess the male hormone profile. ED and LUTS were evaluated with the International Index of Erectile Function 5 (IIEF-5) and the International Prostate Symptom Score (IPSS), respectively. Results: The follicle-stimulating hormone (FSH) (3.819±1.515 IU/L), luteinizing hormone (LH) (4.023±1.792 IU/L), prolactin (PRL) [12.60 (10.72-15.20) ng/mL], and testosterone (T) [4.345 (3.565-5.525) ng/mL] levels were at normal range in all males enrolled in the study. Levels of semen volume (control: 2.5 mL vs. COVID-19: 1.9 mL; P<0.05) and sperm concentration (control: 59×106/mL vs. COVID-19: 41.5×106/mL; P<0.005) were significantly lower in males recovered from COVID-19, but still technically well within normal regardless of WHO edition. All variables were examined through logistic regression analysis, demonstrating that only sperm concentration was an independent variable associated with men recovered from COVID-19 [odds ratio (OR) =1; 95% confidence interval (CI): 0.999-1.098; P=0.016]. According to correlation analysis, there was no correlation between sperm concentration and other semen parameters and sex-related hormone profiles. Furthermore, an absence of ED and LUTS in men who recovered from COVID-19 was evidenced using the IIEF-5 and IPSS, respectively. Conclusions: Reproductive-age males recovered from COVID-19 have normal sperm concentration. Sperm concentration did not correlate with other semen parameters, sex-related hormones, IIEF-5, and IPSS. Further studies should be performed to evaluate whether the lower sperm concentration and semen volume that were still within the normal range are a transient or prolonged downregulation resulting from the COVID-19 attack.

Encephalitis Associated With SARS-CoV-2 Infection in a Child With Chiari Malformation Type I.

BACKGROUND/AIM: There is increasing evidence that patients infected with SARS-CoV-2 develop neurological manifestations such as encephalitis. The purpose of this article was to present a case of viral encephalitis associated with SARS-CoV-2 in a 14-year-old child with Chiari malformation type I. CASE REPORT: The patient manifested frontal headache, nausea, vomiting, skin pallor, right side Babinski sign and was diagnosed with Chiari malformation type I. He was admitted with generalized seizures and suspected encephalitis. Brain inflammation and viral RNA in the cerebrospinal fluid suggested SARS-CoV-2 encephalitis. These findings indicate that the SARS-CoV-2 test in CSF of patients with neurological manifestations, confusion, and fever during the COVID-19 pandemic should be carried out even when there is no evidence of respiratory infection. To our knowledge, this presentation of encephalitis associated with COVID-19 has not yet been reported in a patient with a congenital syndrome such as Chiari malformation type I. CONCLUSION: Further clinical data are needed to determine the complications of encephalitis due to SARS-CoV-2 in patients with Chiari malformation type I to standardize diagnosis and treatment.

Nucleo-Cytoplasmic Transport of ZIKV Non-Structural 3 Protein Is Mediated by Importin-α/ß and Exportin CRM-1.

Flaviviruses have a cytoplasmic replicative cycle, and crucial events, such as genome translation and replication, occur in the endoplasmic reticulum. However, some viral proteins, such as C, NS1, and NS5 from Zika virus (ZIKV) containing nuclear localization signals (NLSs) and nuclear export signals (NESs), are also located in the nucleus of Vero cells. The NS2A, NS3, and NS4A proteins from dengue virus (DENV) have also been reported to be in the nucleus of A549 cells, and our group recently reported that the NS3 protein is also located in the nucleus of Huh7 and C636 cells during DENV infection. However, the NS3 protease-helicase from ZIKV locates in the perinuclear region of infected cells and alters the morphology of the nuclear lamina, a component of the nuclear envelope. Furthermore, ZIKV NS3 has been reported to accumulate on the concave face of altered kidney-shaped nuclei and may be responsible for modifying other elements of the nuclear envelope. However, nuclear localization of NS3 from ZIKV has not been substantially investigated in human host cells. Our group has recently reported that DENV and ZIKV NS3 alter the nuclear pore complex (NPC) by cleaving some nucleoporins. Here, we demonstrate the presence of ZIKV NS3 in the nucleus of Huh7 cells early in infection and in the cytoplasm at later times postinfection. In addition, we found that ZIKV NS3 contains an NLS and a putative NES and uses the classic import (importin-α/ß) and export pathway via CRM-1 to be transported between the cytoplasm and the nucleus. IMPORTANCE Flaviviruses have a cytoplasmic replication cycle, but recent evidence indicates that nuclear elements play a role in their viral replication. Viral proteins, such as NS5 and C, are imported into the nucleus, and blocking their import prevents replication. Because of the importance of the nucleus in viral replication and the role of NS3 in the modification of nuclear components, we investigated whether NS3 can be localized in the nucleus during ZIKV infection. We found that NS3 is imported into the nucleus via the importin pathway and exported to the cytoplasm via CRM-1. The significance of viral protein nuclear import and export and its relationship with infection establishment is highlighted, emphasizing the development of new host-directed antiviral therapeutic strategies.

Leukocyte glucose index as a novel biomarker for COVID-19 severity.

The severity of coronavirus disease 2019 (COVID-19) quickly progresses with unfavorable outcomes due to the host immune response and metabolism alteration. Hence, we hypothesized that leukocyte glucose index (LGI) is a biomarker for severe COVID-19. This study involved 109 patients and the usefulness of LGI was evaluated and compared with other risk factors to predict COVID 19 severity. LGI was identified as an independent risk factor (odds ratio [OR] = 1.727, 95% confidence interval [CI]: 1.026-3.048, P = 0.041), with an area under the curve (AUC) of 0.749 (95% CI: 0.642-0.857, P < 0.0001). Interestingly, LGI was a potential risk factor (OR = 2.694, 95% CI: 1.575-5.283, Pcorrected < 0.05) for severe COVID-19 in female but not in male patients. In addition, LGI proved to be a strong predictor of the severity in patients with diabetes (AUC = 0.915 (95% CI: 0.830-1), sensitivity = 0.833, and specificity = 0.931). The AUC of LGI, together with the respiratory rate (LGI + RR), showed a considerable improvement (AUC = 0.894, 95% CI: 0.835-0.954) compared to the other biochemical and respiratory parameters analyzed. Together, these findings indicate that LGI could potentially be used as a biomarker of severity in COVID-19 patients.

The Usefulness of Peripheral Blood Cell Counts to Distinguish COVID-19 from Dengue during Acute Infection.

COVID-19 and dengue disease are challenging to tell apart because they have similarities in clinical and laboratory features during the acute phase of infection, leading to misdiagnosis and delayed treatment. The present study evaluated peripheral blood cell count accuracy to distinguish COVID-19 non-critical patients from non-severe dengue cases between the second and eleventh day after symptom onset. A total of 288 patients infected with SARS-CoV-2 (n = 105) or dengue virus (n = 183) were included in this study. Neutrophil, platelet, and lymphocyte counts were used to calculate the neutrophil-lymphocyte ratio (NLR), the platelet-lymphocyte ratio (PLR), and the neutrophil-lymphocyte*platelet ratio (NLPR). The logistic regression and ROC curves analysis revealed that neutrophil and platelet counts, NLR, LPR, and NLPR were higher in COVID-19 than dengue. The multivariate predictive model showed that the neutrophils, platelets, and NLPR were independently associated with COVID-19 with a good fit predictive value (p = 0.1041). The neutrophil (AUC = 0.95, 95% CI = 0.84-0.91), platelet (AUC = 0.89, 95% CI = 0.85-0.93) counts, and NLR (AUC = 0.88, 95% CI = 0.84-0.91) were able to discriminate COVID-19 from dengue with high sensitivity and specificity values (above 80%). Finally, based on predicted probabilities on combining neutrophils and platelets with NLR or NLPR, the adjusted AUC was 0.97 (95% CI = 0.94-0.98) to differentiate COVID-19 from dengue during the acute phase of infection with outstanding accuracy. These findings might suggest that the neutrophil, platelet counts, and NLR or NLPR provide a quick and cost-effective way to distinguish between dengue and COVID-19 in the context of co-epidemics in low-income tropical regions.

Anti-flavivirus Properties of Lipid-Lowering Drugs.

Although Flaviviruses such as dengue (DENV) and zika (ZIKV) virus are important human pathogens, an effective vaccine or antiviral treatment against them is not available. Hence, the search for new strategies to control flavivirus infections is essential. Several studies have shown that the host lipid metabolism could be an antiviral target because cholesterol and other lipids are required during the replicative cycle of different Flaviviridae family members. FDA-approved drugs with hypolipidemic effects could be an alternative for treating flavivirus infections. However, a better understanding of the regulation between host lipid metabolism and signaling pathways triggered during these infections is required. The metabolic pathways related to lipid metabolism modified during DENV and ZIKV infection are analyzed in this review. Additionally, the role of lipid-lowering drugs as safe host-targeted antivirals is discussed.

Case Report: Extrapulmonary Manifestations of COVID-19 and Dengue Coinfection.

The risk of coronavirus disease 2019 (COVID-19) and dengue coinfection is increased in tropical countries; however, the extrapulmonary clinical manifestations have not been fully characterized. We report a 42-year-old woman whose clinical manifestations began with fever, diarrhea, headache, chest pain, myalgia, odynophagia, and arthralgia. Despite mild respiratory symptoms and normal chest computed tomography scan results, she was diagnosed with real-time reverse-transcription polymerase chain reaction (RT-PCR)-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Because she had erythema and petechiae with a decreased platelet count, the dengue NS1 antigen and anti-dengue IgM/IgG test were performed, and the Centers for Disease Control and Prevention RT-PCR assay detected the dengue virus serotype 1 infection. Additionally, increased liver enzyme serum levels were found in the patient, who later developed hepatomegaly. Hence, the mechanism of hepatic pathology associated with SARS-CoV-2 and dengue coinfection needs further research.

The importance of viral and cellular factors on flavivirus entry.

The flavivirus are emerging and re-emerging arthropod-borne pathogens responsible for significant mortality and morbidity worldwide. The genus comprises more than 70 viruses, and despite genomic and structural similarities, infections by different flaviviruses result in different clinical presentations. In the absence of a safe and effective vaccine against these infections, the search for new strategies to inhibit viral infection is necessary. The life cycle of arboviruses begins with the entry process composed of multiple steps: attachment, internalization, endosomal escape and capsid uncoating. This mini-review describes factors and mechanisms involved in the viral entry as events required to take over the cellular machinery and host factors and cellular pathways commonly used by flaviviruses as possible approaches for developing broad-spectrum antiviral drugs.

The antiviral effect of metformin on zika and dengue virus infection.

The Dengue (DENV) and zika (ZIKV) virus infections are currently a public health concern. At present, there is no treatment or a safe and effective vaccine for these viruses. Hence, the development of new strategies as host-directed therapy is required. In this sense, Metformin (MET), an FDA-approved drug used for the treatment of type 2 diabetes, has shown an anti-DENV effect in vitro by activating AMPK and reducing HMGCR activity. In this study, MET treatment was evaluated during in vitro and in vivo ZIKV infection and compared to MET treatment during DENV infection. Our results demonstrated that MET has a broad in vitro antiviral spectrum. MET inhibited ZIKV infection in different cell lines, but it was most effective in inhibiting DENV and yellow fever virus (YFV) infection in Huh-7 cells. However, the drug failed to protect against ZIKV infection when AG129 immunodeficient mice were used as in vivo model. Interestingly, MET increased DENV-infected male mice's survival time, reducing the severe signs of the disease. Together, these findings indicate that, although MET was an effective antiviral agent to inhibit in vitro and in vivo DENV infection, it could only inhibit in vitro ZIKV infection.

The Nuclear Pore Complex Is a Key Target of Viral Proteases to Promote Viral Replication.

Various viruses alter nuclear pore complex (NPC) integrity to access the nuclear content favoring their replication. Alteration of the nuclear pore complex has been observed not only in viruses that replicate in the nucleus but also in viruses with a cytoplasmic replicative cycle. In this last case, the alteration of the NPC can reduce the transport of transcription factors involved in the immune response or mRNA maturation, or inhibit the transport of mRNA from the nucleus to the cytoplasm, favoring the translation of viral mRNAs or allowing access to nuclear factors necessary for viral replication. In most cases, the alteration of the NPC is mediated by viral proteins, being the viral proteases, one of the most critical groups of viral proteins that regulate these nucleus-cytoplasmic transport changes. This review focuses on the description and discussion of the role of viral proteases in the modification of nucleus-cytoplasmic transport in viruses with cytoplasmic replicative cycles and its repercussions in viral replication.

Nuclear localization of non-structural protein 3 (NS3) during dengue virus infection.

Although dengue virus (DENV) replication occurs in the cytoplasm, the nucleus plays an essential role during infection. Both the capsid protein (C) and non-structural protein 5 (NS5) are translocated into the infected cell nucleus to favor viral replication. Previously, our group reported the nuclear localization of the NS3 protein during DENV infection of mosquito cells; however, the nuclear localization of the DENV NS3 protein in human host cells has not been described. Here, we demonstrated that NS3 is present in the nucleus of Huh7 cells at early infection times, and later, it is mainly located in the cytoplasm.

Cholesterol-Rich Lipid Rafts as Platforms for SARS-CoV-2 Entry.

Since its appearance, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), the causal agent of Coronavirus Disease 2019 (COVID-19), represents a global problem for human health that involves the host lipid homeostasis. Regarding, lipid rafts are functional membrane microdomains with highly and tightly packed lipid molecules. These regions enriched in sphingolipids and cholesterol recruit and concentrate several receptors and molecules involved in pathogen recognition and cellular signaling. Cholesterol-rich lipid rafts have multiple functions for viral replication; however, their role in SARS-CoV-2 infection remains unclear. In this review, we discussed the novel evidence on the cholesterol-rich lipid rafts as a platform for SARS-CoV-2 entry, where receptors such as the angiotensin-converting enzyme-2 (ACE-2), heparan sulfate proteoglycans (HSPGs), human Toll-like receptors (TLRs), transmembrane serine proteases (TMPRSS), CD-147 and HDL-scavenger receptor B type 1 (SR-B1) are recruited for their interaction with the viral spike protein. FDA-approved drugs such as statins, metformin, hydroxychloroquine, and cyclodextrins (methyl-ß-cyclodextrin) can disrupt cholesterol-rich lipid rafts to regulate key molecules in the immune signaling pathways triggered by SARS-CoV-2 infection. Taken together, better knowledge on cholesterol-rich lipid rafts in the SARS-CoV-2-host interactions will provide valuable insights into pathogenesis and the identification of novel therapeutic targets.