DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target.

Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication.

Effect of Omicron on the prevalence of COVID-19 in international travelers at the Mexico city international airport. December 16th, 2021 to January 31st, 2022.

INTRODUCTION: SARS-CoV-2 continues to have a high rate of contagion worldwide. The new variant of concern, Omicron, has mutations that decrease the effectiveness of vaccines and evade antibodies from previous infections resulting in a fourth wave of the pandemic. It was identified in Mexico in December 2021. METHODS: The Traveler's Preventive Care Clinic from the Faculty of Medicine UNAM at Mexico City International Airport has performed rapid antigen and PCR SARS CoV2 tests since January 2021 to comply with the new travel requirements. Demographic and clinical characteristics were collected from each passenger and the fourth wave of the pandemic in Mexico mainly caused by Omicron was analyzed in the travelers. RESULTS: A total of 5176 travelers attended the clinic between the second half of December 2021 and January 2022. Ten percent of all the tests performed were positive (13% of PCR and 9.3% of antigens, p = 0.001). Most of the SARS CoV2 positive cases were asymptomatic (78%), with a ratio of 3.5:1 over the symptomatic. By age groups, this ratio was higher for those under 20 years old (8.7:1). DISCUSSION: This study shows the rapid escalation of positivity that occurred in Mexico, detected in travelers, from the second half of December 2020 and throughout the month of January 2021. The incidence of COVID-19 was extremely high in travelers who were mostly asymptomatic for the period under study.

Microglia HMC3 cells are highly susceptible to Rhinovirus infection.

Human rhinovirus (HRV) affects the lower and upper respiratory tract, however, some studies suggest that HRV infection can lead to extrapulmonary complications in critical illness. Moreover, some reports have shown the presence of HRV in patients with Central Nervous System (CNS) disease. During a CNS infection, the microglia cells are the first line of defense against pathogens. In this study, the susceptibility of the human microglial clone 3 cell line (HMC3) to HRV infection was analyzed. Our findings demonstrate for the first time that HRV is capable of completing the entire viral cycle in microglial cells.

Mosquito cells persistently infected with dengue virus produce viral particles with host-dependent replication.

Dengue viruses (DENV) are important arboviruses that can establish a persistent infection in its mosquito vector Aedes. Mosquitoes have a short lifetime in nature which makes trying to study the processes that take place during persistent viral infections in vivo. Therefore, C6/36 cells have been used to study this type of infection. C6/36 cells persistently infected with DENV 2 produce virions that cannot infect BHK -21 cells. We hypothesized that the following passages in mosquito cells have a deleterious impact on DENV fitness in vertebrate cells. Here, we demonstrated that the viral particles released from persistently infected cells were infectious to mosquito but not to vertebrate cells. This host restriction occurs at the replication level and is associated with several mutations in the DENV genome. In summary, our findings provide new information about viral replication fitness in a host-dependent manner.

The ß4 subunit of Cav1.2 channels is required for an optimal interferon response in cardiac muscle cells.

The auxiliary ß4 subunit of the cardiac Cav1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the ß4 subunit in H9c2 rat cardiac cells on the abundances of Ifnb mRNA [which encodes interferon-ß (IFN-ß)] and of the IFN-ß-related genes Ddx58, Ifitm3, Irf7, Stat2, Ifih1, and Mx1, as well as on the abundances of the antiviral proteins DDX58, IRF7, STAT2, and IFITM3. Knocking down the ß4 subunit in H9c2 cells reduced the expression of IFN-ß-stimulated genes. In response to inhibition of the kinase JAK1, the abundances of ß4 subunit mRNA and protein were decreased. ß4 subunit abundance was increased, and it translocated to the nucleus, in cells treated with IFN-ß, infected with dengue virus (DENV), or transfected with poly(I:C), a synthetic analog of double-stranded RNA. Cells that surrounded the virus-infected cells showed translocation of ß4 subunit proteins to nuclei in response to spreading infection. We showed that the ß4 subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter-driven reporter was increased in cells overexpressing the ß4 subunit. Last, overexpressing ß4 in undifferentiated and differentiated H9c2 cells reduced DENV infection and decreased the abundance of the viral proteins NS1, NS3, and E-protein. DENV infection and poly(I:C) also increased the concentration of intracellular Ca2+ in these cells. These findings suggest that the ß4 subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection.

Ezetimibe inhibits dengue virus infection in Huh-7 cells by blocking the cholesterol transporter Niemann-Pick C1-like 1 receptor.

Despite the importance of Dengue virus (DENV) infection in human health, there is not a fully effective vaccine or antiviral treatment against the infection. Since lipids such as cholesterol are required during DENV infection, its uptake and synthesis are increased in infected cells. Ezetimibe is an FDA-approved drug that reduces cholesterol uptake by inhibiting the endocytosis through Niemman-Pick C1-Like 1 (NPC1L1) receptor, expressed on the membrane of enterocytes and hepatocytes. Our results indicate that an increase in the amount of NPC1L1 occurs on the surface of Huh-7 cells during DENV infection, which correlates with an increase in cholesterol levels. Blockage of NPC1L1 with ezetimibe in concentrations up to 50 µM does not reduce cell viability but diminished total cellular cholesterol, the percentage of infected cells, viral yield, viral RNA and protein synthesis without affecting DENV binding and/or entry to Huh-7 cells. Moreover, ezetimibe inhibited DENV replicative complex formation and lipid droplets accumulation. All these results indicate that ezetimibe is an excellent drug to inhibit DENV infection and confirm that cholesterol is a key target to inhibit viral infection.

The calmodulin antagonist W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride) inhibits DENV infection in Huh-7 cells.

Dengue virus (DENV) replicative cycle occurs in the endoplasmic reticulum where calcium ions play an important role in cell signaling. Calmodulin (CaM) is the primary sensor of intracellular Ca2+ levels in eukaryotic cells. In this paper, the effect of the calmodulin antagonist W-7 in DENV infection in Huh-7 cells was evaluated. W7 inhibited viral yield, NS1 secretion and viral RNA and protein synthesis. Moreover, luciferase activity, encoded by a DENV replicon, was also reduced. A decrease in the replicative complexes formation was clearly observed in W7 treated cells. Docking simulations suggest 2 possible mechanisms of action for W7: the direct inhibition of NS2B-NS3 activity and/or inhibition of the interaction between NS2A with Ca2+-CaM complex. This last possibility was supported by the in vitro interaction observed between recombinant NS2A and CaM. These results indicate that Ca2+-CaM plays an important role in DENV replication.

An embryonic heart cell line is susceptible to dengue virus infection.

Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.

Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells.

Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.