Claudin-4 Modulates Autophagy via SLC1A5/LAT1 as a Mechanism to Regulate Micronuclei.

Genome instability is a hallmark of cancer crucial for tumor heterogeneity and is often a result of defects in cell division and DNA damage repair. Tumors tolerate genomic instability, but the accumulation of genetic aberrations is regulated to avoid catastrophic chromosomal alterations and cell death. In ovarian cancer tumors, claudin-4 is frequently upregulated and closely associated with genome instability and worse patient outcomes. However, its biological association with regulating genomic instability is poorly understood. Here, we used CRISPR interference and a claudin mimic peptide to modulate the claudin-4 expression and its function in vitro and in vivo. We found that claudin-4 promotes a tolerance mechanism for genomic instability through micronuclei generation in tumor cells. Disruption of claudin-4 increased autophagy and was associated with the engulfment of cytoplasm-localized DNA. Mechanistically, we observed that claudin-4 establishes a biological axis with the amino acid transporters SLC1A5 and LAT1, which regulate autophagy upstream of mTOR. Furthermore, the claudin-4/SLC1A5/LAT1 axis was linked to the transport of amino acids across the plasma membrane as one of the potential cellular processes that significantly decreased survival in ovarian cancer patients. Together, our results show that the upregulation of claudin-4 contributes to increasing the threshold of tolerance for genomic instability in ovarian tumor cells by limiting its accumulation through autophagy. SIGNIFICANCE: Autophagy regulation via claudin-4/SLC1A5/LAT1 has the potential to be a targetable mechanism to interfere with genomic instability in ovarian tumor cells.

NOD2 Responds to Dengue Virus Type 2 Infection in Macrophage-like Cells Interacting with MAVS Adaptor and Affecting IFN-α Production and Virus Titers.

In pathogen recognition, the nucleotide-binding domain (NBD) and leucine rich repeat receptors (NLRs) have noteworthy functions in the activation of the innate immune response. These receptors respond to several viral infections, among them NOD2, a very dynamic NLR, whose role in dengue virus (DENV) infection remains unclear. This research aimed to determine the role of human NOD2 in THP-1 macrophage-like cells during DENV-2 infection. NOD2 levels in DENV-2 infected THP-1 macrophage-like cells was evaluated by RT-PCR and Western blot, and an increase was observed at both mRNA and protein levels. We observed using confocal microscopy and co-immunoprecipitation assays that NOD2 interacts with the effector protein MAVS (mitochondrial antiviral signaling protein), an adaptor protein promoting antiviral activity, this occurring mainly at 12 h into the infection. After silencing NOD2, we detected increased viral loads of DENV-2 and lower levels of IFN-α in supernatants from THP-1 macrophage-like cells with NOD2 knock-down and further infected with DENV-2, compared with mock-control or cells transfected with Scramble-siRNA. Thus, NOD2 is activated in response to DENV-2 in THP-1 macrophage-like cells and participates in IFN-α production, in addition to limiting virus replication at the examined time points.

Molecular survey of Zika virus in the animal-human interface in traditional farming.

Backyard animal husbandry is common in rural communities in developing countries and, given the conditions in which it occurs, it can increase the risk of disease transmission, such as arboviruses. To determine the presence of the Zika virus (ZIKV) and abundance of its arthropod vectors we evaluated the socioeconomic implications involved in its transmission in two highly vulnerable Mayan communities in the state of Yucatan that practice backyard farming. An analytical cross-sectional study was carried out throughout 2016 to understand socioeconomic variables and seasonal patterns in mosquito populations. We selected 20 households from each community. Social exclusion indicators were analyzed, human and domestic animals were sampled, and mosquitoes were collected and identified. Four out of eight indicators of social exclusion were higher than the reported national averages. We captured 5,825 mosquitoes from 16 species being Culex quinquefasciatus and Aedes aegypti the most abundant. The presence of chickens and human overcrowding in dwellings were the most significant factors (P = 0.026) associated with the presence of Ae. aegypti. Septic tanks (odds ratio = 6.64) and chickens (odds ratio = 27.41) in backyards were the main risk factors associated with the presence of immature states of Ae. aegypti in both communities. Molecular analysis to detect ZIKV was performed in blood samples from 416 humans, 1,068 backyard animals and 381 mosquito pools. Eighteen humans and 10 pig pools tested positive for ZIKV. Forty-three mosquito pools tested positive for flavivirus. Ten of the 43 pools of positive mosquitoes were sequenced, corresponding 3/10 to ZIKV and 1/10 to Dengue virus type 2. The findings obtained indicate the continuous circulation of Flavivirus (including ZIKV) in backyard environments in vulnerable communities, highlighting the importance of studying their transmission and maintenance in these systems, due that backyard animal husbandry is a common practice in these vulnerable communities with limited access to health services.

Evidence of Coinfections between SARS-CoV-2 and Select Arboviruses in Guerrero, Mexico, 2020-2021.

We provide evidence of concurrent and close sequential infections between SARS-CoV-2 and select arboviruses-namely, chikungunya virus (CHIKV); dengue viruses 1, 2, and 3 (DENV1-3), and Zika virus (ZIKV)-in patients in Guerrero, southwest Mexico, in 2020-2021. The study population consisted of 176 febrile patients with laboratory evidence of SARS-CoV-2 infection. Sera from all patients were serologically and antigenically tested for seven arboviruses known to occur in Guerrero. Eighteen patients contained CHIKV IgM, six of whom also contained CHIKV RNA. Another 16 patients contained flavivirus antigen. The flaviviruses responsible for the infections were identified by plaque reduction neutralization test as DENV1 (two patients), DENV2 (five patients), DENV3 (three patients), ZIKV (three patients), and an undetermined flavivirus (three patients). In summary, we identified patients in Guerrero, Mexico, with concurrent or recent sequential infections between SARS-CoV-2 and select arboviruses, exemplifying the importance of performing differential diagnosis in regions where these viruses cocirculate.

The host range restriction of bat-associated no-known-vector flaviviruses occurs post-entry.

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.

Chikungunya in Guerrero, Mexico, 2019 and Evidence of Gross Underreporting in the Region.

The local public health authorities reported nine cases of chikungunya in Mexico in 2019, none of which occurred in Guerrero, a coastal state in the southwest. To test the hypothesis that chikungunya is grossly underreported in Mexico, acute sera were collected from 639 febrile patients from low-income households in Guerrero in 2019 and serologically assayed for chikungunya virus (CHIKV). Analysis of the sera by plaque reduction neutralization test revealed that 181 (28.3%) patients were seropositive for CHIKV. To identify patients with acute CHIKV infections, a subset of serum samples were tested for CHIKV-specific IgM by ELISA. Serum samples from 21 of 189 (11.1%) patients were positive. These patients met the chikungunya case definition established by the WHO. In conclusion, we provide evidence that CHIKV remains an important public health problem in Mexico and that the true number of cases is severely underestimated.

Co-Circulation of All Four Dengue Viruses and Zika Virus in Guerrero, Mexico, 2019.

A clinical and entomological investigation was performed to identify flavivirus infections in humans and mosquitoes in impoverished areas of Guerrero, a coastal state in southwestern Mexico. A total of 639 patients with acute febrile illness and 830 resting female mosquitoes in low-income communities of Guerrero in 2019 were tested for evidence of flavivirus infection. Sera were collected from all patients and screened at a dilution of 1:20 by plaque reduction neutralization test (PRNT) using dengue virus (DENV)2. A total of 431 (67.4%) patients were seropositive. Sera from a subset of seropositive patients (n = 263) were tested for flavivirus NS1 by enzyme-linked immunosorbent assay. Forty-eight (18.3%) sera contained viral antigen. All NS1-positive sera were titrated and further tested by PRNT using DENV-1 to -4, St. Louis encephalitis virus, West Nile virus, and Zika virus (ZIKV). Seven patients were seropositive for DENV-1, five patients were seropositive for DENV-2, one patient was seropositive for DENV-3, and two patients each were seropositive for DENV-4 and ZIKV. The remainder had secondary flavivirus infections or antibodies to an undetermined flavivirus. Comparative PRNTs were also performed on 60 randomly selected NS1-negative sera, identifying patients seropositive for DENV-2, DENV-3, and ZIKV. The entomological investigation yielded 736 Aedes aegypti and 94 Culex quinquefasciatus that were sorted into 183 pools and 20 pools, respectively. Mosquitoes were assayed for flavivirus RNA by RT-PCR and Sanger sequencing. DENV-2 RNA was detected in three pools of A. aegypti. In summary, we provide evidence for the concurrent circulation of all four DENVs and ZIKV in Guerrero, Mexico. The public health authorities reported no cases of DENV-3, DENV-4, and ZIKV in Guerrero in 2019 and thus, we provide evidence of under-reporting in the region.

Chimeric Zika viruses containing structural protein genes of insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions.

Long Pine Key virus (LPKV) and Lammi virus are insect-specific flaviviruses that phylogenetically affiliate with dual-host flaviviruses. The goal of this study was to provide insight into the genetic determinants that condition this host range restriction. Chimeras were initially created by replacing select regions of the Zika virus genome, including the premembrane and envelope protein (prM-E) genes, with the corresponding regions of the LPKV genome. Of the four chimeras produced, one (the prM-E swap) yielded virus that replicated in mosquito cells. Another chimeric virus with a mosquito replication-competent phenotype was created by inserting the prM-E genes of Lammi virus into a Zika virus genetic background. Vertebrate cells did not support the replication of either chimeric virus although trace to modest amounts of viral antigen were produced, consistent with suboptimal viral entry. These data suggest that dual-host affiliated insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions.

Evidence of Zika Virus Infection in Pigs and Mosquitoes, Mexico.

Evidence suggests that pigs seroconvert after experimental exposure to Zika virus and are potential sentinels. We demonstrate that pigs are also susceptible to natural Zika virus infection, shown by the presence of antibodies in domestic pigs in Yucatan, Mexico. Zika virus RNA was detected in 5 species of mosquitoes collected inside pigpens.

Dengue Virus Induces the Release of sCD40L and Changes in Levels of Membranal CD42b and CD40L Molecules in Human Platelets.

Platelets are considered as significant players in innate and adaptive immune responses. The adhesion molecules they express, including P-selectin, CD40L, and CD42b, facilitate interactions with many cellular effectors. Upon interacting with a pathogen, platelets rapidly express and enhance their adhesion molecules, and secrete cytokines and chemokines. A similar phenomenon occurs after exposure of platelets to thrombin, an agonist extensively used for in vitro activation of these cells. It was recently reported that the dengue virus not only interacts with platelets but possibly infects them, which triggers an increased expression of adhesion molecule P-selectin as well as secretion of IL-1ß. In the present study, surface molecules of platelets like CD40L, CD42b, CD62P, and MHC class I were evaluated at 4 h of interaction with dengue virus serotype 2 (DENV-2), finding that DENV-2 induced a sharp rise in the membrane expression of all these molecules. At 2 and 4 h of DENV-2 stimulation of platelets, a significantly greater secretion of soluble CD40L (sCD40L) was found (versus basal levels) as well as cytokines such as GM-CSF, IL-6, IL-8, IL-10, and TNF-α. Compared to basal, DENV-2 elicited more than two-fold increase in these cytokines. Compared to the thrombin-induced response, the level generated by DENV-2 was much higher for GM-CSF, IL-6, and TNF-α. All these events induced by DENV end up in conspicuous morphological changes observed in platelets by confocal microscopy and transmission electron microscopy, very different from those elicited by thrombin in a more physiological scenery.

NOD2: Activation During Bacterial and Viral Infections, Polymorphisms and Potential as Therapeutic Target.

Nucleotide-binding domain (NBD) leucine-rich repeat (LRR)-containing receptors or NLRs are a family of receptors that detect both, molecules associated to pathogens and alarmins, and are located mainly in the cytoplasm. NOD2 belongs to the NLR family and is a dynamic receptor capable of interacting with multiple proteins and modulate immune responses in a stimuli-dependent manner. The experimental evidence shows that interaction between NOD2 structural domains and the effector proteins shape the overall response against bacterial or viral infections. Other reports have focused on the importance of NOD2 not only in infection but also in maintaining tissue homeostasis. However, not only protein interactions relate to function but also certain polymorphisms in the gene that encodes NOD2 have been associated with inflammatory diseases, such as Crohn's disease. Here, we review the importance and general characteristics of NOD2, discussing its participation in infections caused by bacteria and viruses as well as its interaction with other pathogen recognition receptors or effectors to induce antibacterial and antiviral responses. Finally, the role of NOD2 in chronic inflammatory conditions and its potential to be targeted therapeutically are examined.

Chikungunya: Molecular Aspects, Clinical Outcomes and Pathogenesis.

The alarming worldwide emergence of the chikungunya virus began in the last decade. Since the first autochthonous transmission in Mexico in November of 2014, the virus has spread throughout the country, resulted in multiple outbreaks. This virus produces an acute and self-limiting disease characterized by fever, polyarthralgia, myalgia, exanthema, and general malaise. It is transmitted to humans by the bite of Aedes aegypti and A. albopictus mosquitoes. The fact that the clinical presentation is similar to that produced by other arboviruses complicates its clinical diagnosis. The chronic stage of the disease can cause severe consequences lasting months or years, from local arthralgia to rheumatoid arthritis. In this review, we emphasize the public health threat posed by this highly disabling emerging disease, the clinical outcomes, and its possible physiopathological process. We outline the diagnosis and the impact that this virus has had in Mexico since its introduction.