Isradipine, an L-type calcium channel blocker, attenuates cocaine effects in mice by reducing central glutamate release.

Substance abuse disorder is a chronic condition for which pharmacological treatment options remain limited. L-type calcium channels (LTCC) have been implicated in drug-related plasticity and behavior. Specifically, dopaminergic neurons in the mesocorticolimbic pathway express Cav1.2 and Cav1.3 channels, which may regulate dopaminergic activity associated with reward behavior. Therefore, this study aimed to investigate the hypothesis that pre-administration of the LTCC blocker, isradipine can mitigate the effects of cocaine by modulating central glutamatergic transmission. For that, we administered isradipine at varying concentrations (1, 7.5, and 15 µg/µL) via intracerebroventricular injection in male Swiss mice. This pretreatment was carried out prior to subjecting animals to behavioral assessments to evaluate cocaine-induced locomotor sensitization and conditioned place preference (CPP). The results revealed that isradipine administered at a concentration of 1 µg/µL effectively attenuated both the sensitization and CPP induced by cocaine (15 mg/kg, via i. p.). Moreover, mice treated with 1 µg/µL of isradipine showed decreased presynaptic levels of glutamate and calcium in the cortex and hippocampus as compared to control mice following cocaine exposure. Notably, the gene expression of ionotropic glutamate receptors, AMPA, and NMDA, remained unchanged, as did the expression of Cav1.2 and Cav1.3 channels. Importantly, these findings suggest that LTCC blockage may inhibit behavioral responses to cocaine, most likely by decreasing glutamatergic input in areas related to addiction.

High capacity of Brazilian Aedes aegypti populations to transmit a locally circulating lineage of Chikungunya virus.

The global incidence of chikungunya has surged in recent decades, with South America, particularly Brazil, experiencing devastating outbreaks. The primary vector for transmitting CHIKV in urban areas is the mosquito species Aedes aegypti, which is very abundant in Brazil. However, little is known about the impact of locally circulating CHIKV genotypes and specific combinations of mosquito populations on vector competence. In this study, we analyzed and compared the infectivity and transmissibility of a recently isolated CHIKV-ECSA lineage from Brazil among four Ae. aegypti populations collected from different regions of the country. When exposed to CHIKV-infected mice for blood feeding, all mosquito populations showed high infection rates and dissemination efficiency. Moreover, using a mouse model to assess transmission rates in a manner that better mirrors natural cycles, we observed that these populations exhibit highly efficient transmission rates of CHIKV-ECSA. Our findings underscore the robust capability of Brazilian Ae. aegypti populations to transmit the locally circulating CHIKV-ECSA lineage, potentially explaining its higher prevalence compared to the Asian lineage also introduced in Brazil.

Chronic hyperpalatable diet induces impairment of hippocampal-dependent memories and alters glutamatergic and fractalkine axis signaling.

Chronic consumption of hyperpalatable and hypercaloric foods has been pointed out as a factor associated with cognitive decline and memory impairment in obesity. In this context, the integration between peripheral and central inflammation may play a significant role in the negative effects of an obesogenic environment on memory. However, little is known about how obesity-related peripheral inflammation affects specific neurotransmission systems involved with memory regulation. Here, we test the hypothesis that chronic exposure to a highly palatable diet may cause neuroinflammation, glutamatergic dysfunction, and memory impairment. For that, we exposed C57BL/6J mice to a high sugar and butter diet (HSB) for 12 weeks, and we investigated its effects on behavior, glial reactivity, blood-brain barrier permeability, pro-inflammatory features, glutamatergic alterations, plasticity, and fractalkine-CX3CR1 axis. Our results revealed that HSB diet induced a decrease in memory reconsolidation and extinction, as well as an increase in hippocampal glutamate levels. Although our data indicated a peripheral pro-inflammatory profile, we did not observe hippocampal neuroinflammatory features. Furthermore, we also observed that the HSB diet increased hippocampal fractalkine levels, a key chemokine associated with neuroprotection and inflammatory regulation. Then, we hypothesized that the elevation on glutamate levels may saturate synaptic communication, partially limiting plasticity, whereas fractalkine levels increase as a strategy to decrease glutamatergic damage.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus.

BACKGROUND: The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES: Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS: Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS: MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS: Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus

BACKGROUND The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis.

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

Nitric oxide synthase expression correlates with death in an experimental mouse model of dengue with CNS involvement.

BACKGROUND: The clinical presentation of dengue is classified by the World Health Organization into dengue without warning signs, dengue with warning signs and severe dengue. Reports of neurological disease caused by Dengue virus (DENV) are becoming frequent, with symptoms that include reduced consciousness, severe headache, neck stiffness, focal neurological signs, tense fontanelle and convulsions. However, the immune mechanisms involved in neurovirulence remain poorly understood. Here we present a mouse model in which one genotype of DENV is inoculated by the intracranial route and infects C57/BL6 mice and replicates in the brain, causing death of mice. METHODS: Mice were infected with different serotypes/genotypes of DENV by the intracranial route to evaluate viral replication, host cytokine and nitric oxide synthase 2 (Nos2) expression in the brain via real-time PCR. Histological analysis of the brain tissues was also performed. An analysis of which cells were responsible for the expression of cytokines and Nos2 was performed using flow cytometry. Survival curves of infected animals were also generated RESULTS: DENV 3 genotype I infected mice and replicated in the brain, causing death in our murine model. The increased levels of NOS2 could be the cause of the death of infected mice, as viral replication correlates with increased Nos2 and cytokine expression in the brain of C57BL/6 mice. In Nos2-/- mice that were infected with DENV, no clinical signs of infection were observed and cytokines were expressed at low levels, with the exception of interferon gamma (Ifng). Additionally, the Ifng-/- mice infected with DENV exhibited a severe and lethal disease, similar to the disease observed in C57BL/6 mice, while the DENV- infected Nos2-/- mice did not display increased mortality. Analyses of the brains from infected C57BL/6 mice revealed neuronal degeneration and necrosis during histopathologic examination. IFNg and NOS2 were produced in the brains of infected mice by CD4+ T cells and macrophages, respectively. CONCLUSION: The neurovirulence of DENV 3 genotype I is associated with a deleterious role of NOS2 in the brain, confirming this murine model as an appropriate tool to study DENV neurovirulence.

dsRNA sensing during viral infection: lessons from plants, worms, insects, and mammals.

Host defense systems often rely on direct and indirect pattern recognition to sense the presence of invading pathogens. Patterns can be molecules directly produced by the pathogen or indirectly generated by changes in host parameters as a consequence of infection. Viruses are intracellular pathogens that hijack the cellular machinery to synthesize their own molecules making direct recognition of viral molecules a great challenge. Antiviral systems in prokaryotes and eukaryotes commonly exploit aberrant nucleic acid sensing to recognize virus infection as host and viral nucleic acid metabolism can greatly differ. Indeed, the generation of dsRNA is often associated with viral infection. In this review, we discuss current knowledge on the mechanisms of viral dsRNA sensing utilized by 2 important antiviral defense systems, RNA interference (RNAi) and the vertebrate immune system. The major viral sensors of the vertebrate immune systems are RIG-like receptors, while RNAi pathways depend on Dicer proteins. These 2 families of sensors share a similar helicase domain with high specificity for dsRNA, which is necessary, but not sufficient for efficient recognition by these receptors. Additional intrinsic features to the dsRNA molecule are also necessary for activation of antiviral systems. Studies utilizing synthetic ligands, in vitro biochemistry and reporter systems have greatly helped increase our knowledge on intrinsic features of dsRNA recognition. However, characteristics such as subcellular localization are extrinsic to the dsRNA itself, but certainly influence the recognition in vivo. Thus, mechanisms of viral dsRNA recognition must address how cellular sensors are recruited to nucleic acids or vice versa. Accessory proteins are likely important for in vivo recognition of extrinsic features of viral RNA, but have mostly remained undiscovered due to the limitations of previous strategies. Hence, the identification of novel components of antiviral systems must take into account the complexities involved in viral recognition in vivo.