Chikungunya and Mayaro Viruses Induce Chronic Skeletal Muscle Atrophy Triggered by Pro-Inflammatory and Oxidative Response.

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, nuclear magnetic resonance imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced inflammation promote a rapid loss of muscle mass and reduction in fiber cross-sectional area by upregulation of muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1 expression, both key regulators of SkM fibers atrophy. Despite a reduction in inflammation and clearance of infectious viral particles, SkM atrophy persists until 30 days post-infection. The genomic CHIKV and MAYV RNAs were still detected in SkM in the late phase, along with the upregulation of chemokines and anti-inflammatory cytokine expression. In agreement with the involvement of inflammatory mediators on induced atrophy, the neutralization of TNF and a reduction in oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogen expression and atrophic fibers while increasing weight gain in treated mice. These data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphavirus pathogenesis and with a description of potentially new targets of therapeutic intervention.

Immunosuppression-induced Zika virus reactivation causes brain inflammation and behavioral deficits in mice.

Zika virus (ZIKV) is a neurotropic flavivirus that can persist in several tissues. The late consequences of ZIKV persistence and whether new rounds of active replication can occur, remain unaddressed. Here, we investigated whether neonatally ZIKV-infected mice are susceptible to viral reactivation in adulthood. We found that when ZIKV-infected mice are treated with immunosuppressant drugs, they present increased susceptibility to chemically induced seizures. Levels of subgenomic flavivirus RNAs (sfRNAs) were increased, relative to the amounts of genomic RNAs, in the brains of mice following immunosuppression and were associated with changes in cytokine expression. We investigated the impact of immunosuppression on the testicles and found that ZIKV genomic RNA levels are increased in mice following immunosuppression, which also caused significant testicular damage. These findings suggest that ZIKV can establish new rounds of active replication long after acute stages of disease, so exposed patients should be monitored to ensure complete viral eradication.

TNF-α blockage prevents late neurological consequences of Zika virus infection in mice.

Zika virus (ZIKV) is a neurotropic Orthoflavivirus that causes a myriad of neurological manifestations in newborns exposed in uterus. Despite the devastating consequences of ZIKV on the developing brain, strategies to prevent or treat the consequences of viral infection are not yet available. We previously showed that short-term treatment with the TNF-α neutralizing monoclonal antibody. Infliximab could prevent seizures at acute and chronic stages of ZIKV infection, but had no impact on long-term cognitive and motor dysfunction. Due to the central role of inflammation in ZIKV-neuropathology, we hypothesized that prolonged treatment with the anti-TNF-α monoclonal antibody Infliximab could provide complete rescue of long-term behavioral deficits associated with neonatal ZIKV infection in mice. Here, neonatal (post-natal day 3) Swiss mice were submitted to subcutaneous (s.c.) injection of 106 PFU of ZIKV or mock medium and were then treated with Infliximab (20 µg/day) or sterile saline intraperitoneally (i.p.), for 40 days starting on the day of infection, and behavioral assessment started at 60 days post-infection (dpi). Infliximab prevented ZIKV-induced cognitive and motor impairments in mice. In addition, microgliosis and cell death found in mice following ZIKV infection were partially reversed by TNF-α blockage. Altogether, these results suggest that TNF-α-mediated inflammation is central for late ZIKV-induced behavioral deficits and cell death and strategies targeting this cytokine may be promising approaches to treat subjects exposed to the virus during development.

ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection.

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-ß, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Zika virus infection impairs synaptogenesis, induces neuroinflammation, and could be an environmental risk factor for autism spectrum disorder outcome.

Zika virus (ZIKV) infection was first associated with Central Nervous System (CNS) infections in Brazil in 2015, correlated with an increased number of newborns with microcephaly, which ended up characterizing the Congenital Zika Syndrome (CZS). Here, we investigated the impact of ZIKV infection on the functionality of iPSC-derived astrocytes. Besides, we extrapolated our findings to a Brazilian cohort of 136 CZS children and validated our results using a mouse model. Interestingly, ZIKV infection in neuroprogenitor cells compromises cell migration and causes apoptosis but does not interfere in astrocyte generation. Moreover, infected astrocytes lost their ability to uptake glutamate while expressing more glutamate transporters and secreted higher levels of IL-6. Besides, infected astrocytes secreted factors that impaired neuronal synaptogenesis. Since these biological endophenotypes were already related to Autism Spectrum Disorder (ASD), we extrapolated these results to a cohort of children, now 6-7 years old, and found seven children with ASD diagnosis (5.14 %). Additionally, mice infected by ZIKV revealed autistic-like behaviors, with a significant increase of IL-6 mRNA levels in the brain. Considering these evidence, we inferred that ZIKV infection during pregnancy might lead to synaptogenesis impairment and neuroinflammation, which could increase the risk for ASD.

Antiviral, Cytoprotective, and Anti-Inflammatory Effect of Ampelozizyphus amazonicus Ducke Ethanolic Wood Extract on Chikungunya Virus Infection.

Chikungunya fever, a debilitating disease caused by Chikungunya virus (CHIKV), is characterized by a high fever of sudden onset and an intense arthralgia that impairs individual regular activities. Although most symptoms are self-limited, long-term persistent arthralgia is observed in 30-40% of infected individuals. Currently, there is no vaccine or specific treatment against CHIKV infection, so there is an urgent need for the discovery of new therapeutic options for CHIKF chronic cases. This present study aims to test the antiviral, cytoprotective, and anti-inflammatory activities of an ethanol extract (FF72) from Ampelozizyphus amazonicus Ducke wood, chemically characterized using mass spectrometry, which indicated the major presence of dammarane-type triterpenoid saponins. The major saponin in the extract, with a deprotonated molecule ion m/z 897 [M-H]-, was tentatively assigned as a jujubogenin triglycoside, a dammarane-type triterpenoid saponin. Treatment with FF72 resulted in a significant reduction in both virus replication and the production of infective virions in BHK-21-infected cells. The viability of infected cells was assessed using an MTT, and the result indicated that FF72 treatment was able to revert the toxicity mediated by CHIKV infection. In addition, FF72 had a direct effect on CHIKV, since the infectivity was completely abolished in the presence of the extract. FF72 treatment also reduced the expression of the major pro-inflammatory mediators overexpressed during CHIKV infection, such as IL-1ß, IL-6, IL-8, and MCP-1. Overall, the present study elucidates the potential of FF72 to become a promising candidate of herbal medicine for alphaviruses infections.

Age-dependent memory impairment induced by co-exposure to nicotine and a synthetic cannabinoid in mice.

Co-use of marijuana and tobacco products is the second most common drug combination among adolescents. Nicotine (NIC) and cannabinoid use during adolescence induce similar detrimental changes, raising the hypothesis that simultaneous exposure could result in even more severe outcomes. Thus, we investigated whether the co-exposure to NIC and the synthetic cannabinoid WIN 55,212-2 (WIN) in adolescent mice causes behavioral outcomes different from those observed after exposure to a single drug. Male Swiss mice were exposed twice daily to NIC, WIN, or NIC + WIN during adolescence (PND28-47) or adulthood (PND70-89). Drug combination led to a greater reduction in weight gain in adolescent mice, while NIC-induced weight loss was observed in adults. During administration, NIC provoked hypothermia, and WIN produced hyperlocomotion in adolescent and adult mice. Animals exposed to NIC + WIN presented a profile of changes similar to those exposed to NIC. After drug exposure, changes in locomotion, thigmotaxis, social preference, prepulse inhibition, and working and recognition memory were evaluated. Adolescent but not adult mice exposed to NIC showed withdrawal-related hyperlocomotion unaffected by WIN co-administration. An age-specific impairment in object recognition memory was induced only by drug co-exposure during adolescence, which resolved spontaneously before reaching early adulthood. A transient decrease in hippocampal α7 nAChR subunit and CB1 receptor mRNA levels was induced by NIC exposure, which may be involved but is not enough to explain the memory impairment. Our work confirms the potential of NIC and cannabinoids association to aggravate some of the individual drug effects during critical neurodevelopmental periods.

Integrated NMR and MS Analysis of the Plasma Metabolome Reveals Major Changes in One-Carbon, Lipid, and Amino Acid Metabolism in Severe and Fatal Cases of COVID-19.

Brazil has the second-highest COVID-19 death rate worldwide, and Rio de Janeiro is among the states with the highest rate in the country. Although vaccine coverage has been achieved, it is anticipated that COVID-19 will transition into an endemic disease. It is concerning that the molecular mechanisms underlying clinical evolution from mild to severe disease, as well as the mechanisms leading to long COVID-19, are not yet fully understood. NMR and MS-based metabolomics were used to identify metabolites associated with COVID-19 pathophysiology and disease outcome. Severe COVID-19 cases (n = 35) were enrolled in two reference centers in Rio de Janeiro within 72 h of ICU admission, alongside 12 non-infected control subjects. COVID-19 patients were grouped into survivors (n = 18) and non-survivors (n = 17). Choline-related metabolites, serine, glycine, and betaine, were reduced in severe COVID-19, indicating dysregulation in methyl donors. Non-survivors had higher levels of creatine/creatinine, 4-hydroxyproline, gluconic acid, and N-acetylserine, indicating liver and kidney dysfunction. Several changes were greater in women; thus, patients' sex should be considered in pandemic surveillance to achieve better disease stratification and improve outcomes. These metabolic alterations may be useful to monitor organ (dys) function and to understand the pathophysiology of acute and possibly post-acute COVID-19 syndromes.

Different outcomes of neonatal and adult Zika virus infection on startle reflex and prepulse inhibition in mice.

Zika virus (ZIKV) infection causes severe neurological consequences in both gestationally-exposed infants and adults. Sensorial gating deficits strongly correlate to the motor, sensorial and cognitive impairments observed in ZIKV-infected patients. However, no startle response or prepulse inhibition (PPI) assessment has been made in patients or animal models. In this study, we identified different outcomes according to the age of infection and sex in mice: neonatally infected animals presented an increase in PPI and delayed startle latency. However, adult-infected male mice presented lower startle amplitude, while a PPI impairment was observed 14 days after infection in both sexes. Our data further the understanding of the functional impacts of ZIKV on the developing and mature nervous system, which could help explain other behavioral and cognitive alterations caused by the virus. With this study, we support the startle reflex testing in ZIKV-exposed patients, especially infants, allowing for early detection of functional neuromotor damage and early intervention.

Yellow fever vaccine protects mice against Zika virus infection.

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak.

Skeletal Muscle Is an Early Site of Zika Virus Replication and Injury, Which Impairs Myogenesis.

Zika virus (ZIKV) infection became a worldwide concern due to its correlation with the development of microcephaly and other neurological disorders. ZIKV neurotropism is well characterized, but the role of peripheral viral amplification to brain infection remains unknown. Here, we found that ZIKV replicates in human primary skeletal muscle myoblasts, impairing its differentiation into myotubes but not interfering with the integrity of the already-formed muscle fibers. Using mouse models, we showed ZIKV tropism to muscle tissue either during embryogenesis after maternal transmission or when infection occurred after birth. Interestingly, ZIKV replication in the mouse skeletal muscle started immediately after ZIKV inoculation, preceding viral RNA detection in the brain and causing no disruption to the integrity of the blood brain barrier, and remained active for more than 2 weeks, whereas replication in the spleen and liver were not sustained over time. In addition, ZIKV infection of the skeletal muscle induces necrotic lesions, inflammation, and fiber atrophy. We also found a reduction in the expression of regulatory myogenic factors that are essential for muscle repair after injury. Taken together, our results indicate that the skeletal muscle is an early site of viral amplification and lesion that may result in late consequences in muscle development after ZIKV infection. IMPORTANCE Zika Virus (ZIKV) neurotropism and its deleterious effects on central nervous system have been well characterized. However, investigations of the initial replication sites for the establishment of infection and viral spread to neural tissues remain underexplored. A complete description of the range of ZIKV-induced lesions and others factors that can influence the severity of the disease is necessary to prevent ZIKV's deleterious effects. ZIKV has been shown to access the central nervous system without significantly affecting blood-brain barrier permeability. Here, we demonstrated that skeletal muscle is an earlier site of ZIKV replication, contributing to the increase of peripheral ZIKV load. ZIKV replication in muscle promotes necrotic lesions and inflammation and also impairs myogenesis. Overall, our findings showed that skeletal muscle is involved in pathogenesis and opens new fields in the investigation of the long-term consequences of early infection.

The immunodominant antibody response to Zika virus NS1 protein is characterized by cross-reactivity to self.

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.

Common Dysregulation of Innate Immunity Pathways in Human Primary Astrocytes Infected With Chikungunya, Mayaro, Oropouche, and Zika Viruses.

Arboviruses pose a major threat throughout the world and represent a great burden in tropical countries of South America. Although generally associated with moderate febrile illness, in more severe cases they can lead to neurological outcomes, such as encephalitis, Guillain-Barré syndrome, and Congenital Syndromes. In this context astrocytes play a central role in production of inflammatory cytokines, regulation of extracellular matrix, and control of glutamate driven neurotoxicity in the central nervous system. Here, we presented a comprehensive genome-wide transcriptome analysis of human primary astrocytes infected with Chikungunya, Mayaro, Oropouche, or Zika viruses. Analyses of differentially expressed genes (DEGs), pathway enrichment, and interactomes have shown that Alphaviruses up-regulated genes related to elastic fiber formation and N-glycosylation of glycoproteins, with down-regulation of cell cycle and DNA stability and chromosome maintenance genes. In contrast, Oropouche virus up-regulated cell cycle and DNA maintenance and condensation pathways while down-regulated extracellular matrix, collagen metabolism, glutamate and ion transporters pathways. Zika virus infection only up-regulated eukaryotic translation machinery while down-regulated interferon pathways. Reactome and integration analysis revealed a common signature in down-regulation of innate immune response, antiviral response, and inflammatory cytokines associated to interferon pathway for all arboviruses tested. Validation of interferon stimulated genes by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) corroborated our transcriptome findings. Altogether, our results showed a co-evolution in the mechanisms involved in the escape of arboviruses to antiviral immune response mediated by the interferon (IFN) pathway.

Mayaro Virus Replication Restriction and Induction of Muscular Inflammation in Mice Are Dependent on Age, Type-I Interferon Response, and Adaptive Immunity.

Mayaro virus (MAYV) is an emergent arbovirus first described in forest regions of the American continent, with recent and increasing notification of urban area circulation. Similar to Chikungunya (CHIKV) and other arthritogenic Alphavirus, MAYV-induced disease shows a high prevalence of persistent arthralgia, and myalgia. Despite this, knowledge regarding pathogenesis and characteristics of host immune response of MAYV infections are still limited. Here, using different ages of wild-type (WT), adult Type I Interferon receptor deficient (IFNAR-/-), and adult recombination activation gene-1 deficient (RAG-/-) mice, we have investigated the dependence of age, innate and adaptive immunity for the control of MAYV replication, tissue damage, and inflammation in mice. We have found that MAYV induces clinical signal and replicates in young WT mice, which gain the ability to restrict MAYV replication with aging. In addition, we observed that mice age and type I interferon response are related to restriction of MAYV infection and muscular inflammation in mice. Moreover, MAYV continues to replicate persistently in RAG-/- mice, being detected at blood and tissues 40 days post infection, indicating that adaptive immunity is essential to MAYV clearance. Despite chronic replication, infected adult RAG-/- mice did not develop an apparent signal of muscle damage in early and late infection. On the other hand, MAYV infection in young WT and adult IFNAR-/- mice triggers an increase in the expression of pro-inflammatory mediators, such as TNF, IL-6, KC, IL-1ß, MCP-1, and RANTES, in muscle tissue, and decreases TGF-ß expression, that were not significantly modulated in adult WT and RAG-/- mice. Taken together, our data demonstrated that age, innate and adaptive immunity are important to restrict MAYV replication and that adaptive immunity is also involved in MAYV-induced tissue damage. These results contribute to the comprehension of MAYV pathogenesis, and describe translational mice models for further studies of MAYV infection, vaccine tests, and therapeutic strategies against this virus.

Zika virus replicates in adult human brain tissue and impairs synapses and memory in mice.

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

Comprehensive characterisation of polyphenols in leaves and stems of three anti-dengue virus type-2 active Brazilian Faramea species (Rubiaceae) by HPLC-DAD-ESI-MS/MS.

INTRODUCTION: The methanol (MeOH) leaf extracts of the species Faramea bahiensis, F. hyacinthina and F. truncata (Rubiaceae) have previously shown in vitro non-cytotoxic and anti-dengue virus serotype 2 (DENV2) activities in human hepatocarcinoma cell lineage (HepG2). Chemical studies have led to the isolation of major flavonoids, but quite complex fractions of phenolic compounds still remain. OBJECTIVE: To complete the study of phenolic compounds in the leaves and to access the presence of these compounds in the stems of these Faramea spp. by online high-performance liquid chromatography-diode array detector-electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI-MS/MS), as well as to evaluate the in vitro cytotoxic and anti-DENV2 activities of their MeOH stem extracts. METHODOLOGY: The identification was performed by comparing retention times, UV and mass spectra with those of available standards and by using the mechanisms and fragmentation patterns established in previous studies. The effects of the extracts in DENV2 infected HepG2 cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The virus titer was quantified by plaque assay. RESULTS: The study led to the characterisation of 31 phenolic compounds including flavonoid O- and C-glycosides, phenolic acids and one coumarin. The stem extracts from F. hyacinthina and F. bahiensis presented a similar bioactivity to those of their leaves but a loss of cytoprotective activity of F. bahiensis and a higher cytotoxicity of F. truncata were observed. CONCLUSIONS: This research allowed a detailed phenolic composition of three bioactive Faramea species to be achieved, thus contributing to the study of this genus and providing valuable information for further phytotherapeutic applications.

Acute and chronic neurological consequences of early-life Zika virus infection in mice.

Although congenital Zika virus (ZIKV) exposure has been associated with microcephaly and other neurodevelopmental disorders, long-term consequences of perinatal infection are largely unknown. We evaluated short- and long-term neuropathological and behavioral consequences of neonatal ZIKV infection in mice. ZIKV showed brain tropism, causing postnatal-onset microcephaly and several behavioral deficits in adulthood. During the acute phase of infection, mice developed frequent seizures, which were reduced by tumor necrosis factor-α (TNF-α) inhibition. During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures, neurodegeneration, and brain calcifications. Altogether, the results show that neonatal ZIKV infection has long-term neuropathological and behavioral complications in mice and suggest that early inhibition of TNF-α-mediated neuroinflammation might be an effective therapeutic strategy to prevent the development of chronic neurological abnormalities.

Co-protoporphyrin IX and Sn-protoporphyrin IX inactivate Zika, Chikungunya and other arboviruses by targeting the viral envelope.

The global situation of diseases transmitted by arthropod-borne viruses such as Dengue (DENV), Yellow Fever (YFV), Chikungunya (CHIKV) and Zika (ZIKV) viruses is alarming and treatment of human infection by these arboviruses faces several challenges. The discovery of broad-spectrum antiviral molecules, able to inactivate different groups of viruses, is an interesting approach. The viral envelope is a common structure among arboviruses, being a potential target for antivirals. Porphyrins are amphipathic molecules able to interact with membranes and absorb light, being widely used in photodynamic therapy. Previously, we showed that heme, Co-protoporphyrin IX (CoPPIX) and Sn-protoporphyrin IX (SnPPIX) directly inactivate DENV and YFV infectious particles. Here we demonstrate that the antiviral activity of these porphyrins can be broadened to CHIKV, ZIKV, Mayaro virus, Sindbis virus and Vesicular Stomatitis virus. Porphyrin treatment causes viral envelope protein loss, affecting viral morphology, adsorption and entry into target cells. Also, light-stimulation enhanced the SnPPIX activity against all tested arboviruses. In summary, CoPPIX and SnPPIX were shown to be efficient broad-spectrum compounds to inactivate medically and veterinary important viruses.

Antiviral Activity of Faramea hyacinthina and Faramea truncata Leaves on Dengue Virus Type-2 and Their Major Compounds.

The defatted fractions of the Faramea hyacinthina and F. truncata (Rubiaceae) leaf MeOH extracts showed in vitro non-cytotoxic and anti-dengue virus serotype 2 (DENV2) activity in human hepatocarcinoma cell lineage (HepG2). Submitting these fractions to the developed RP-SPE method allowed isolating the antiviral flavanone (2S)-isosakuranetin-7-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (1) from both species and yielded less active sub-fractions. The new diastereoisomeric epimer pair (2S) + (2R) of 5,3',5'-trihydroxyflavanone-7-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (2a/2b) from F. hyacinthina; the known narigenin-7-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (3) from both species; rutin (4) and quercetin-4'-ß-d-O-glucopyranosyl-3-O-rutinoside (5) from F. hyacinthina, and kaempferol-3-O-rutinoside (6), erythroxyloside A (7) and asperuloside (8) from F. truncata have been isolated from these sub-fractions. Compounds 4 - 8 are reported for the first time in Faramea spp.

Development of standard methods for Zika virus propagation, titration, and purification.

The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7days post infection in C6/36 cells (7×107 and 2×108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5×107PFU/ml at 72hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.