Saxitoxin potentiates human neuronal cell death induced by Zika virus while sparing neural progenitors and astrocytes.

The Zika virus (ZIKV) epidemic declared in Brazil between 2015 and 2016 was associated with an increased prevalence of severe congenital malformations, including microcephaly. The distribution of microcephaly cases was not uniform across the country, with a disproportionately higher incidence in the Northeast region (NE). Our previous work demonstrated that saxitoxin (STX), a toxin present in the drinking water reservoirs of the NE, exacerbated the damaging effects of ZIKV on the developing brain. We hypothesized that the impact of STX might vary among different neural cell types. While ZIKV infection caused severe damages on astrocytes and neural stem cells (NSCs), the addition of STX did not exacerbate these effects. We observed that neurons subjected to STX exposure were more prone to apoptosis and displayed higher ZIKV infection rate. These findings suggest that STX exacerbates the harmful effects of ZIKV on neurons, thereby providing a plausible explanation for the heightened severity of ZIKV-induced congenital malformations observed in Brazil's NE. This study highlights the importance of understanding the interactive effects of environmental toxins and infectious pathogens on neural development, with potential implications for public health policies.

Structural and dynamics characterization of the Zika virus NS2B using nuclear magnetic resonance and RosettaMP: A challenge for transmembrane protein studies.

Zika virus (ZIKV) is an emergent flavivirus that represents a global public health concern due to its association with severe neurological disorders. NS2B is a multifunctional viral membrane protein primarily used to regulate viral protease activity and is crucial for virus replication, making it an appealing target for antiviral drugs. This study presents the structural elucidation of full-length ZIKV NS2B in sodium dodecyl sulfate (SDS) micelles using solution nuclear magnetic resonance experimental data and RosettaMP. The protein structure has four transmembrane α-helices, two amphipathic α-helices, and a ß-hairpin in the hydrophilic region. NS2B presented secondary and tertiary stability in different concentrations of SDS. Furthermore, we studied the dynamics of NS2B in SDS micelles through relaxation parameters and paramagnetic relaxation enhancement experiments. The findings were consistent with the structural calculations. Our work will be essential in understanding the role of NS2B in viral replication and screening for inhibitors against ZIKV.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.

Shared Molecular Signatures Across Zika Virus Infection and Multiple Sclerosis Highlight AP-1 Transcription Factor as a Potential Player in Post-ZIKV MS-Like Phenotypes.

Zika virus (ZIKV) is an arbovirus of the Flaviviridae genus that has rapidly disseminated from across the Pacific to the Americas. Robust evidence has indicated a crucial role of ZIKV in congenital virus syndrome, including neonatal microcephaly. Moreover, emerging evidence suggests an association between ZIKV infection and the development of an extensive spectrum of central nervous system inflammatory demyelinating diseases (CNS IDD), such as multiple sclerosis-like clinical phenotypes. However, the underlying mechanisms of host-pathogen neuro-immune interactions remain to be elucidated. This study aimed to identify common transcriptional signatures between multiple sclerosis (MS) and ZIKV infection to generate molecular interaction networks, thereby leading to the identification of deregulated processes and pathways, which could give an insight of these underlying molecular mechanisms. Our investigation included publicly available transcriptomic data from MS patients in either relapse or remission (RR-MS) and datasets of subjects acutely infected by ZIKV for both immune peripheral cells and central nervous system cells. The protein-protein interaction (PPI) analysis showed upregulated AP-1 transcription factors (JUN and FOS) among the top hub and bottleneck genes in RR-MS and ZIKV data. Gene enrichment analysis retrieved a remarkable presence of ontologies and pathways linked to oxidative stress responses, immune cell function, inflammation, interleukin signaling, cell division, and transcriptional regulation commonly enriched in both scenarios. Considering the recent findings concerning AP-1 function in immunological tolerance breakdown, regulation of inflammation, and its function as an oxidative stress sensor, we postulate that the ZIKV trigger may contribute as a boost for the activation of such AP-1-regulated mechanisms that could favor the development of MS-like phenotypes following ZIKV infection in a genetically susceptible individual.

Big Data to Knowledge Analytics Reveals the Zika Virus Epidemic as Only One of Multiple Factors Contributing to a Year-Over-Year 28-Fold Increase in Microcephaly Incidence.

During the 2015-2016 Zika Virus (ZIKV) epidemic in Brazil, the geographical distributions of ZIKV infection and microcephaly outbreaks did not align. This raised doubts about the virus as the single cause of the microcephaly outbreak and led to research hypotheses of alternative explanatory factors, such as environmental variables and factors, agrochemical use, or immunizations. We investigated context and the intermediate and structural determinants of health inequalities, as well as social environment factors, to determine their interaction with ZIKV-positive- and ZIKV-negative-related microcephaly. The results revealed the identification of 382 associations among 382 nonredundant variables of Zika surveillance, including multiple determinants of environmental public health factors and variables obtained from 5565 municipalities in Brazil. This study compared those factors and variables directly associated with microcephaly incidence positive to ZIKV and those associated with microcephaly incidence negative to ZIKV, respectively, and mapped them in case and control subnetworks. The subnetworks of factors and variables associated with low birth weight and birthweight where birth incidence served as an additional control were also mapped. Non-significant differences in factors and variables were observed, as were weights of associations between microcephaly incidence, both positive and negative to ZIKV, which revealed diagnostic inaccuracies that translated to the underestimation of the scope of the ZIKV outbreak. A detailed analysis of the patterns of association does not support a finding that vaccinations contributed to microcephaly, but it does raise concerns about the use of agrochemicals as a potential factor in the observed neurotoxicity arising from the presence of heavy metals in the environment and microcephaly not associated with ZIKV. Summary: A comparative network inferential analysis of the patterns of variables and factors associated with Zika virus infections in Brazil during 2015-2016 coinciding with a microcephaly epidemic identified multiple contributing determinants. This study advances our understanding of the cumulative interactive effects of exposures to chemical and non-chemical stressors in the built, natural, physical, and social environments on adverse pregnancy and health outcomes in vulnerable populations.

Epilepsy in children with Congenital Zika Syndrome: A systematic review and meta-analysis.

OBJECTIVES: To estimate the overall frequency of epilepsy in children with congenital Zika syndrome (CZS) and describe the profile of seizures and the response rate to anti-epileptic treatment in this group of patients. METHODS: A systematic review and meta-analysis were conducted following the Cochrane Handbook and preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. PubMed/MEDLINE, Scopus, Cochrane Library, SciELO, and LILACS were searched until June 23, 2020. Observational studies that evaluated the frequency of epilepsy in children diagnosed with CZS according to international criteria were included in the study. RESULTS: Fourteen studies evaluating 903 patients diagnosed with CZS were pooled in a meta-analysis. All studies were conducted in Brazil, with reports published between 2016 and 2020, and included children diagnosed with CSZ from 0 to 40 months of age. The overall rate of epilepsy in children diagnosed with CZS was estimated at 60% (95% confidence interval [CI] 0.51-0.68). The studies included in this review show that the frequency of epilepsy in patients with CSZ varies with age, with higher rates in older children. Epileptic spasms was the primary type of seizure observed in this group, followed by focal and generalized crisis. The response rate to anti-epileptic drugs was considerably low, ranging from 20% of seizure control in the first year and 30% in the second year. SIGNIFICANCE: Children with CZS presented a high cumulative incidence of epilepsy episodes with increased severity and a low response to anti-epileptic therapy, which is associated with the extensive damage caused by the Zika virus on the cortical structures of patients.

Accurate detection of Zika virus IgG using a novel immune complex binding ELISA.

OBJECTIVES: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection. METHODS: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252). RESULTS: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations. CONCLUSIONS: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating.

Zika virus-spread, epidemiology, genome, transmission cycle, clinical manifestation, associated challenges, vaccine and antiviral drug development.

Zika Virus (ZIKV) is a Flavivirus transmitted primarily via the bite of infected Aedes aegypti mosquitoes. Globally, 87 countries and territories have recorded autochthonous mosquito-borne transmission of ZIKV as at July 2019 and distributed across four of the six WHO Regions. Outbreaks of ZIKV infection peaked in 2016 and declined substantially throughout 2017 and 2018 in the Americas region. There is the likely risk for ZIKV to spread to more countries. There is also the potential for the re-emergence of ZIKV in all places with prior reports of the virus transmission. The current status of ZIKV transmission and spread is, however, a global health threat, and from the aforementioned, has the potential to re-emerge as an epidemic. This review summarizes the past and present spread of ZIKV outbreak-2007-2019, the genome, transmission cycle, clinical manifestations, vaccine and antiviral drug advancement.

A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

Geographical trends of chikungunya and Zika in the Colombian Amazonian gateway department, Caqueta, 2015-2018 - Implications for public health and travel medicine.

BACKGROUND: Chikungunya (CHIKV) and Zika (ZIKV) significantly affected Latin America in the period 2015-2017. Most studies were reported from urban areas of Brazil and Colombia. In this paper we estimate Incidence rates for CHIKV and ZIKV in Caqueta, the Amazonian gateway area of Colombia, from 2015 to 2018. METHODS: Using surveillance data of CHIKV and ZIKV in Caqueta, Colombia, incidence rates were estimated (cases/100,000 population). Sixteen geographical information systems (GIS)-based municipal maps were developed. GIS software used was Kosmo 3.0®. RESULTS: From 1st of January 2015 to the 24th of November 2018, 825 cases of CHIK and 1079 of ZIKV were reported, yielding cumulated incidence rates of 169.42 and 221.59 cases/100,000 population respectively. In 2016, 48.7% of the CHIKV cases (402) and 96.6% of the ZIKV cases (1042) were reported. The highest number of both arboviral diseases occurred at Florencia (capital department city), 225 cases for CHIKV (127.17 cases/100,000 pop.) and 611 for ZIKV (345.34 cases/100,000 pop.). DISCUSSION: The temporo-spatial distribution of CHIKV and ZIKV infections in Caquetá reflected the pattern of concurrent epidemics, especially in 2016. Studies using GIS-linked maps are necessary to attain accurate epidemiological analyses for public health decisions. That is also useful for an epidemiologically based assessment of traveler risks when visiting specific areas in destination countries.

Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review.

The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.

Spatial distribution of Zika in Honduras during 2016-2017 using geographic information systems (GIS) - Implications for public health and travel medicine.

BACKGROUND: Zika virus (ZIKV) infection has significantly affected Latin America in 2015-2017. Most studies have been reported from Brazil and Colombia, and only a few from Central America. For these reasons, we analyzed the incidence, incidence rates and evolution of cases in Honduras from 2016 to 2017. METHODS: Using epidemiological weeks (EW) surveillance data on the ZIKV epidemics in Honduras, we estimated incidence rates (cases/100,000 population), and developed maps at national, departmental and municipal levels. RESULTS: From 1 January 2016 to 31 December 2017, a total of 32,607 cases of ZIKV were reported (98.5% in 2016 for an incidence rate of 36.85 cases/100,000 pop; 1% confirmed by RT-PCR). The highest peak was reached on the EW 6°, 2016 (2559 cases; 29.34 cases/100,000 pop). The department with the highest number of cases and incidence rate was Cortés (13,128 cases, 791.08 cases/100,000 pop in 2016). DISCUSSION: The pattern and evolution of ZIKV infection in Honduras have been similar to that which occurred for chikungunya in 2015. As previously reported, infection with chikungunya involved predominantly the central and capital area of the country, reaching incidences there >750 cases/100,000 pop. Studies using geographical information systems linked with clinical disease characteristics are necessary to attain accurate epidemiological data for public health systems. Such information is also useful for assessment of risk for travelers who visit specific areas in a destination country.

Knowledge, perceptions and practices regarding Zika virus of university students in northern Colombia (Santa Marta, 2016) / Conocimientos, percepciones y prácticas sobre el virus del Zika en estudiantes universitarios del norte de Colombia (Santa Marta, 2016)

This study focuses on the knowledge, perceptions and practices of 171 university students regarding Zika virus and its vector in Santa Marta (Colombia) in 2016. A survey was conducted, and the answers about causative agent and mode of transmission were classified into three levels of knowledge. Altogether, 32.1% of the students stated that they had suffered from Zika. A total of 60% stated that they knew what the disease was; however, only 29.2% knew what the causative agent was, and 45.6% knew the mode of transmission. Regarding the level of knowledge, only 14.6% knew the causative agent and the mode of transmission (Level 2). In general, the students recognize the symptoms of Zika virus, and 53.8% of them consider Zika to be very serious. More than half of them believe that they, the community, and the government are responsible for controlling the vector. Even though more than half the students know the most important strategies to control the vector, they do not apply them; the reasons for this might be their everyday habits, the lack of organization in their communities, a deficient public health system, and climate change. It is recommended to implement permanent strategies for vector control that take into account the sociocultural characteristics of at-risk populations.

Este estudio analiza el nivel de conocimiento, percepciones y prácticas de 171 estudiantes universitarios sobre el virus del Zika y su vector en Santa Marta (Colombia) en 2016. Se aplicó una encuesta y las respuestas sobre el agente causativo y el modo de transmisión se clasificaron en tres niveles de conocimiento. Se encontró que el 32,1% de los estudiantes manifiestan haber sufrido Zika. Un 60% afirmó que conocían la enfermedad, sin embargo, solo el 29,2% conocía el agente causal y el 45,6% el modo de transmisión. Sólo el 14,6% conocía el agente causal y el modo de transmisión (Nivel 2). En general, los estudiantes reconocen los síntomas del Zika. Para el 53,8% es una enfermedad muy grave y más de la mitad considera que ellos, la comunidad y el gobierno son responsables de controlar el vector. Aunque más de la mitad de los estudiantes conocen las estrategias más importantes para controlar el vector, no las practican; esto puede explicarse debido a sus prácticas cotidianas, falta de organización comunitaria, deficiencia en el sistema de salud pública y el cambio climático. Se recomienda implementar estrategias permanentes de control de vectores que consideren las características socioculturales de las poblaciones en riesgo.

Emerging arboviruses in Rio Grande do Sul, Brazil: Chikungunya and Zika outbreaks, 2014-2016.

QUESTIONS INVESTIGATED: The recent emergence of arboviruses such as Chikungunya virus (CHIKV) and Zika virus (ZIKV) in Brazil has posed a threat to human health and to the country's economy. Outbreaks occur mainly in tropical areas; however, increasing number of cases have been observed in Rio Grande do Sul (RS), the Southernmost state; therefore, surveillance of these arboviruses is essential for public health measures. DESIGN: In this study, we analyzed 1276 samples from patients with clinically suspected arboviral diseases between 2014 and 2016. Demographic and clinical data were collected and described; cases of microcephaly associated with congenital infection were analyzed. ESSENTIAL FINDINGS: Results show that CHIKV and ZIKV entered RS in 2014 and 2015, respectively, with imported cases confirmed. Autochthonous infections occurred in 2016 for both viruses, with a total of 5 autochthonous cases for CHIKV and 44 for ZIKV. Most patients were older than 21 years; the main symptoms were fever, arthralgia, myalgia, and headache; rash, conjunctivitis, and pruritus were also reported in ZIKV cases. Three cases of congenital Zika syndrome were confirmed in our study, while another 20 cases of microcephaly associated with congenital infection were confirmed (10 positive for syphilis, 6 for toxoplasmosis and 4 for cytomegalovirus). MAIN CONCLUSIONS: Considering co-circulation of different arbovirus in RS, including Dengue virus, CHIKV, and ZIKV, and the presence of Aedes aegypti and Aedes albopictus in the area, surveillance of patients infected by these viruses contributes to the control and prevention of such diseases. Practical difficulties in diagnosing these infections are discussed.

Mapping the ecoepidemiology of Zika virus infection in urban and rural areas of Pereira, Risaralda, Colombia, 2015-2016: Implications for public health and travel medicine.

OBJECTIVE: Geographical information systems (GIS) have been demonstrated earlier to be of great use to inform public health action against vector-borne infectious diseases. METHODS: Using surveillance data on the ongoing ZIKV outbreak from Pereira, Colombia (2015-2016), we estimated incidence rates (cases/100,000 population), and developed maps correlating with the ecoepidemiology of the area. RESULTS: Up to October 8, 2016, 439 cases of ZIKV were reported in Pereira (93 cases/100,000 pop.), with highest rates in the South-West area. At the corregiments (sub-municipalities) of Pereira, Caimalito presented the highest rate. An urban area, Cuba, has 169 cases/100,000 pop., with a low economical level and the highest Aedic index (9.1%). Entomological indexes were associated with ZIKV incidence at simple and multiple non-linear regressions (r2 > 0.25; p < 0.05). CONCLUSIONS: Combining entomological, environmental, human population density, travel patterns and case data of vector-borne infections, such as ZIKV, leads to a valuable tool that can be used to pinpoint hotspots also for infections such as dengue, chikungunya and malaria. Such a tool is key to planning mosquito control and the prevention of mosquito-borne diseases in local populations. Such data also enable microepidemiology and the prediction of risk for travelers who visit specific areas in a destination country.

Emerging arboviruses in Rio Grande do Sul, Brazil: chikungunya and zika outbreaks, 2014­2016

Questions investigated: The recent emergence of arboviruses such as Chikungunya virus (CHIKV) and Zika virus (ZIKV) in Brazil has posed a threat to human health and to the country's economy. Outbreaks occur mainly in tropical areas; however, increasing number of cases have been observed in Rio Grande do Sul (RS), the Southernmost state; therefore, surveillance of these arboviruses is essential for public health measures. Design: In this study, we analyzed 1276 samples from patients with clinically suspected arboviral diseases between 2014 and 2016. Demographic and clinical data were collected and described; cases of microcephaly associated with congenital infection were analyzed. Essential findings: Results show that CHIKV and ZIKV entered RS in 2014 and 2015, respectively, with imported cases confirmed. Autochthonous infections occurred in 2016 for both viruses, with a total of 5 autochthonous cases for CHIKV and 44 for ZIKV. Most patients were older than 21 years; the main symptoms were fever, arthralgia, myalgia, and headache; rash, conjunctivitis, and pruritus were also reported in ZIKV cases. Three cases of congenital Zika syndrome were confirmed in our study, while another 20 cases of microcephaly associated with congenital infection were confirmed (10 positive for syphilis, 6 for toxoplasmosis and 4 for cytomegalovirus). Main conclusions: Considering co­circulation of different arbovirus in RS, including Dengue virus, CHIKV, and ZIKV, and the presence of Aedes aegypti and Aedes albopictus in the area, surveillance of patients infected by these viruses contributes to the control and prevention of such diseases. Practical difficulties in diagnosing these infections are discussed. (AU)