Zika virus (ZIKV) was first discovered in 1947 in Uganda but was not considered a public health threat until 2007 when it found to be the source of epidemic activity in Asia. Epidemic activity spread to Brazil in 2014 and continued to spread throughout the tropical and subtropical regions of the Americas. Despite ZIKV being zoonotic in origin, information about transmission, or even exposure of non-human vertebrates and mosquitoes to ZIKV in the Americas, is lacking. Accordingly, from February 2017 to March 2018, we sought evidence of sylvatic ZIKV transmission by sampling whole blood from approximately 2000 domestic and wild vertebrates of over 100 species in West-Central Brazil within the active human ZIKV transmission area. In addition, we collected over 24,300 mosquitoes of at least 17 genera and 62 species. We screened whole blood samples and mosquito pools for ZIKV RNA using pan-flavivirus primers in a real-time reverse-transcription polymerase chain reaction (RT-PCR) in a SYBR Green platform. Positives were confirmed using ZIKV-specific envelope gene real-time RT-PCR and nucleotide sequencing. Of the 2068 vertebrates tested, none were ZIKV positive. Of the 23,315 non-engorged mosquitoes consolidated into 1503 pools tested, 22 (1.5%) with full data available showed some degree of homology to insect-specific flaviviruses. To identify previous exposure to ZIKV, 1498 plasma samples representing 62 species of domestic and sylvatic Viruses 2019, 11, 11643 of 18 vertebrates were tested for ZIKV-neutralizing antibodies by plaque reduction neutralization test (PRNT 90 ). From these, 23 (1.5%) of seven species were seropositive for ZIKV and negative for dengue virus serotype 2, yellow fever virus, and West Nile virus, suggesting potential monotypic reaction for ZIKV. Results presented here suggest no active transmission of ZIKV in non-human vertebrate populations or in alternative vector candidates, but suggest that vertebrates around human populations have indeed been exposed to ZIKV in West-Central Brazil.

Successful SARS-CoV-2 inactivation allows its safe use in Biosafety Level 2 facilities, and the use of the whole viral particle helps in the development of analytical methods and a more reliable immune response, contributing to the development and improvement of in vitro and in vivo assays. In order to obtain a functional product, we evaluated several inactivation protocols and observed that 0.03% beta-propiolactone for 24 h was the best condition tested, as it promoted SARS-CoV-2 inactivation above 99.99% and no cytopathic effect was visualized after five serial passages. Moreover, RT-qPCR and transmission electron microscopy revealed that RNA quantification and viral structure integrity were preserved. The antigenicity of inactivated SARS-CoV-2 was confirmed by ELISA using different Spike-neutralizing monoclonal antibodies. K18-hACE2 mice immunized with inactivated SARS-CoV-2, formulated in AddaS03TM, presented high neutralizing antibody titers, no significant weight loss, and longer survival than controls from a lethal challenge, despite RNA detection in the oropharyngeal swab, lung, and brain. This work emphasizes the importance of using different techniques to confirm viral inactivation and avoid potentially disastrous contamination. We believe that an efficiently inactivated product can be used in several applications, including the development and improvement of molecular diagnostic kits, as an antigen for antibody production as well as a control for non-clinical trials.

Introdução:O vírus ZIKA atualmente está disseminado em países da África, Ásia e Américas. Os sintomas são semelhantes aos de Dengue. Entretanto, após sua emergência no Brasil, observou-se o aumento de casos de microcefalia e síndrome de Guillain-Barré. Ao nível de diagnóstico, recomenda-se o Teste de Neutralização por Placas de Lise (PRNT, do inglês Plaque Reduction Neutralization Test) como teste confirmatório ao ELISA, uma vez que este método é considerado padrão ouro para quantificação de anticorpos neutralizantes. Entretanto, estudos preliminares demonstram a ocorrência de falsos positivos em pacientes que apresentaram infecção prévia por outros flavivírus. Sendo assim, avaliar estratégias para minimizar reações cruzadas é indispensável para a obtenção de um resultado confiável. Objetivo:O principal objetivo do estudo é minimizar reações cruzadas com flavivírus no PRNT de ZIKA, a partir da análise de endpoint , 50% e 90%, e do tempo de neutralização do teste. Metodologia:Para esta avaliação foram utilizadas quinze amostras, divididas em grupos, sendo: duas amostras positivas para ZIKA e negativas para Dengue (Z+/D-); duas amostras positivas para ZIKA e Dengue (Z+/D+); onze amostras coletadas no período anterior à circulação do vírus no Brasil, logo, negativas para ZIKA, porém entre estas, oito são positivas para Dengue (Z-/D+) e três são negativas para estes dois flavivírus , no entanto, positivas para Febre Amarela (Z-/D-/F+). Esses grupos foram todos previamente testados por PRNT de Dengue, e Febre Amarela no caso do último grupo. Além disso, os grupos positivos para ZIKA foram selecionados por quadros clínicos característicos e confirmados por PRNT.

Introduction: Two most important forms of diagnostic testing available for SARS-CoV-2 are molecular and serological tests. Among those, the serum neutralization assay stands out as the gold standard for evaluation of the effectiveness of neutralizing antibodies (NAbs) against viral infections. In this regard, it is important the standardization of neutralization-based assay to validate the specificity and sensitivity of current immunoassays against SARS-CoV-2 to avoid bias outcomes.Objective: The present study aims to develop a test for the evaluation of NAbs against SARS-CoV-2 through universal serum neutralization platform by plaque reduction neutralization test (PRNT-SARS-CoV-2).Methodology: Briefly, 24-well plates were seeded with two different Vero cell concentrations (1.2 x 10 5 or 2 x 10 5 cells/well). In addition, dilutions were assessed with approximately 60-100 PFU of SARS-CoV-2 (PV004/20 CoV-2-P4; 1,71 X 10 6 TCID50/mL) per well, and plates with virus and serum as well as mock plates followed by incubation at 37°C for 1h. Thereafter, the supernatant was transferred to definitive plates with cell monolayers and incubated at 37°C for 1 h. After this time, media was discarded; cells were overlaid with 1 mL of E199 medium with 1.5 or 2% of CMC and incubated for 3 days at 37°C in 5% CO2. Cells were then fixed with 10% formalin, stained with crystal violet and plaques were counted. Neutralizing antibody titers were expressed by 50% or 90% of plaque reduction. Results:We found that 2 x 105 cells/well and 1.5% of CMC, besides the 1:12.000 of virus dilution revealed to be the better conditions to perform the assay. Our early results showed that the majority of specimens from COVID-19 positive donor presented low neutralizing antibodies levels (Median 1:36.5; titers calculated by reciprocal dilution). Conclusion:In perspective, this project aims to contribute to elucidate the role of NAb levels in the protection and/or severity of COVID-19. Considering that SARS-CoV-2 infection is a public health concern, besides the imminent vaccination, the available of a neutralization assay, standardized and validated, could help to answer important gaps related to epidemiologic perspective on surveillance policies. The authors thank the Multi-user Research Facility of Biosafety Platform NB3-

Introduction: Plaque Reduction Neutralization Test (PRNT) is considered the "gold standard" by the World Health Organization for the confirmatory diagnosis of Flavivirus infection from the determination of neutralizing antibodies. Zika virus (ZIKV), a Flavivirus widely circulating in Brazil has caused considerable epidemic impact. Although 80% of cases are asymptomatic, virus infection in symptomatic cases causes headache, low fever, mild joint pain, red spots on the skin, itching and redness in the eyes. Other less common symptoms are swelling in the body, sore throat, coughing and vomiting. Infection by ZIKV has great importance especially for pregnant women, since the virus is a potential cause for the birth of children with a congenital malformation, in which the brain does not develop properly, called microcephaly. The Virological Technology Laboratory (LATEV, Bio-Manguinhos/Fiocruz) performs PRNT assays for different Flavivirus species, not only as confirmatory differential diagnosis, but mainly in the evaluation of the immunogenicity of commercial vaccines in development. Bio-Manguinhos currently participates in the development of three different vaccine proposals for the Zika virus. Therefore, considering potential increase in the demand for neutralizing antibody titers for diagnostic and vaccine evaluation, the standardization of a neutralization test for the Zika virus with high sample processing capacity meets the needs of LATEV and, consequently, of Institution and public health. Objective: The objective of this work was to standardize the micro-PRNT for ZIKV, methodology with greater capacity of sample processing, performed in 96-well plates. Methodology: The methodology of this work was based on the determination of the protocol of execution of the micro-PRNT for ZIKV. Although the rationale and test steps have already been determined for other flaviviruses, it was necessary to evaluate the variables of the micro-PRNT methodology and their different conditions specifically for the determination of neutralizing antibodies to ZIKV, ie standardize the test for ZIKV. Therefore, the standardization process involved the main steps/variables of the test: cell density, final incubation time, ideal virus dilution and concentration of semisolid medium. Results: Monolayers prepared at 2.0 x 10 5 cells / ml 24 hours before virus infection and incubated for 4 days with 2.0% semisolid medium resulted in the best PFU profile for ZIKV in 96-well plates. The ideal virus dilution to obtain on average 80 PFU / well was previously determined. Conclusion: As a result of the standardization of this gold-standard methodology in 96-well plates, LATEV becomes capable of increasing its sample processing capacity and, consequently, efficiently meet the increasing demand for the determination of neutralizing antibody titers for the ZIKV. Thus, the next step will be to validate the test according to Anvisa's regulatory standards.