Co-circulation of Chikungunya virus, Zika virus, and serotype 1 of Dengue virus in Western Bahia, Brazil

Chikungunya, mayaro, dengue, zika, and yellow fever are mosquito-borne viral diseases caused, respectively, by Chikungunya virus, Mayaro virus (CHIKV and MAYV, respectively: Togaviridae: Alphavirus), Dengue virus, Zika virus, and Yellow fever virus (DENV, ZIKV, and YFV, respectively: Flaviviridae: Flavivirus). These viruses have an important epidemiological impact worldwide, especially in Brazil. Western Bahia is one of the less studied regions in that country regarding the circulation of these pathogens. In this study, we aimed to apply molecular biology assays to better know the mosquito-borne viruses circulating in Barreiras and Luís Eduardo Magalhães, two main cities of Western Bahia. From March to June 2021, we enrolled 98 patients with the clinical diagnosis of dengue. Personal information (gender and age) were retrieved at the moment of enrollment. Serum samples were obtained from volunteers and used in molecular detection of CHIKV, MAYV, DENV, ZIKV, and YFV by reverse transcription followed by real-time polymerase chain reaction as well as in genome sequencing aiming phylogenetic analysis. As the main result, we found that from the 98 patients 45 were infected by CHIKV, 32 were infected by serotype 1 of DENV (DENV-1) and six were infected by ZIKV, while 15 were negative for all arboviruses tested. In addition, phylogenetic analysis revealed that all CHIKV-positive samples were of the East/Central/South African (ECSA) genotype, while all DENV-1-positive samples were of the V genotype. These results clearly show that epidemiological surveillance cannot be based only on clinical evaluations. Laboratory diagnosis is important in arbovirus infection that are prevalent in a particular area. These findings also demonstrate the co-circulation of many arboviruses in Western Bahia in 2021

Development and validation of an in-house method for whole genome amplification and sequencing of Zika virus.

O zika é um arbovírus emergente. Há evidências para a relação entre o zika e a microcefalia congênita e também com a síndrome de Guillain-Barre. Várias características do vírus são importantes, como a persistência do vírus no sêmen por vários meses, transmissão sexual e evidência de transmissão pré-natal. As mães grávidas infectadas com zika podem dar à luz crianças aparentemente saudáveis que podem apresentar manifestações e complicações tardias. Existe uma clara necessidade de diagnosticar e sequenciar amostras clínicas do ZIKV que circulam na América do Sul, especificamente no Brasil. No entanto, as baixas cargas virais observadas que são observadas comumente em amostras humanas constituem um fator complicador para detecção, amplificação e sequenciamento. Neste projeto, propor projetar um fluxo de trabalho otimizado para o sequenciamento completo do genoma com base no pré-enriquecimento por PCR (reação em cadeia da polimerase) e pools de amplicons.Zika is an emerging arbovirus. There is enough evidence for the relation between Zika and congenital microcephaly and also with the Guillain-Barre syndrome. Several characteristics of the virus are important, such as persistence of the virus in semen for several months, sexual transmission and evidence of prenatal transmission. Zika infected pregnant mothers may give birth to apparently healthy children that may show late manifestations and complications. There is a clear necessity of diagnosing and sequencing clinical samples of ZIKV circulating in South America, specifically in Brazil. Nevertheless, the observed low viral loads that are commonly in human samples constitute a complicating factor for detection, amplification and sequencing. In this project, we aim to design an optimized workflow for full genome sequencing based on pre-enrichment by PCR (polymerase chain reaction) and amplicon pools

Chikungunya Virus E2 Structural Protein B-Cell Epitopes Analysis

The Togaviridae family comprises a large and diverse group of viruses responsible for recurrent outbreaks in humans. Within this family, the Chikungunya virus (CHIKV) is an important Alphavirus in terms of morbidity, mortality, and economic impact on humans in different regions of the world. The objective of this study was to perform an IgG epitope recognition of the CHIKV’s structural proteins E2 and E3 using linear synthetic peptides recognized by serum from patients in the convalescence phase of infection. The serum samples used were collected in the state of Sergipe, Brazil in 2016. Based on the results obtained using immunoinformatic predictions, synthetic B-cell peptides corresponding to the epitopes of structural proteins E2 and E3 of the CHIKV were analyzed by the indirect peptide ELISA technique. Protein E2 was the main target of the immune response, and three conserved peptides, corresponding to peptides P3 and P4 located at Domain A and P5 at the end of Domain B, were identified. The peptides P4 and P5 were the most reactive and specific among the 11 epitopes analyzed and showed potential for use in serological diagnostic trials and development and/or improvement of the Chikungunya virus diagnosis and vaccine design

Ultrasound-guided minimally invasive autopsy as a tool for rapid post-mortem diagnosis in the 2018 Sao Paulo yellow fever epidemic: Correlation with conventional autopsy.

BackgroundNew strategies for collecting post-mortem tissue are necessary, particularly in areas with emerging infections. Minimally invasive autopsy (MIA) has been proposed as an alternative to conventional autopsy (CA), with promising results. Previous studies using MIA addressed the cause of death in adults and children in developing countries. However, none of these studies was conducted in areas with an undergoing infectious disease epidemic. We have recently experienced an epidemic of yellow fever (YF) in Brazil. Aiming to provide new information on low-cost post-mortem techniques that could be applied in regions at risk for infectious outbreaks, we tested the efficacy of ultrasound-guided MIA (MIA-US) in the diagnosis of patients who died during the epidemic.Methodology/principal findingsIn this observational study, we performed MIA-US in 20 patients with suspected or confirmed YF and compared the results with those obtained in subsequent CAs. Ultrasound-guided biopsies were used for tissue sampling of liver, kidneys, lungs, spleen, and heart. Liver samples from MIA-US and CA were submitted for RT-PCR and immunohistochemistry for detection of YF virus antigen. Of the 20 patients, 17 had YF diagnosis confirmed after autopsy by histopathological and molecular analysis. There was 100% agreement between MIA-US and CA in determining the cause of death (panlobular hepatitis with hepatic failure) and main disease (yellow fever). Further, MIA-US obtained samples with good quality for molecular studies and for the assessment of the systemic involvement of the disease. Main extrahepatic findings were pulmonary hemorrhage, pneumonia, acute tubular necrosis, and glomerulonephritis. One patient was a 24-year-old, 27-week pregnant woman; MIA-US assessed the placenta and provided adequate placental tissue for analysis.ConclusionsMIA-US is a reliable tool for rapid post-mortem diagnosis of yellow fever and can be used as an alternative to conventional autopsy in regions at risk for hemorrhagic fever outbreaks with limited resources to perform complete diagnostic autopsy

Molecular characterization of the sequenced yellow fever cases, showing the number of YFV RNA copies according to each of the 8 tissues analyzed for positive YFV-sylvatic patients.

The different colors represent the different tissues analyzed. The results indicate that both patients present a positive correlation for the quantification of genetic material.</p

Correlation between genetic divergence and sampling time was obtained by a root-to-tips analysis using the YFV sequences.

Root-to-tips distances were inferred using a maximum likelihood phylogeny for the genotype South American I, Clade II, (dataset-2).</p

Highest posterior probability migration paths for the YFV Clade II from 2015 to 2019 towards and beyond the MRSP, based on the analysis of 91 complete genomes (dataset 2).

(A) Migration on the full map of Brazil, and (B) migration being viewed up close. The spatiotemporal spread was visualized with spreaD3 v.0.9.7.1, using a GeoJSON file, (MIT license https://www.kaggle.com/datasets/moiseslima/majson?resource=download) to visualize the spatial YFV spread.</p

S1 Text -

Fig A in S1 Text. Combined coverage (normalized by the sample average) along the two sequenced Yellow Fever virus (YFV) genomes generated in this study. The genomic position reflects the genomic organization of the YFV, which is organized as follows: a single polyprotein cleaved into three structural, including capsid (C), membrane (M), and envelope (E), and seven non-structural (NS) proteins named NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The polyprotein is flanked by the 5’ and 3’ ends, which are non-coding. Table A in S1 Text. Complete YFV polyprotein sequences used in the phylogenetic analysis (dataset-1) (n = 264). Table B in S1 Text. Complete YFV polyprotein sequences used in the phylogeographic analysis (dataset-2) (n = 91). Table C in S1 Text. Model comparison of the relaxed molecular clock and demographic growth models through path sampling (PS) and stepping stone (SS) methods. Bold numbers indicate the best fitting model. Table D in S1 Text. Comparison among continuous diffusion models for Brazilian sequences. (DOCX)</p

Epidemiological situation of the YFV outbreak in São Paulo, 2016–2019.

(A) The number of YFV positive NHPs in São Paulo, from 2016–2019. (B) The number of YFV-positive human cases in São Paulo, from 2016–2019. (C) Location of the two fatal human cases studied in the transition from 2018 to 2019. We used R software, geobr package, (MIT license https://ipeagit.github.io/geobr/) to create the maps produced in this Fig to visualize the spatial location.</p

Maximum likelihood phylogenetic trees for YFV based on full-length polyprotein sequences (n = 264) indicate that the sequences associated with the outbreak in Brazil between 2016–2019 grouped within the genotype South American I.

The tree is midpoint-rooted and the black circles on the main nodes represent bootstrap support values greater than 0.7. The distinct colors represent distinct genotypes: (i) EAfr—East African; (ii) WAfr—West African; (iii) SA1—South American I; (iv) SA2—South American II.</p