Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga, and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama.

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological diseases in human and equine hosts. Detecting them during the acute phase is hindered by non-specific symptoms and limited diagnostic tools. We designed and clinically assessed real-time reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015 to 2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021 to 2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in five suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in three Culex (Melanoconion) vomerifer pools, leading to VEEV isolates in two. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests that this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions.

Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2.

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.

Short report: Introduction of chikungunya virus ECSA genotype into the Brazilian Midwest and its dispersion through the Americas.

Since introduction into Brazil in 2014, chikungunya virus (CHIKV) has presented sustained transmission, although much is unknown about its circulation in the midwestern states. Here, we analyze 24 novel partial and near complete CHIKV genomes from Cuiaba, an urban metropolis located in the Brazilian midwestern state of Mato Grosso (MT). Nanopore technology was used for sequencing CHIKV complete genomes. Phylogenetic and epidemiological approaches were used to explore the recent spatio-temporal evolution and spread of the CHIKV-ECSA genotype in Midwest Brazil as well as in the Americas. Epidemiological data revealed a reduction in the number of reported cases over 2018-2020, likely as a consequence of a gradual accumulation of herd-immunity. Phylogeographic reconstructions revealed that at least two independent introductions of the ECSA lineage occurred in MT from a dispersion event originating in the northeastern region and suggest that the midwestern Brazilian region appears to have acted as a source of virus transmission towards Paraguay, a bordering South American country. Our results show a complex dynamic of transmission between epidemic seasons and suggest a possible role of Brazil as a source for international dispersion of the CHIKV-ECSA genotype to other countries in the Americas.

First report of Aedes albopictus infected by Dengue and Zika virus in a rural outbreak in Brazil.

In Brazil, Dengue (DENV) and Zika (ZIKV) viruses are reported as being transmitted exclusively by Aedes aegypti in urban settings. This study established the vectors and viruses involved in an arbovirus outbreak that occurred in 2019 in a rural area of Espírito Santo state, Brazil. Mosquitoes collected were morphologically identified, sorted in samples, and submitted to molecular analysis for arboviruses detection. Phylogenetic reconstruction was performed for the viral sequence obtained. All 393 mosquitoes were identified as Aedes albopictus. DENV-1 genotype V was present in one sample and another sample was positive for ZIKV. The DENV-1 clustered with viruses that have circulated in previous years in large urban centers of different regions in Brazil. This is the first report of A. albopictus infected by DENV and ZIKV during an outbreak in a rural area in Brazil, indicating its involvement in arboviral transmission. The DENV-1 strain found in the A. albopictus was not new in Brazil, being involved previously in epidemics related to A. aegypti, suggesting the potential to A. albopictus in transmitting viruses already circulating in the Brazilian population. This finding also indicates the possibility of these viruses to disperse across urban and rural settings, imposing additional challenges for the control of the diseases.

Predictors of mortality in patients with yellow fever: an observational cohort study.

BACKGROUND: Yellow fever virus infection results in death in around 30% of symptomatic individuals. The aim of this study was to identify predictors of death measured at hospital admission in a cohort of patients admitted to hospital during the 2018 outbreak of yellow fever in the outskirts of São Paulo city, Brazil. METHODS: In this observational cohort study, we enrolled patients with yellow fever virus from two hospitals in São Paolo-the Hospital das Clínicas, University of São Paulo and the Infectious Diseases Institute "Emilio Ribas". Patients older than 18 years admitted to hospital with fever or myalgia, headache, arthralgia, oedema, rash, or conjunctivitis were consecutively screened for inclusion in the present study. Consenting patients were included if they had travelled to geographical areas in which yellow fever virus cases had been previously confirmed. Yellow fever infection was confirmed by real-time PCR in blood collected at admission or tissues at autopsy. We sequenced the complete genomes of yellow fever virus from infected individuals and evaluated demographic, clinical, and laboratory findings at admission and investigated whether any of these measurements correlated with patient outcome (death). FINDINGS: Between Jan 11, 2018, and May 10, 2018, 118 patients with suspected yellow fever were admitted to Hospital das Clínicas, and 113 patients with suspected yellow fever were admitted to Infectious Diseases Institute "Emilio Ribas". 95 patients with suspected yellow fever were included in the study, and 136 patients were excluded. Three (3%) of 95 patients with suspected yellow fever who were included in the study were excluded because they received a different diagnosis, and 16 patients with undetectable yellow fever virus RNA were excluded. Therefore, 76 patients with confirmed yellow fever virus infection, based on detectable yellow fever virus RNA in blood (74 patients) or yellow fever virus confirmed only at the autopsy report (two patients), were included in our analysis. 27 (36%) of 76 patients died during the 60 day period after hospital admission. We generated 14 complete yellow fever virus genomes from the first 15 viral load-detectable samples. The genomes belonged to a single monophyletic clade of the South America I genotype, sub-genotype E. Older age, male sex, higher leukocyte and neutrophil counts, higher alanine aminotransferase, aspartate transaminase (AST), bilirubin, and creatinine, prolonged prothrombin time, and higher yellow fever virus RNA plasma viral load were associated with higher mortality. In a multivariate regression model, older age, elevated neutrophil count, increased AST, and higher viral load remained independently associated with death. All 11 (100%) patients with neutrophil counts of 4000 cells per mL or greater and viral loads of 5·1 log10 copies/mL or greater died (95% CI 72-100), compared with only three (11%) of 27 (95% CI 2-29) among patients with neutrophil counts of less than 4000 cells per mL and viral loads of less than 5·1 log10 copies/mL. INTERPRETATION: We identified clinical and laboratory predictors of mortality at hospital admission that could aid in the care of patients with yellow fever virus. Identification of these prognostic markers in patients could help clinicians prioritise admission to the intensive care unit, as patients often deteriorate rapidly. Moreover, resource allocation could be improved to prioritise key laboratory examinations that might be more useful in determining whether a patient could have a better outcome. Our findings support the important role of the virus in disease pathogenesis, suggesting that an effective antiviral could alter the clinical course for patients with the most severe forms of yellow fever. FUNDING: São Paulo Research Foundation (FAPESP).