Circulation of Chikungunya virus East-Central-South African genotype during the 2020­21 outbreak in São Paulo State, Brazil

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. The Asian and East-Central-South African (ECSA) genotypes were introduced in Brazil in 2014, in Bahia State and Northeast region. CHIKV virus rapidly spread, causing epidemics in urban areas, and the ECSA genotype was detected also in other regions. In the last five years, more than 700,000 cases of Chikungunya fever were reported in Brazil. Nevertheless, there is limited information about the genomic epidemiology of CHIKV from surveillance studies. In São Paulo (SP), the most populous Brazilian state, until 2015 only imported cases were identified. In 2020­21, an outbreak was detected in the coastal region of SP, accounting for 98% of confirmations in SP. To better understand the disease dynamics in SP, we generated 53 nearcomplete genomes of CHIKV isolates from Baixada Santista, obtained from clinical samples. Our results demonstrate that SP-CHIKV clade belongs to a distinct clade from those previously found in Brazil, supporting the local circulation of a specific lineage in the period. None of the SP-CHIKV carry the substitutions A226V (E1 protein) and L210Q (E2 protein) associated with increased transmission in Aedes albopictus mosquitoes. The results confirm that the ECSA lineage continues to spread across the country and reinforce that genomic data can provide information about virus genetic diversity and transmission dynamics, assisting the establishment of a more effective surveillance framework. (AU)

Anti-DENV-NS1 monoclonal antibody for the differential histopathological diagnosis of hemorrhagic fever caused by dengue

Dengue is the most prevalent arboviral disease in humans in tropical and subtropical regions, especially in urban areas, and can cause major epidemics. Although a self-limiting illness, it may sometimes have serious hemorrhagic manifestations, and the outcome of dengue hemorrhagic fever has similar clinical manifestations as in other infections, which could result in death. Therefore, autopsy procedures are required under certain circumstances such as in hemorrhagic fevers, sometimes to confirm or to clarify the diagnosis that may have epidemiological consequences. Normally, the Immunohistochemistry Laboratory of the Pathology Center of Adolfo Lutz Institute receives autopsy samples from different hospitals in Sao Paulo State to confirm a previous diagnosis, especially hemorrhagic fever of infectious etiology. For this diagnosis, we have been using a mouse polyclonal antibody to dengue virus that often does not provide a clear conclusion, because of background staining or no relevant immunostaining, which hampers the histopathological analysis. Accordingly, in the present study, anti-DENV-NS1 monoclonal antibody (4H2) was tested to determine its accuracy in immunohistochemical analysis. Twenty-four autopsy cases of hemorrhagic febrile syndrome showing histopathological alterations compatible with dengue disease were studied: twenty cases were confirmed by RT-PCR for DENV-2 and in four by RT-PCR for yellow fever virus. Samples from autopsied cases of deaths caused by other infectious diseases (two meningitis C and two severe acute respiratory syndrome caused by influenza A H1N1) were included as negative control cases. Positive immunostaining for DENV-NS1 was detected in 16/20 (80%) liver samples and 11/15 (73%) spleen samples from autopsied hemorrhagic dengue patients, whereas the polyclonal antibody detected DENV antigens in 12/20 (60%) liver and in 6/15 (40%) spleen samples from the same cases. Positive results were not obtained with liver biopsy samples from yellow fever or Neisseria meningitides and Flu-A cases. 4H2 mAb recognizes the native protein of the four DENV serotypes in infected cells and did not cross-react with native ZIKV- or CHKV-infected cells by immunohistochemical assay, so it is a useful tool for differential histopathological conclusion of acute febrile hemorrhagic deaths.