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.

Guapiaçu virus, a new insect-specific flavivirus isolated from two species of Aedes mosquitoes from Brazil.

Classical insect-flaviviruses (cISFVs) and dual host-related insect-specific flavivirus (dISFV) are within the major group of insect-specific flavivirus. Remarkably dISFV are evolutionarily related to some of the pathogenic flavivirus, such as Zika and dengue viruses. The Evolutionary relatedness of dISFV to flavivirus allowed us to investigate the evolutionary principle of host adaptation. Additionally, dISFV can be used for the development of flavivirus vaccines and to explore underlying principles of mammalian pathogenicity. Here we describe the genetic characterization of a novel putative dISFV, termed Guapiaçu virus (GUAPV). Distinct strains of GUAPV were isolated from pools of Aedes terrens and Aedes scapularis mosquitoes. Additionally, we also detected viral GUAPV RNA in a plasma sample of an individual febrile from the Amazon region (North of Brazil). Although GUAPV did not replicate in tested mammalian cells, 3'UTR secondary structures duplication and codon usage index were similar to pathogenic flavivirus.