Dengue virus 3 genotype I (GI) lineage 1 (L1) isolates elicit differential cytopathic effect with syncytium formation in human glioblastoma cells (U251).

BACKGROUND: Dengue virus (DENV) is a Flaviviridae member classified into four antigenically distinct serotypes (DENV 1, 2, 3, and 4) and further subdivided genotypes. DENV3 is subdivided into four or five genotypes, depending on the classification adopted. Despite their high genetic proximity, as revealed by phylogenetic complete polyprotein analysis, DENV3 MG-20 and DENV3 PV_BR showed different neurovirulence in mice models. Our group identified six amino acid mutations in protein E, including the E62K and E123Q, which may affect interactions of hydrophobic clusters on domain II, thus leading to the observed differences in the studied viruses. METHODS: Human glioblastoma cells (U251) derived from a malignant glioblastoma tumor by explant technique were infected by the DENV3 GIL1 isolates DENV3 MG-20 and DENV3 PV_BR and analyzed by plaque assays and titration, optical, immunofluorescence, and transmission electronic microscopy. RESULTS: The two isolates showed different cytopathic effects (CPE) and fusogenic patterns, further confirmed by indirect immunofluorescence. Transmission electron microscopy revealed intense cytopathic effects in DENV3 MG-20 infected U251 cells, displaying endoplasmic reticulum hypertrophy and turgid vesicles with proteins and multiple viruses, distinct from DENV3 PV_BR infected cells. It is hypothesized that the different amino acids in the DENV3 MG-20 isolate are related to an increased membrane fusion ability in viral infection, thus facilitating immune system evasion and increased chances of central nervous system cell infection. CONCLUSION: These results emphasize the biological differences between the isolates, which could be a critical factor in host-virus interaction and severe dengue development. Our study presents comparative results of highly similar isolates with the potential to generate more subsidies for a deeper understanding of the DENV pathogenesis. The neurotropism of the isolate DENV3 MG-20 (belonging to the DENV3 GI L1 genotype) showing infection of nervous system cells (U251) could contribute to understanding neurological dengue disease.

Potential use of high-resolution melting analyses for SARS-CoV-2 genomic surveillance.

The pandemic caused by COVID-19 and the emergence of new variants of SARS-CoV-2 have generated clinical and epidemiological impacts on a global scale. The use of strategies for monitoring viral circulation and identifying mutations in genomic regions involved in host interaction are important measures to mitigate viral dissemination and reduce its likely complications on population health. In this context, the objective of this work was to explore the potential of high-resolution melting (HRM) analysis combined with one-step real-time reverse transcription PCR in a closed-tube system, as a fast and convenient method of screening for SARS-CoV-2 mutations with possible implications on host-pathogen interactions. The HRM analyses allowed the distinction of the Gamma, Zeta, Alpha, Delta, and Omicron variants against the predecessors (B.1.1.28, B.1.1.33) of occurrence in Brazil. It is concluded that the molecular tool standardized here has the potential to optimize the genomic surveillance of SARS-CoV-2, and could be adapted for genomic surveillance of other pathogens, due to its ability to detect, prior to sequencing, samples suggestive of new variants, selecting them more assertively and earlier for whole genome sequencing when compared to random screening.

Influence of SARS-CoV-2 inactivation by different chemical reagents on the humoral response evaluated in a murine model.

Viral inactivation for antibody induction purposes, among other applications, should ensure biosafety, completely avoiding the risk of infectivity, and preserving viral immunogenicity. ß-propiolactone (BPL) is one of the most used reagents for viral inactivation, despite its high toxicity and recent difficulties related to importation, experienced in Brazil during the SARS-CoV-2 pandemic. In this context, the main objectives of this work were to test different inactivation procedures for SARS-CoV-2 and to evaluate the induction of neutralizing antibodies in mice immunized with antigenic preparations obtained after viral treatment with formaldehyde (FDE), glutaraldehyde (GDE), peroxide hydrogen (H2O2), as well as with viral proteins extract (VPE), in parallel with BPL. Verification of viral inactivation was performed by subsequent incubations of the inactivated virus in Vero cells, followed by cytopathic effect and lysis plaques observation, as well as by quantification of RNA load using reverse transcription-quantitative real time polymerase chain reaction. Once viral inactivation was confirmed, cell culture supernatants were concentrated and purified. In addition, an aliquot inactivated by BPL was also subjected to viral protein extraction (VPE). The different antigens were prepared using a previously developed microemulsion as adjuvant, and were administered in a four-dose immunization protocol. Antibody production was comparatively evaluated by ELISA and Plaque Reduction Neutralization Tests (PRNT). All immunogens evaluated showed some level of IgG anti-SARS-CoV-2 antibodies in the ELISA assay, with the highest levels presented by the group immunized with FDE-inactivated viral antigen. In the PRNT results, except for VPE-antigen, all other immunogens evaluated induced some level of neutralizing anti-SARS-CoV-2 antibodies, and the FDE-antigen stood out again with the most expressive values. Taken together, the present work shows that FDE can be an efficient and affordable alternative to BPL for the production of inactivated SARS-CoV-2 viral antigen.

Central and peripheral nervous system involvement in Zika virus infection in a child.

A 7-year-old boy that presented an encephalomyeloradiculitis and no classic symptoms of arboviruses. Zika virus (ZIKV) was confirmed by molecular analyses of cerebrospinal fluid and 1 year later by plaque reduction neutralization test. This case demonstrates that ZIKV can be associated with diffuse nervous system infection in children.