Curcumin, a traditional Chinese and Indian treatment for many diseases, has recently been found to alter the in vitro infection processes of various viruses, including hepatitis C virus, human immunodeficiency virus, coxsackievirus, and Japanese encephalitis virus. The present study evaluated the cellular effects of curcumin in an in vitro (cellular) infection model of dengue virus. Within a dose range of 10 to 30 μM and a treatment period of 24 hours, the cytotoxicity of curcumin was low, as determined by MTT assays. Cells infected with dengue virus type 2 at a multiplicity of infection of 5 were treated with various concentrations of curcumin or the proteasome inhibitor MG132. Plaque assays, immunofluorescence analysis, western blots, and in-cell western assays were then performed. Treatment with 10, 15, and 20 μM curcumin decreased the number of plaques produced, caused an intracellular accumulation of viral proteins, and increased the level of Lys48 ubiquitin-conjugated proteins. At 20 μM curcumin, changes in cell and nuclear morphology and alterations in the actin cytoskeleton were also observed. Treatment with MG132 also reduced plaque production. These results show that curcumin can interfere with the infection processes of dengue virus and that this interference may not occur through direct effects on viral particle production but may result from curcumin's effects on various cellular systems such as the cytoskeleton, the ubiquitin-proteasome system, or the apoptosis process.
Dengue virus (DENV) is a member of the Flaviviridae family, which is transmitted to mammalian species through arthropods, and causes dengue fever or severe dengue fever in humans. The DENV genome encodes for multiple nonstructural (NS) proteins including NS1. NS1 plays an essential role in replication by interacting with other viral proteins including NS4B, however how these interactions are regulated during virus infection is not known. By using bioinformatics, mass spectrometry analysis, and co-immunoprecipitation assays, here we show that DENV-NS1 is ubiquitinated on multiples lysine residues during DENV infection, including K189, a lysine residue previously shown to be important for efficient DENV replication. Data from in vitro and cell culture experiments indicate that dengue NS1 undergoes modification with K48-linked polyubiquitin chains, which usually target proteins to the proteasome for degradation. Furthermore, ubiquitinated NS1 was detected in lysates as well as in supernatants of human and mosquito infected cells. Ubiquitin deconjugation of NS1 using the deubiquitinase OTU resulted in increased interaction with the viral protein NS4B suggesting that ubiquitinated NS1 has reduced affinity for NS4B. In support of these data, a K189R mutation on NS1, which abrogates ubiquitination on amino acid residue 189 of NS1, also increased NS1-NS4B interactions. Our work describes a new mechanism of regulation of NS1-NS4B interactions and suggests that ubiquitination of NS1 may affect DENV replication.
Paraguay has registered no human cases of rabies since 2004, and the last case in dogs, reported in 2009, was due to a variant maintained in the common vampire bat "Desmodus rotundus". In 2014, a dog was diagnosed as positive for rabies with aggression towards a boy and all required measures of control were successfully adopted. Epidemiological investigation revealed that the dog was not vaccinated and had been attacked by a crab-eating fox, "zorro" (Cerdocyon thous). The sample was diagnosed by the Official Veterinary Service of the Country and sent to the Center on Rabies Research from the University of São Paulo, Brazil, for antigenic and genetic characterization. A second sample from a dog positive for rabies in the same region in 2015 and 11 samples from a rabies outbreak from Asuncion in 1996 were also characterized. The antigenic profile of the samples, AgV2, was compatible with one of the variants maintained by dogs in Latin America. In genetic characterization, the samples segregated in the canine (domestic and wild species)-related group in an independent subgroup that also included samples from Argentina. These results and the epidemiology of the case indicate that even with the control of rabies in domestic animals, the virus can still circulate in wildlife and may be transmitted to domestic animals and humans, demonstrating the importance of continuous and improved surveillance and control of rabies, including in wild species, to prevent outbreaks in controlled areas.
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