Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication.

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.

Molecular and biological characterization of an Asian-American isolate of Chikungunya virus.

Chikungunya virus is an arthropod-transmitted virus that causes chikungunya fever, a disease characterized by severe muscle and joint pain. In 2013, the virus was introduced to the Americas and caused approximately 2.7 million cases of infection during the subsequent two years. The lack of knowledge regarding the biological behavior of the viral strains circulating during the outbreak motivated the characterization of an isolate from the Colombian outbreak, starting from analysis of the complete genome to the biological behavior in vitro. The full genome was retrieved using next-generation sequencing. The infective and replicative capacities were evaluated in HEK293T, Huh-7, and MRC-5 cell lines. The infection rates were determined by flow cytometry, and the cytopathic effect was assessed by a resazurin fluorescent metabolic assay. The viral yield was quantified using the virus plaque formation assay, while the viral proteins and genomic RNA kinetics were subsequently evaluated by western-blot and RT-qPCR. The COL7624 isolate clustered with other American and Caribbean sequences in the Asian American lineage. The T669A substitution in E2 protein distinguished it from other Colombian sequences reported in 2014. After 48 h post infection (hpi), the three cell lines analyzed reached infection percentages exceeding 65%, generating a high load of infectious viral progeny. The infection kinetics indicated that the replication peak of this CHIKV isolate is around 24 hpi, although gRNA is detectable in the culture supernatant from 4 hpi onwards. The infection caused the overexpression of interferon and pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-8. The COL7624 CHIKV isolate exhibited a high infective and replicative capacity as well as activation of cellular immune responses, similar to isolates belonging to the other genotypes.

Activity of the Giardia intestinalis proteasome during encystation and its connection with the expression of the cyst wall protein 1 (CWP1).

Giardia is a parasite whose life cycle is composed of two stages: replicative trophozoites, responsible for the symptoms of the disease, and infective cysts, resistant to adverse environments outside of hosts. Proteasomes are multicatalytic peptidase complexes responsible for the specific degradation of proteins in eukaryotic cells. This study assessed the proteasome activity in the trophozoite and during encystation. Strong activation of the proteasome was observed during the differentiation of trophozoites into cysts, reaching its maximum level 24 h after the stimulus. We also found that the Giardia proteasome presents unusual characteristics related to higher eukaryotic proteasomes, making it an eventual therapeutic target. Here we tested the effects on the synthesis of a cyst wall protein by chemical inactivation of the proteasome and by overexpression or partial inhibition of the deubiquitinating protein RPN11 in transfected cells. Moreover, an analysis of the intracellular localization of RPN11 (an integral part of the proteasome regulatory particle) revealed major changes associated with the differentiation of trophozoites into cysts. This evidence further supports the important role of the proteasome in Giardia encystation.

Easy and inexpensive molecular detection of dengue, chikungunya and zika viruses in febrile patients.

Dengue (DENV), chikungunya (CHIKV) and zika (ZIKV) are arthropod-borne viruses (arboviruses) sharing a common vector, the mosquito Aedes aegypti. At initial stages, patients infected with these viruses have similar clinical manifestations, however, the outcomes and clinical management of these diseases are different, for this reason early and accurate identification of the causative virus is necessary. This paper reports the development of a rapid and specific nested-PCR for detection of DENV, CHIKV and ZIKV infection in the same sample. A set of six outer primers targeting the C-preM, E1, and E gene respectively was used in a multiplex one-step RT-PCR assay, followed by the second round of amplification with specific inner primers for each virus. The specificity of the present assay was validated with positive and negative serum samples for viruses and supernatants of infected cells. The assay was tested using clinical samples from febrile patients. In these samples, we detected mono and dual infections and a case of triple co-infection DENV-CHIKV-ZIKV. This assay might be a useful and an inexpensive tool for detection of these infections in regions where these arboviruses co-circulate.

Zika, dengue, and chikungunya co-infection in a pregnant woman from Colombia.

The clinical findings of a pregnant woman from Colombia with a triple co-infection caused by dengue, chikungunya, and Zika viruses are described. Weekly obstetric ultrasounds from 14.6 to 29 weeks of gestation were normal. She remains under follow-up and management according to the standard guidelines for the management of Zika virus-infected pregnant women.