A study of the MAYV replication cycle: Correlation between the kinetics of viral multiplication and viral morphogenesis.

Mayaro virus (MAYV) is mainly found in Central and South America and causes a febrile illness followed by debilitating arthritis and arthralgia similar to chikungunya virus (CHIKV). Infection leads to long-term sequelae with a direct impact on the patient's productive capacity, resulting in economic losses. Mayaro fever is a neglected disease due to the limited epidemiological data. In Brazil, it is considered a potential public health risk with the number of cases increasing every year. Most of our knowledge about MAYV biology is inferred from data obtained from other alphaviruses as well as more recent studies on MAYV. Here, we analyzed the kinetics of viral replication through standard growth curves, quantification of intracellular and extracellular particles, and RNA quantification. We compared transmission electron microscopy data during different stages of infection. This approach allowed us to establish a chronological order of events during MAYV replication and its respective timepoints including cell entry through clathrin-mediated endocytosis occurring at 15-30 min, genome replication at 2-3 h, morphogenesis at 4 hpi, and release at 4-6 hpi. We also present evidence of uncharacterized events such as ribosome reorganization as well as clusters of early viral precursors and release through exocytosis in giant forms. Our work sheds new and specific light on the MAYV replication cycle and may contribute to future studies on the field.

In-Depth Characterization of the Chikungunya Virus Replication Cycle.

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (it was first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found infection cellular activities other than those usually described for the chikungunya virus replication cycle, i.e., we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area, and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages, as follows: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

Evaluation of kinase inhibitors as potential therapeutics for flavivirus infections.

The recent introduction of Zika virus (ZIKV), the recurrence of dengue virus (DENV), and the lethality of yellow fever virus (YFV) have had a significant impact on Brazilian society and public health. Here, we targeted two cellular kinases implicated in cell proliferation and cancer that are also important for viral replication: mitogen-activated protein kinase kinase (MEK) and Src. We used two MEK inhibitors - trametinib and selumetinib - and two Src inhibitors - saracatinib and bosutinib - to inhibit ZIKV, DENV, and YFV replication in cell culture. The cytotoxicity of the four inhibitors was determined by the observation of abnormal morphology and quantification of adherent cells by crystal violet staining. The antiviral activity of these drugs was assessed based on the reduction of plaque-forming units in cell culture as evidence of the inhibition of the replication of the selected flaviviruses. All four inhibitors showed antiviral activity, but among them, trametinib was the safest and most efficacious against all of the viruses, inhibiting the replication of ZIKV and YFV by 1000-fold, and DENV2/3 by nearly 100-fold. This pan-antiviral effect shows that trametinib could be repurposed for the treatment of flaviviral infections.