Zika Virus Infection Alters the Circadian Clock Expression in Human Neuronal Monolayer and Neurosphere Cultures.

Rhythmic regulations are virtually described in all physiological processes, including central nervous system development and immunologic responses. Zika virus (ZIKV), a neurotropic arbovirus, has been recently linked to a series of birth defects and neurodevelopmental disorders. Given the well-characterized role of the intrinsic cellular circadian clock within neurogenesis, cellular metabolism, migration, and differentiation among other processes, this study aimed to characterize the influence of ZIKV infection in the circadian clock expression in human neuronal cells. For this, in vitro models of human-induced neuroprogenitor cells (hiNPCs) and neuroblastoma cell line SH-SY5Y, cultured as monolayer and neurospheres, were infected by ZIKV, followed by RNA-Seq and RT-qPCR investigation, respectively. Targeted circadian clock components presented mRNA oscillations only after exogenous synchronizing stimuli (Forskolin) in SH-SY5Y monolayer culture. Interestingly, when these cells were grown as 3D-arranged neurospheres, an intrinsic oscillatory expression pattern was observed for some core clock components without any exogenous stimulation. The ZIKV infection significantly disturbed the mRNA expression pattern of core clock components in both neuroblastoma cell culture models, which was also observed in hiNPCs infected with different strains of ZIKV. The ZIKV-mediated desynchronization of the circadian clock expression in human cells might further contribute to the virus impairment of neuronal metabolism and function observed in adults and ZIKV-induced congenital syndrome. In vitro models of Zika virus (ZIKV) neuronal infection. Human neuroprogenitor cells were cultured as monolayer and neurospheres and infected by ZIKV. Monolayer-cultured cells received forskolin (FSK) as a coupling factor for the circadian clock rhythmicity, while 3D-arranged neurospheres showed an intrinsic oscillatory pattern in the circadian clock expression. The ZIKV infection affected the mRNA expression pattern of core clock components in both cell culture models. The ZIKV-mediated desynchronization of the circadian clock machinery might contribute to the impairment of neuronal metabolism and function observed in both adults (e.g., Guillain-Barré syndrome) and ZIKV-induced congenital syndrome (microcephaly). The graphical abstract has been created with Canva at the canva.com website.

The translation initiation factor EIF4E5 from Leishmania: crystal structure and interacting partners.

The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5' end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in Leishmania species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in L. infantum. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in Trypanosoma brucei, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the Leishmania-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in Leishmania (W45) did not substantially impair the identified interactions. Finally, the crystal structure of Leishmania EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its Trypanosoma orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation.

Chikungunya virus infection induces ultrastructural changes and impaired neuronal differentiation of human neurospheres.

Chikungunya virus (CHIKV) is an arthropod-borne virus recently associated with large outbreaks in many parts of the world. Infection is typically manifested as a febrile and self-limited illness, characterized by joint pain and myalgia, albeit severe neurological manifestations are also reported. Although CHIKV is not recognized as a truly neurotropic virus, neurons, astrocytes, and oligodendrocytes are susceptible to infection in vitro. Here we employed a model of 3D cell culture to obtain neurospheres from ATRA/BNDF differentiated human neuroblastoma cells. We demonstrate that CHIKV is able to establish a productive infection, resulting in ultrastructural changes in cell morphology and impaired neuronal differentiation. Ultrastructural analysis of neurospheres infected with CHIKV during neuronal differentiation revealed diminished neuron dendrite formation, accumulation of viral particles associated with the plasma membrane, numerous cell vacuoles, and swollen mitochondria. Apoptotic cells were significantly increased at 72 h post-infection. Compared to Zika virus, a well-characterized neurotropic arbovirus, CHIKV infection resulted in a more discrete, albeit detectable upregulation of IL-6 levels. Finally, we found that CHIKV infection resulted in an altered profile expression, mainly downregulation, of a group of transcription factors named Hox genes. Altogether our findings highlight important features of CHIKV in the CNS, as well as the feasibility of neurospheres as robust experimental models that can support further studies for novel pharmacological interventions.

A Review on Chikungunya Virus Epidemiology, Pathogenesis and Current Vaccine Development.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that recently re-emerged in many parts of the world causing large-scale outbreaks. CHIKV infection presents as a febrile illness known as chikungunya fever (CHIKF). Infection is self-limited and characterized mainly by severe joint pain and myalgia that can last for weeks or months; however, severe disease presentation can also occur in a minor proportion of infections. Among the atypical CHIKV manifestations that have been described, severe arthralgia and neurological complications, such as encephalitis, meningitis, and Guillain-Barré Syndrome, are now reported in many outbreaks. Moreover, death cases were also reported, placing CHIKV as a relevant public health disease. Virus evolution, globalization, and climate change may have contributed to CHIKV spread. In addition to this, the lack of preventive vaccines and approved antiviral treatments is turning CHIKV into a major global health threat. In this review, we discuss the current knowledge about CHIKV pathogenesis, with a focus on atypical disease manifestations, such as persistent arthralgia and neurologic disease presentation. We also bring an up-to-date review of the current CHIKV vaccine development. Altogether, these topics highlight some of the most recent advances in our understanding of CHIKV pathogenesis and also provide important insights into the current development and clinical trials of CHIKV potential vaccine candidates.

Identification of a Virulent Newcastle Disease Virus Strain Isolated from Pigeons (Columbia livia) in Northeastern Brazil Using Next-Generation Genome Sequencing.

Newcastle disease virus (NDV), also known as avian paramyxoviruses 1 (APMV-1) is among the most important viruses infecting avian species. Given its widespread circulation, there is a high risk for the reintroduction of virulent strains into the domestic poultry industry, making the surveillance of wild and domestic birds a crucial process to appropriately respond to novel outbreaks. In the present study, we investigated an outbreak characterized by the identification of sick pigeons in a large municipality in Northeastern Brazil in 2018. The affected pigeons presented neurological signs, including motor incoordination, torticollis, and lethargy. Moribund birds were collected, and through a detailed histopathological analysis we identified severe lymphoplasmacytic meningoencephalitis with perivascular cuffs and gliosis in the central nervous system, and lymphoplasmacytic inflammation in the liver, kidney, and intestine. A total of five pigeons tested positive for NDV, as assessed by rRT-PCR targeted to the M gene. Laboratory virus isolation on Vero E6 cells confirmed infection, after the recovery of infectious NVD from brain and kidney tissues. We next characterized the isolated NDV/pigeon/PE-Brazil/MP003/2018 by next-generation sequencing (NGS). Phylogenetic analysis grouped the virus with other NDV class II isolates from subgenotype VI.2.1.2, including two previous NDV isolates from Brazil in 2014 and 2019. The diversity of aminoacid residues at the fusion F protein cleavage site was analyzed identifying the motif RRQKR↓F, typical of virulent strains. Our results all highlight the importance of virus surveillance in wild and domestic birds, especially given the risk of zoonotic NDV.