Flaviviruses such as dengue (DENV), yellow fever (YFV), West Nile (WNV), and Zika (ZIKV) are human pathogens of global significance. In particular, DENV causes the most prevalent mosquito-borne viral diseases in humans, and ZIKV emerged from obscurity into the spotlight in 2016 as the etiologic agent of congenital Zika syndrome. Owing to the recent emergence of ZIKV as a global pandemic threat, the roles of the immune system during ZIKV infections are as yet unclear. In contrast, decades of DENV research implicate a dual role for the immune system in protection against and pathogenesis of DENV infection. As DENV and ZIKV are closely related, knowledge based on DENV studies has been used to prioritize investigation of ZIKV immunity and pathogenesis, and to accelerate ZIKV diagnostic, therapeutic, and vaccine design. This review discusses the following topics related to innate and adaptive immune responses to DENV and ZIKV: the interferon system as the key mechanism of host defense and viral target for immu...

https://www.annualreviews.org/doi/pdf/10.1146/annurev-immunol-042617-053142

Immune Response to Dengue and Zika.

Coronavirus possesses the largest RNA genome among all the RNA viruses. Its genome encodes about 29 proteins. Most of the viral proteins are non-structural proteins (NSP) except envelop (E), membrane (M), nucleocapsid (N) and Spike (S) proteins that constitute the viral nucleocapsid, envelop and surface. We have recently cloned all the 29 SARS-CoV-2 genes into vectors for their expressions in mammalian cells except NSP11 that has only 14 amino acids (aa). We are able to express all the 28 cloned SARS-CoV-2 genes in human cells to characterize their subcellular distributions. The proteins of SARS-CoV-2 are mostly cytoplasmic but some are both cytoplasmic and nuclear. Those punctate staining proteins were further investigated by immunofluorescent assay (IFA) using specific antibodies or by co-transfection with an organelle marker-expressing plasmid. As a result, we found that NSP15, ORF6, M and ORF7a are related to Golgi apparatus, and that ORF7b, ORF8 and ORF10 colocalize with endoplasmic reticulum (ER). Interestingly, ORF3a distributes in cell membrane, early endosome, endosome, late endosome and lysosome, which suggests that ORF3a might help the infected virus to usurp endosome and lysosome for viral use. Furthermore, we revealed that NSP13 colocalized with SC35, a protein standing for splicing compartments in the nucleus. Our studies for the first time visualized the subcellular locations of SARS-CoV-2 proteins and might provide novel insights into the viral proteins’ biological functions.

/pdf/a-systemic-and-molecular-study-of-subcellular-localization-jgtdgax84u.pdf

A systemic and molecular study of subcellular localization of SARS-CoV-2 proteins.

Global Interactomics Uncovers Extensive Organellar Targeting by Zika Virus.

Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG-I-like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG-I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/eji.201847483

Infection with a Brazilian isolate of Zika virus generates RIG-I stimulatory RNA and the viral NS5 protein blocks type I IFN induction and signaling.

Functional RNA during Zika virus infection.

Molecular cloning and characterization of the genes encoding the proteins of Zika virus.

Two lineages of Zika virus (ZIKV) have been classified according to the phylogenetic analysis: African and Asian lineages. It is unclear whether differences exist between the two strains in host cell permissiveness, this information is important for understanding viral pathogenesis and designing anti-viral strategies. In the present study, we comparatively studied the permissive spectrum of human cells for both the African (MR766) and Asian strains (PRVABC59) using an RNA in situ hybridization (RISH) to visualize RNA replication, an immunofluorescence technology, and a western blot assay to determine viral protein production, and a real-time RT-PCR to examine viral RNA multiplication level. The experiments were undertaken in the condition of cell culture. We identified several human cell lines, including fibroblast, epithelial cells, brain cells, stem cells, and blood cells that are susceptible for the infection of both Asian and African strains. We did not find any differences between the MR766 and the PRVABC59 in the permissiveness, infection rate, and replication modes. Inconsistent to a previous report (Hamel et al. JVI 89:8880–8896, 2015), using RISH or real-time RT-PCR, we found that human foreskin fibroblast cells were not permissive for ZIKV infection. Instead, human lung fibroblast cells (MRC-5) were fully permissive for ZIKV infection. Surprisingly, a direct interaction of ZIKV RNA with envelop (E) protein (a structure protein) was demonstrated by an RNA chromatin immunoprecipitation (ChIP) assay. Three binding sites were identified in the ZIKV RNA genome for the interaction with the E protein. Our results imply that the E protein may be important for viral RNA replication, and provide not only the information of ZIKV permissiveness that guides the usage of human cells for the ZIKV studies, but also the insight into the viral RNA-E protein interaction that may be targeted for intervention by designing small molecule drugs.

/pdf/determination-of-the-cell-permissiveness-spectrum-mode-of-1ruew6jcij.pdf

Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus

Zika virus increases mind bomb 1 levels, causing degradation of pericentriolar material 1 (PCM1) and dispersion of PCM1-containing granules from the centrosome.



Dengue viruses (DENV) and Zika virus (ZIKV) are transmitted among humans, or from non-human primates to humans, through mosquito bites. The interaction of the virus with mosquito cells is a key step in the viral life cycle. Therefore, the objective of this study was to determine how DENV and ZIKV interact with mosquito cells.



Immunofluorescence assays and a direct visualization system were combined to monitor the syncytial or congregative effects of DENV and ZIKV strains on C6/36 cells. We examined the cytopathic effects of the strains on C6/36 mosquito cells, a widely used laboratory model for studying infection with these viruses.



Our results indicated that all strains of DENV-1 and DENV-2, most DENV-4 strains, and some DENV-3 strains caused syncytial effects on C6/36 cells, whereas some DENV-3 and DENV-4 strains, and all tested ZIKV strains, caused cell congregation after infection but no cell fusion. In addition, we detected a range of environmental pH values from 6.0 to 8.0 supporting virus-induced cell fusion. The optimal pH condition was 7.5, at which viral production was also highest. Furthermore, the UV-inactivated virus did not cause cell fusion, thus suggesting that viral replication may be required for DENV’s syncytial effects on C6/36 cells.



Syncytial and congregative effects of DENV and ZIKV on Aedes albopictus cells differ among viral strains. Syncytial effects of DENV on C6/36 are important for viral replication.


https://www.scienceopen.com/document_file/81919bff-f948-4adc-a00d-58d8ff71e337/ScienceOpen/Zoonoses20230002.pdf

Lilian Akello Obwolo

Papers

Molecular cloning and characterization of the genes encoding the proteins of Zika virus.

Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus

Zika virus increases mind bomb 1 levels, causing degradation of pericentriolar material 1 (PCM1) and dispersion of PCM1-containing granules from the centrosome.

Syncytial and Congregative Effects of Dengue and Zika Viruses on the Aedes Albopictus Cell Line Differ among Viral Strains