Tropism and immune response of chikungunya and zika viruses: An overview.

Zika virus (ZIKV) and chikungunya virus (CHIKV) are two medically important vector-borne viruses responsible for causing significant disease burden in humans, including neurological sequelae/complications. Besides sharing some common clinical features, ZIKV has major shares in causing microcephaly and brain malformations in developing foetus, whereas CHIKV causes chronic joint pain/swelling in infected individuals. Both viruses have a common route of entry to the host body. i.e., dermal site of inoculation through the bite of an infected mosquito and later taken up by different immune cells for further dissemination to other areas of the host body that lead to a range of immune responses via different pathways. The immune responses generated by both viruses have similar characteristics with varying degrees of inflammation and activation of immune cells. However, the overall response of immune cells is not fully explored in the context of ZIKV and CHIKV infection. The knowledge of cellular tropism and the immune response is the key to understanding the mechanisms of viral immunity and pathogenesis, which may allow to develop novel therapeutic strategies for these viral infections. This review aims to discuss recent advancements and identify the knowledge gaps in understanding the mechanism of cellular tropism and immune response of CHIKV and ZIKV.

Where all the Roads Meet? A Crossover Perspective on Host Factors Regulating SARS-CoV-2 infection.

COVID-19 caused by SARS-CoV-2 is the latest pandemic which has thrown the world into an unprecedented social and economic uncertainties along with huge loss to humanity. Identification of the host factors regulating the replication of SARS-CoV-2 in human host may help in the development of novel anti-viral therapies to combat the viral infection and spread. Recently, some research groups used genome-wide CRISPR/Cas screening to identify the host factors critical for the SARS-CoV-2 replication and infection. A comparative analysis of these significant host factors (p < 0.05) identified fifteen proteins common in these studies. Apart from ACE2 (receptor for SARS-CoV-2 attachment), other common host factors were CSNK2B, GDI2, SLC35B2, DDX51, VPS26A, ARPP-19, C1QTNF7, ALG6, LIMA1, COG3, COG8, BCOR, LRRN2 and TLR9. Additionally, viral interactome of these host factors revealed that many of them were associated with several SARS-CoV-2 proteins as well. Interestingly, some of these host factors have already been shown to be critical for the pathogenesis of other viruses suggesting their crucial role in virus-host interactions. Here, we review the functions of these host factors and their role in other diseases with special emphasis on viral diseases.

Dengue Virus Dysregulates Master Transcription Factors and PI3K/AKT/mTOR Signaling Pathway in Megakaryocytes.

Dengue virus (DENV) infection can cause either self-limited dengue fever or hemorrhagic complications. Low platelet count is one of the manifestations of dengue fever. Megakaryocytes are the sole producers of platelets. However, the role of both host and viral factors in megakaryocyte development, maturation, and platelet production is largely unknown in DENV infection. PI3K/AKT/mTOR pathway plays a significant role in cell survival, maturation, and megakaryocyte development. We were interested to check whether pathogenic insult can impact this pathway. We observed decreased expression of most of the major key molecules associated with the PI3K/AKT/mTOR pathway in DENV infected MEG-01 cells. In this study, the involvement of PI3K/AKT/mTOR pathway in megakaryocyte development and maturation was confirmed with the use of specific inhibitors in infected MEG-01 cells. Our results showed that direct pharmacologic inhibition of this pathway greatly impacted megakaryopoiesis associated molecule CD61 and some essential transcription factors (GATA-1, GATA-2, and NF-E2). Additionally, we observed apoptosis in megakaryocytes due to DENV infection. Our results may suggest that DENV impairs PI3K/AKT/mTOR axis and molecules involved in the development and maturation of megakaryocytes. It is imperative to investigate the role of these molecules in the context of megakaryopoiesis during DENV infection to better understand the pathways and mechanisms, which in turn might provide insights into the development of antiviral strategies.

Dengue virus induces interferon-ß by activating RNA sensing pathways in megakaryocytes.

Activation of innate receptors in megakaryocytes (MKs) may affect the ability to produce functional platelets. Low platelet count is one of the clinical manifestations of dengue virus (DENV) infection. In MKs, the effect of innate receptors during DENV-infection is not well studied. Here we used MEG-01 cells to investigate DENV serotype 2 induced innate receptors in these cells. DENV RNA was estimated by qRT-PCR in the culture supernatant. The expression of innate receptors was determined by western blot and qPCR. DENV infection led to increased expression of RIG-I at 24 hrs post-infection (hpi) and MDA-5 at 48 and 72 hpi (p<0.05). However, no change in the expression of TLR3 at protein level was observed. Activation of MDA-5 resulted in increased expression of IFN-ß and ISG-15 in DENV infected MEG-01 cells, which was further confirmed by MDA-5 siRNA treatment. Apart from inducing innate receptors, DENV significantly decreases the expression of CD61, an activation marker of megakaryocyteson MEG-01 cells as observed by flow cytometry analysis (p<0.01). Results from this study confirm that DENV infection activates the type-I interferon in megakaryocytes and may play a significant role in maturation and development.

Dengue Virus Degrades USP33-ATF3 Axis via Extracellular Vesicles to Activate Human Microglial Cells.

Dengue virus (DENV) infection disrupts host innate immune signaling at various checkpoints. Cellular levels and stability of intermediate signaling molecules are a crucial hijacking point for a successful viral pathogenesis. Stability and turnover of all the cellular proteins including intermediate signaling molecules are principally regulated by proteasomal degradation pathway. In this study, we show that how DENV infection and particularly DENV-NS1 can modulate the host extracellular vesicle (EV) cargo to manipulate the deubiquitination machinery of the human microglial cell (CHME3). We have performed EV harvesting, size analysis by nanoparticle tracking analysis, identification of cargo microRNA via quantitative PCR, microRNA target validation by overexpression, and knockdown via mimics and anti-miRs, immunoblotting, dual luciferase reporter assay, in vivo ubiquitination assay, chase assay, and promoter activity assay to reach the conclusion. In this study, we show that DENV-infected monocytes and DENV-NS1-transfected cells release high amounts of EVs loaded with miR-148a. These EVs get internalized by human microglial cells, and miR-148a suppresses the ubiquitin-specific peptidase 33 (USP33) protein expression levels via binding to its 3' untranslated region. Reduced USP33 in turn decreases the stability of cellular ATF3 protein via deubiquitylation. ATF3 acts as a suppressor of major proinflammatory gene expression pathways of TNF-α, NF-κB, and IFN-ß. Our mechanistic model explains how DENV uses the EV pathway to transfer miR-148a for modulating USP33 and downstream ATF3 levels in human microglial cells and contributes in neuroinflammation within the CNS.

Dengue viruses infect human megakaryocytes, with probable clinical consequences.

One of the most important clinical signs of dengue virus infection is the reduction of white blood cells and platelets in human peripheral blood (leukopenia and thrombocytopenia, respectively), which may significantly impair the clearance of dengue virus by the immune system. The cause of thrombocytopenia and leukopenia during dengue infection is still unknown, but may be related to severe suppression of bone marrow populations including hematopoietic stem cells and megakaryocytes, the progenitors of white blood cells and platelets respectively. Here, we explored the possibility that bone marrow suppression, including ablation of megakaryocyte populations, is caused by dengue virus infection of megakaryocytes. We used three different models to measure dengue virus infection and replication: in vitro, in a human megakaryocyte cell line with viral receptors, ex vivo, in primary human megakaryocytes, and in vivo, in humanized mice. All three systems support dengue virus infection and replication, including virus strains from serotypes 1, 2, and 3, and clinical signs, in vivo; all assays showed viral RNA and/or infectious viruses 7-14 days post-infection. Although we saw no significant decrease in cell viability in vitro, there was significant depletion of mature megakaryocytes in vivo. We conclude that megakaryocytes can produce dengue viruses in the bone marrow niche, and a reduction of cell numbers may affect bone marrow homeostasis.

Mosquito saliva alone has profound effects on the human immune system.

Mosquito saliva is a very complex concoction of >100 proteins, many of which have unknown functions. The effects of mosquito saliva proteins injected into our skin during blood feeding have been studied mainly in mouse models of injection or biting, with many of these systems producing results that may not be relevant to human disease. Here, we describe the numerous effects that mosquito bites have on human immune cells in mice engrafted with human hematopoietic stem cells. We used flow cytometry and multiplex cytokine bead array assays, with detailed statistical analyses, to detect small but significant variations in immune cell functions after 4 mosquitoes fed on humanized mice footpads. After preliminary analyses, at different early times after biting, we focused on assessing innate immune and subsequent cellular responses at 6 hours, 24 hours and 7 days after mosquito bites. We detected both Th1 and Th2 human immune responses, and delayed effects on cytokine levels in the blood, and immune cell compositions in the skin and bone marrow, up to 7 days post-bites. These are the first measurements of this kind, with human immune responses in whole animals, bitten by living mosquitoes, versus previous studies using incomplete mouse models and salivary gland extracts or needle injected saliva. The results have major implications for the study of hematophagous insect saliva, its effects on the human immune system, with or without pathogen transmission, and the possibility of determining which of these proteins to target for vaccination, in attempts to block transmission of numerous tropical diseases.

Replication of Zika Virus in Human Prostate Cells: A Potential Source of Sexually Transmitted Virus.

Background: While Zika virus (ZIKV) is mainly transmitted by mosquitoes, numerous cases of sexual transmission have been reported during recent outbreaks. Little is known about which host cell types or entry factors aid in mediating this sexual transmission. Methods: In this study, we investigated ZIKV cell tropism by infecting 2 types of human prostate cells with 3 contemporary ZIKV isolates from persons infected in the Americas. We used real-time quantitative polymerase chain reaction and immunofluorescence analyses to measure infection and flow cytometry to detect entry factor expression. Results: Here we show that ZIKV infects, replicates, and produces infectious virus in prostate stromal mesenchymal stem cells, epithelial cells, and organoids made with a combination of these cells. We also show that prostate cells express several well-characterized flavivirus attachment factors. In contrast, dengue virus does not infect or does not replicate in these prostate cells, although it is known to use similar receptors. Conclusions: Our results indicate that ZIKV favors infection of stromal cells more so than epithelial cells in organoids, possibly indicating a preference for stem cells in general. Overall, these results suggest that ZIKV replication occurs in the human prostate and can account for ZIKV secretion in semen, thus leading to sexual transmission.

Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication.

Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations.

Characterization of a Zika Virus Isolate from Colombia.

BACKGROUND: Zika virus (Flavivirus genus) is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments. METHODOLOGY/CLINICAL FINDINGS: We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015). Here, we describe the patient's clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3' un-translated region), to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains. CONCLUSIONS/SIGNIFICANCE: We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas.

Molecular characterization of group B rotavirus circulating in pigs from India: identification of a strain bearing a novel VP7 genotype, G21.

The occurrence of group B rotavirus (RVB) infections in pigs has been reported from different parts of world. However, such infection in the pig population maintained in Indian farms has not been investigated as yet. A total of 187 faecal specimens were collected from pigs reared in different pig farms/pigsties located in western and northern regions of India and tested for the presence of porcine RVB by amplification of the NSP2 gene using conventional RT-PCR. Nine specimens (4.8%) were shown to contain RVB RNA. N2 and N4 genotypes of NSP2 gene were detected in three and six RVB strains respectively. VP7 (G-type) and NSP5 (H-type) genes of selected six RVB strains were characterized to identify the genotypes. Multiple G (G7, G19 and G20) and H (H4 and H5) genotypes detected in the RVB strains indicated circulation of heterogeneous population of RVB strains in pigs of India. Additionally, one strain was proposed to belong to a novel RVB genotype designated as G21 on account of <80% identity of VP7 gene sequence with its counterpart in RVB strains from 20 established genotypes. Deduced amino acid sequence of VP7 gene also displayed the presence of seven unique substitutions in the strain. The study reports for the first time the occurrence of RVB infections in Indian pig herds and provides important epidemiological data useful for better understanding of ecology and evolution of porcine RVBs.

Full genome analysis of group B rotaviruses from western India: genetic relatedness and evolution.

To date, full-genome sequences of only seven human group B rotavirus (RVBs) strains have been described. Such data on more RVBs are necessary to establish the evolutionary relationship and ecological features of RVBs from different geographical regions. The present study was aimed at determining the full-length sequences of all 11 genes of 13 human RVB strains detected during 1995-2010 in sporadic and outbreak cases of acute gastroenteritis from four different cities of western India. This study also included estimation of evolutionary rates and site-specific selection pressure analysis for all gene segments. Nucleotide/deduced amino acid sequence analyses of structural and non-structural genes showed 95.1-99.8/94.1-100 % identity with the counterparts of RVB strains isolated in India, Bangladesh and Myanmar. Phylogenetic analyses of all gene segments revealed formation of a monophyletic clade of the western Indian RVB strains, reflecting their highly conserved nature. All gene segments were also found to be under negative/purifying selection pressure. These data suggest that RVB is circulating in the natural host as a series of stable viral clones. Estimates of rates of nucleotide substitution in all RVBs ranged from 1.36-4.78×10(-3) substitutions per site per year. The rate for human RVB VP7 and NSP2 genes were comparable, respectively, with the evolution kinetics of genotype G9/G12 and N1 group A rotavirus strains. The time of the most recent common ancestor of the extant human RVBs was estimated to be during 1915-1974. Evolutionary and genetic analyses carried out in this study provide data that is useful for the elucidation of evolutionary relationship/timescale, stasis or dynamics existing in the RVB population.

Diversity in the enteric viruses detected in outbreaks of gastroenteritis from Mumbai, Western India.

Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6%) were positive, 73 (76.8%) for a single virus and 22 (23.2%) for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40%) and rotavirus A (RVA, 33.3% and 25%). The other viruses detected in these months were norovirus (NoV, 12.1% and 10%), rotavirus B (RVB, 12.1% and 10%), enteric adenovirus (AdV, 6.1% and 7.5%), Aichivirus (AiV, 3% and 7.5%) and human astrovirus (HAstV, 3% and 0%). Mixed viral infections were largely represented by two viruses (84.6% and 88.9%), a small proportion showed presence of three (7.7% and 11%) and four (7.7% and 0%) viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage), NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India.

Molecular characterization of VP4, VP6, VP7 and NSP4 genes of group B rotavirus strains from outbreaks of gastroenteritis.

OBJECTIVE: To characterize VP4, VP6, VP7 and NSP4 genes of representative GBR strains (NIV-005625, NIV-04622 and NIV-094456) detected as the major etiologic agent in the outbreaks of gastroenteritis in western India. METHODS: Fecal specimens collected during the outbreaks of gastroenteritis were processed for RNA isolation, RT-PCR using GBR VP4, VP6, VP7 and NSP4 gene specific primers, nucleotide sequencing of the amplicons and phylogenetic analysis of the sequences. RESULTS: Phylogenetic analysis of all of the VP4, VP6, VP7 and NSP4 gene sequences revealed clustering of GBR strains in Indian-Bangladeshi lineage of genotype G2 with 95.8%-99.4% nucleotide and 97.3%-100.0% amino acid identities. However, all three strains showed the presence of unique amino acid substitutions in the VP4 protein suggesting alteration in the antigenicity of outbreak strains of GBR. The VP8* and VP5* regions of VP4 proteins showed respectively 0.5%-6.3% and 0.2%-1.1% amino acid divergence from human GBR strains of Indian-Bangladeshi lineage. CONCLUSIONS: These data confirm the reported variability of VP8* region and suggest the possible role of this region in the perpetuation of GBR infections in the environment. This is the first study to document the phylogenetic relationship of VP4, VP6, VP7 and NSP4 genes of GBR strains detected in the outbreaks of gastroenteritis from India with the GBR strains from other parts of world.