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 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.

Phenotypic analysis of monocytes and CD4+ T cells in hepatitis E patients with or without pregnancy.

High mortality in pregnant women is a characteristic of hepatitis E virus (HEV) infection. Role of monocytes/T cells in HEV infection during pregnancy is still unclear. We compared CD14+monocytes and CD4+T cells by flow-cytometry in hepatitis-E patients including 13 pregnant (Antenatal care, ANC), 25 non-ANC patients and respective controls (12 and 20). Non-ANC-patients showed significantly higher frequency of monocytes with increased expression of CD80, CD86 and HLA-DR than control individuals (p < 0.001). Healthy pregnancy was associated with increased frequency of monocytes with higher CD80 expression and lower levels of HLA-DR (p < 0.05) compared to non-ANC controls. ANC-patients exhibited elevated levels of monocytes (p < 0.01) with higher expression of CD80 (p < 0.001) and reduced levels of HLA-DR and CD86 (p < 0.05) when compared with non-ANC patients. TLR2 and TLR4 surface expression on monocytes was higher in non-ANC-patients (p < 0.00) and lower in the ANC-patients (p < 0.01). Healthy-ANCs exhibited lower TLR4 expression on monocytes (p < 0.05). HEV infection did not change the frequency of CD4+ and CD4+CD28+T cells in patients' group (p > 0.05). Compared to respective controls, CD137+ and CD152+CD4+T cells were higher (p < 0.05) in both patients' categories. Higher levels of CD152+CD4+T cells (p < 0.001) was noted in healthy pregnant women. Among patients' groups, the CD4+T cells and their subpopulation were not different (p > 0.05). We found higher and reduced levels of circulating inflammatory cytokines (IL12, TNFα, IL6 and IL8; miliplex-assay) in non-ANC and ANC-patients respectively. In conclusion, on contrary to the classical activation of CD14+monocytes in the non-ANC-patients, impaired response was evident in the ANC-patients while the CD4+T cell populations were similar in the patient groups.

Association of Toll-like receptor 4 polymorphism with hepatitis E virus-infected Indian patients.

Hepatitis E infection caused by hepatitis E virus (HEV), a major public health concern in developing countries, is responsible for sporadic and epidemic acute viral hepatitis in adults. Pathogenesis of hepatitis E infection is poorly understood. Toll-like receptors (TLRs) are the key players of innate immunity recognize pathogen-associated molecular patterns (PAMPs). Previously, we found higher TLR4 expression (at protein and gene level) with impaired cytokine response upon stimulus of PBMCs with LPS in HEV-infected patients. In view of the earlier observations of the association of polymorphisms in TLR4 genes (A299G, C399T) with liver diseases, we investigated TLR4 polymorphisms in HEV-infected patients. We observed the significant association of TLR4-399CC and CT alleles with hepatitis E (both subclinical and acute patients). Carrier frequency of TLR4-399 CT was lower in patients' categories in comparison with the controls. Higher frequency of allele TLR4-399C significantly correlated with disease progression. Acute hepatitis E patients showed the higher frequency of CG and TA haplotypes, while the rare haplotype (TG) was more frequent in controls. The other single nucleotide polymorphism (SNP) at TLR4-299 (A>G) did not show any difference. We report here for the first time the association of TLR4 polymorphism with hepatitis E and suggest that TLR 4 hyporesponsiveness during HEV infection might be related to its polymorphism.

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.

Consensus M2e peptide conjugated to gold nanoparticles confers protection against H1N1, H3N2 and H5N1 influenza A viruses.

The extracellular domain of influenza A ion channel membrane matrix protein 2 (M2e) is considered to be a potential candidate to develop a universal influenza A vaccine. However poor immunogenicity of M2e presents a significant roadblock. We have developed a vaccine formulation comprising of the consensus M2e peptide conjugated to gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). We demonstrate that intranasal delivery of AuNP-M2e + sCpG in mice induces lung B cell activation and robust serum anti-M2e immunoglobulin G (IgG) response, with stimulation of both IgG1 and IgG2a subtypes. Using Madin-Darby canine kidney (MDCK) cells infected with A/California/04/2009 (H1N1pdm) pandemic strain, or A/Victoria/3/75 (H3N2), or the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) as immunosorbants we further show that the antibodies generated are also capable of binding to the homotetrameric form of M2 expressed on infected cells. Lethal challenge of vaccinated mice with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) led to 100%, 92%, and 100% protection, respectively. Overall, this study helps to lay the foundation of a potential universal influenza A vaccine.

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.

Toll like receptors in self-recovering hepatitis E patients with or without pregnancy.

Hepatitis E virus (HEV) causes high mortality among pregnant women. Pathogenesis of HEV, especially during pregnancy, is poorly understood. Our aim was to assess the role of Toll-like-receptors (TLRs) in hepatitis E patients with pregnancy (Antenatal care, ANC) or without pregnancy (non-ANC). The patient categories included acute-phase, non-ANC (n=46) and ANC patients (2nd/3rd trimesters, n=13) and non-ANC patients (n=31) during convalescence. Controls included apparently healthy non-ANC (n=30) and ANC subjects in the first (n=10) and later (2nd/3rd, n=20) trimesters. TLR2/TLR3/TLR4/TLR7/TLR8 levels were determined by flow-cytometry. Cytokine responses induced by TLR-specific-ligands-stimulated-PBMCs from ANC/non-ANC-patients and TLR-signaling-molecules (non-ANC-patients) were measured. PBMCs were used to assess gene expression levels by TaqMan-Low-Density-Array. Compared to the temporal activation of TLR4/TLR7/TLR8 at protein and mRNA levels, the ANC-patients and controls exhibited reduced TLRs indicative of impaired TLR response. Stimulation of PBMCs with TLR-specific ligands led to the induction of type-I interferons, IFNß by the non-ANC group and IFNα by the ANC category. Involvement of MyD88-independent (TLR3/TLR4) and MyD88-dependent (TLR4/TLR7/TLR8) pathways and association of TLR4/TLR7/TLR8 with recovery was documented in the non-ANC-patients. Except for robust type-I-interferon response, HEV infection could not modulate pregnancy-related diminished immune response. The results have implications in the understanding of HEV pathogenesis.

Effect of pregnancy on anti-HEV antibody titres, plasma cytokines and the corresponding gene expression levels in the PBMCs of patients presenting with self-recovering clinical and subclinical hepatitis E.

High mortality in pregnant women (PR) is a characteristic of hepatitis E in developing countries. To understand the pathogenesis of HEV infection in self-limiting disease during pregnancy, we compared clinical (PR-patients) and subclinical-HEV-infections in pregnant women in the first (SC-PR-1) and later (2nd and 3rd, SC-PR-2+3) trimesters with the respective healthy controls and acute non-PR patients. The SC-PR-2+3 exhibited lower ALT, bilirubin levels, anti-HEV-IgM/IgG titres than the acute-PR/non-PR-patients (p<0.05-0.0001). IFNγ/IL4ratios indicated Th2/Th1 bias in non-PR and PR-patients respectively. Raised levels of 10/20 plasma cytokines in the non-PR-patients reflect predominant inflammatory response, unaltered- IFNγ/reduced-IFNα responses and a robust chemokine secretion. On contrary, the acute-PR-patients exhibited drastic reduction in majority of the cytokines relative to in the non-PR-patients. Importantly, diminished or unaltered response was noted in the acute-PR-group when compared to the corresponding controls. The only exception was sIL2RA, increasing in both patient categories. Of the 14 genes evaluated, the expression of IFNγ/IL10/IL1A/IL7/CCL2/CCL3/CXCL8/CXCL10 was higher in the non-PR patients. Of these, the expression of IFNγ/IL10/IL1A/CCL2/CCL3/CXCL8 and, additionally, IL2/IL6/TNF genes was higher in the clinical-PRs. Almost identical pattern was noted in the control-PR-2+3 category indicating no influence of HEV infection. Comparison of patient-categories identified significant elevation of IFNγ(P<0.001), CCL2(p<0.01), CXCL8(P<0.05), IL1B(p<0.05) and IL10(P<0.0001) and decrease in CXCL10(<0.05) in the PR-patients. The results suggest antibody-dependent disease severity and impaired immune response in the PR patients. Higher expression of cytokine-genes in the PBMCs did not correlate with the plasma-cytokine levels in the PR-patients.

Role of host immune response and viral load in the differential outcome of pandemic H1N1 (2009) influenza virus infection in Indian patients.

BACKGROUND: An unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic. METHODOLOGY: The role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs. PRINCIPAL FINDINGS: 13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patient's category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters. CONCLUSIONS: Disease severity was associated with pronounced impairment of host immune response.