Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in ...infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.
Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular ...functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.
JEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.
Viral hepatitis is a major public health concern globally. World health organization aims at eliminating viral hepatitis as a public health threat by 2030. Among the hepatitis causing viruses, ...hepatitis B and C are primarily transmitted via contaminated blood. Hepatitis A and E, which gets transmitted primarily via the feco-oral route, are the leading cause of acute viral hepatitis. Although vaccines are available against some of these viruses, new cases continue to be reported. There is an urgent need to devise a potent yet economical antiviral strategy against the hepatitis-causing viruses (denoted as hepatitis viruses) for achieving global elimination of viral hepatitis. Although zinc was known to mankind for a long time (since before Christ era), it was identified as an element in 1746 and its importance for human health was discovered in 1963 by the pioneering work of Dr. Ananda S. Prasad. A series of follow up studies involving zinc supplementation as a therapy demonstrated zinc as an essential element for humans, leading to establishment of a recommended dietary allowance (RDA) of 15 milligram zinc United States RDA for zinc. Being an essential component of many cellular enzymes and transcription factors, zinc is vital for growth and homeostasis of most living organisms, including human. Importantly, several studies indicate potent antiviral activity of zinc. Multiple studies have demonstrated antiviral activity of zinc against viruses that cause hepatitis. This article provides a comprehensive overview of the findings on antiviral activity of zinc against hepatitis viruses, discusses the mechanisms underlying the antiviral properties of zinc and summarizes the prospects of harnessing the therapeutic benefit of zinc supplementation therapy in reducing the disease burden due to viral hepatitis.
Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis. The disease takes a severe form in pregnant women, leading to around 30% mortality. Zinc is an essential micronutrient that plays a ...crucial role in multiple cellular processes. Our earlier findings demonstrated the antiviral activity of zinc salts against HEV infection. Zinc oxide (ZnO) and its nanostructures have attracted marked interest due to their unique characteristics. Here we synthesized ZnO nanoparticles ZnO(NP) and tetrapod-shaped ZnO nanoparticles ZnO(TP) and evaluated their antiviral activity. Both ZnO(NP) and ZnO(TP) displayed potent antiviral activity against hepatitis E and hepatitis C viruses, with the latter being more effective. Measurement of cell viability and intracellular reactive oxygen species levels revealed that both ZnO(NP) and ZnO(TP) are noncytotoxic to the cells even at significantly higher doses, compared to a conventional zinc salt (ZnSO
4
). Our study paves the way for evaluation of the potential therapeutic benefit of ZnO(TP) against HEV and HCV.
Understanding the dynamics of host innate immune responses against a pathogen marks the first step toward developing intervention strategies against the pathogen. The cytosolic pattern recognition ...receptor retinoic acid-inducible gene I (RIG-I) has been shown to be the major innate immune sensor for hepatitis E virus (HEV). Here, we show that HEV capsid protein (ORF2), a 660 amino acid long protein, interferes with the RIG-I signaling. Interestingly, only the full length ORF2 protein but not the 112-608 ORF2 protein inhibited RIG-I dependent interferon response. Both synthetic agonist and virus induced RIG-I activation was modulated by ORF2. Interference of interferon response was confirmed by reporter assays involving different interferon inducible promoters, qRT PCR, ELISA, and immunofluorescence microscopy. Neither glycosylation nor dimerization of the ORF2 protein had any effect on the observed inhibition. Further analyses revealed that the ORF2 protein antagonized Toll-like receptor (TLR) pathways as well. ORF2 inhibited signaling by RIG-I and TLR adapters, IPS-1, MyD88, and TRIF but was unable to inhibit activation by ectopically expressed IRF3 suggesting that it may be acting at a site upstream of IRF3 and downstream of adapter proteins. Our data uncover a new mechanism by which HEV may interfere with the host antiviral signaling.
Coronavirus‐induced disease‐19 (COVID‐19), caused by SARS‐CoV‐2, is still a major global health challenge. Human endogenous retroviruses (HERVs) represent retroviral elements that were integrated ...into the ancestral human genome. HERVs are important in embryonic development as well as in the manifestation of diseases, including cancer, inflammation, and viral infections. Here, we analyze the expression of several HERVs in SARS‐CoV‐2‐infected cells and observe increased activity of HERV‐E, HERV‐V, HERV‐FRD, HERV‐MER34, HERV‐W, and HERV‐K‐HML2. In contrast, the HERV‐R envelope is downregulated in cell‐based models and PBMCs of COVID‐19 patients. Overexpression of HERV‐R inhibits SARS‐CoV‐2 replication, suggesting its antiviral activity. Further analyses demonstrate the role of the extracellular signal‐regulated kinase (ERK) in regulating HERV‐R antiviral activity. Lastly, our data indicate that the crosstalk between ERK and p38 MAPK controls the synthesis of the HERV‐R envelope protein, which in turn modulates SARS‐CoV‐2 replication. These findings suggest the role of the HERV‐R envelope as a prosurvival host factor against SARS‐CoV‐2 and illustrate a possible advantage of integration and evolutionary maintenance of retroviral elements in the human genome.
Synopsis
HERV‐R‐Env (envelope) levels are reduced upon SARS‐CoV‐2 infection in cell‐based models and COVID‐19 patient samples. HERV‐R‐Env inhibits SARS‐CoV‐2 replication, suggesting its antiviral function, which is mediated by the ERK pathway.
SARS‐CoV‐2 infection reduces HERV‐R‐Env levels.
HERV‐R‐Env inhibits SARS‐CoV‐2 replication.
The HERV‐R‐Env antiviral function against SARS‐CoV‐2 is mediated by activation of the ERK pathway.
SARS‐CoV‐2 antagonizes the antiviral function of HERV‐R‐Env by upregulating the p38 MAPK activity and concomitantly downregulating the ERK activity.
HERV‐R‐Env (envelope) levels are reduced upon SARS‐CoV‐2 infection in cell‐based models and COVID‐19 patient samples. HERV‐R‐Env inhibits SARS‐CoV‐2 replication, suggesting its antiviral function, which is mediated by the ERK pathway.
We previously reported that selective ablation of the nuclear receptors retinoid X receptor (RXR)-α and RXR-β in mouse epidermal keratinocytes (RXR-αβep−/−) or a topical application of active vitamin ...D3 (VD3) and/or all-trans retinoic acid (RA) on wild-type mouse skin induces a human atopic dermatitis-like phenotype that is triggered by an increased expression of the thymic stromal lymphopoietin (TSLP) proinflammatory cytokine. We demonstrate here that in epidermal keratinocytes, unliganded heterodimers of vitamin D receptor (VDR)/RXR-α and retinoic acid receptor-γ (RAR-γ)/RXR-β are bound as repressing complexes to their cognate DNA-binding sequence(s) (DBS) in the TSLP promoter regulatory region. Treatments with either an agonistic VD3 analog or RA dissociate the repressing complexes and recruit coactivator complexes and RNA polymerase II, thereby inducing transcription. Furthermore, we identified several functional NF-κB, activator protein 1 (AP1), STAT, and Smad DBS in the TSLP promoter region. Interestingly, many of these transcription factors and DBS present in the TSLP promoter region are differentially used in intestinal epithelial cell(s) (IEC). Collectively, our study reveals that, in vivo within their heterodimers, the RXR and RAR isotypes are not functionally redundant, and it also unveils the combinatorial mechanisms involved in the tissue-selective regulation of TSLP transcription in epidermal keratinocytes and IEC.
Background & objectives: Nasopharyngeal and oropharyngeal swab (NPS and OPS) collection is widely accepted as the preferred method for obtaining respiratory samples. However, it has certain ...disadvantages which may be overcome by gargling. The primary objective of this study was to assess agreement between gargle lavage and swab as an appropriate respiratory sample for the detection of SARS-CoV-2. The secondary objective was to assess the patient acceptability of the two sampling methods.
Methods: It was a cross-sectional study done at a tertiary care hospital in New Delhi, India, on 50 confirmed COVID-19 patients. Paired swab (NPS and OPS) and gargle samples were taken within 72 h of their diagnosis. Samples were processed by reverse transcription-polymerase chain reaction (RT-PCR) for detection of SARS-CoV-2. Post-sample collection, a 10-point scale was administered to assess the level of discomfort with either of the collection methods.
Results: All gargle samples were positive and comparable to their corresponding swab samples irrespective of the symptoms and duration of illness. The cycle threshold (Ct) values for gargle samples were slightly higher but comparable to those of swabs. Bland-Altman plot showed good agreement between the two methods. Majority (72%) of the patients reported moderate-to-severe discomfort with swab collection in comparison to 24 per cent reporting only mild discomfort with gargle collection.
Interpretation & conclusions: Our preliminary results show that the gargle lavage may be a viable alternative to swabs for sample collection for the detection of SARS-CoV-2. Adoption of gargle lavage for sample collection will have a significant impact as it will enable easy self-collection, relieve healthcare workers and also lead to substantial cost savings by reducing the need for swabs and personal protective equipment.
The newly emerged novel coronavirus, SARS-CoV-2, the causative agent of COVID-19 has proven to be a threat to the human race globally, thus, vaccine development against SARS-CoV-2 is an unmet need ...driving mass vaccination efforts. The receptor binding domain of the spike protein of this coronavirus has multiple neutralizing epitopes and is associated with viral entry. Here we have designed and characterized the SARS-CoV-2 spike protein fragment 330-526 as receptor binding domain 330-526 (RBD
) with two native glycosylation sites (N331 and N343); as a potential subunit vaccine candidate. We initially characterized RBD
biochemically and
investigated its thermal stability, humoral and T cell immune response of various RBD protein formulations (with or without adjuvant) to evaluate the inherent immunogenicity and immunomodulatory effect. Our result showed that the purified RBD immunogen is stable up to 72 h, without any apparent loss in affinity or specificity of interaction with the ACE2 receptor. Upon immunization in mice, RBD generates a high titer humoral response, elevated IFN-γ producing CD4+ cells, cytotoxic T cells, and robust neutralizing antibodies against live SARS-CoV-2 virus. Our results collectively support the potential of RBD
as a promising vaccine candidate against SARS-CoV-2.
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