Japanese encephalitis virus NS5 protein interacts with nucleolin to enhance the virus replication.

Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.

Knockdown of NEAT1 restricts dengue virus replication by augmenting interferon alpha-inducible protein 27 via the RIG-I pathway.

The lncRNA NEAT1 plays a vital role in mitochondrial function and antiviral response. We have previously identified NEAT1 as dysregulated lncRNAs and found an inverse correlation with interferon alpha-inducible protein 27 (IFI27) expression associated with developing dengue severity. However, the role of NEAT1 in dengue virus (DV) infection remains elusive. Here, we undertook a study to evaluate the functional consequences of NEAT1 and IFI27 modulation on antiviral response and viral replication in dengue infection. We observed that the knockdown of NEAT1 augmented IFI27 expression and antiviral response via the RIG-I pathway. Increased antiviral response leads to a decrease in dengue viral replication. Further study suggested that the knockdown of IFI27 augmented expression of the activating transcription factor 3 (ATF3), a negative regulator of antiviral response, and increased dengue virus replication suggesting an important role played by IFI27 in mediating antiviral response. RNA sequencing study confirmed several mitochondrial genes significantly altered upon knockdown of NEAT1 in DV-infected cells. We further verified the effect of NEAT1 knockdown on mitochondrial functions. We observed a reduced level of phospho-DRP1(S616) expression along with elongated mitochondria in DV2-infected cells. Further, NEAT1 knockdown or ectopic expression of IFI27 increased mitochondrial ROS production and cell death via activation of caspase 3. Our study points to the crucial role of NEAT1 and IFI27 in mediating antiviral response and mitochondrial dysfunction in dengue infection.

Insights into the human gut virome by sampling a population from the Indian subcontinent.

Gut virome plays an important role in human physiology but remains poorly understood. This study reports an investigation of the human gut DNA-virome of a previously unexplored ethnic population through metagenomics of faecal samples collected from individuals residing in Northern India. Analysis shows that, similar to the populations investigated earlier, majority of the identified virome belongs to bacteriophages and a smaller fraction (<20 %) consists of viruses that infect animals, archaea, protists, multiple domains or plants. However, crAss-like phages, in this population, are dominated by the genera VI, VII and VIII. Interestingly, it also reveals the presence of a virus family, Sphaerolipoviridae, which has not been detected in the human gut earlier. Viral families, Siphoviridae, Myoviridae, Podoviridae, Microviridae, Herelleviridae and Phycodnaviridae are detected in all of the analysed individuals, which supports the existence of a core virome. Lysogeny-associated genes were found in less than 10 % of the assembled genomes and a negative correlation was observed in the richness of bacterial and free-viral species, suggesting that the dominant lifestyle of gut phage is not lysogenic. This is in contrast to some of the earlier studies. Further, several hundred high-quality viral genomes were recovered. Detailed characterization of these genomes would be useful for understanding the biology of these viruses and their significance in human physiology.

Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain.

Interferon regulatory factor 8 (IRF8), a myeloid lineage transcription factor, emerges as an essential regulator for microglial activation. However, the precise role of IRF8 during Japanese encephalitis virus (JEV) infection in the brain remains elusive. Here, we report that JEV infection enhances IRF8 expression in the infected mouse brain. Comparative transcriptional profiling of whole-brain RNA analysis and validation by quantitative reverse transcription-PCR (qRT-PCR) reveals an impaired interferon gamma (IFN-γ) and related gene expression in Irf8 knockout (Irf8-/-)-infected mice. Further, Ifnγ knockout (Ifnγ-/-) mice exhibit a reduced level of Irf8. Both Ifnγ-/- and Irf8-/- mice exhibit significantly reduced levels of activated (CD11b+ CD45hi, CD11b+ CD45lo, Cd68, and CD86) and infiltrating immune cells (Ly6C+, CD4, and CD8) in the infected brain compared to those of wild-type (WT) mice. However, a higher level of granulocyte cell (Ly6G+) infiltration is evident in Irf8-/- mice as well as the increased concentration of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP1) levels in the brain. Interestingly, neither the Irf8-/- nor the Ifnγ-/- conferred protection against lethal JEV challenge to mice and exhibit augmentation in JEV replication in the brain. The gain of function of Irf8 by overexpressing functional IRF8 in an IRF8-deficient cell line attenuates viral replication and enhances IFN-γ production. Overall, we summarize that in the murine model of JEV encephalitis, IRF8 modulation affects JEV replication. We also show that lack of Irf8 affects immune cell abundance in circulation and the infected brain, leading to a reduction in IFN-γ level and increased viral load in the brain. IMPORTANCE Microglial cells, the resident macrophages in the brain, play a vital role in Japanese encephalitis virus (JEV) pathogenesis. The deregulated activity of microglia can be lethal for the brain. Therefore, it is crucial to understand the regulators that drive microglia phenotype changes and induce inflammation in the brain. Interferon regulatory factor 8 (IRF8) is a myeloid lineage transcription factor involved in microglial activation. However, the impact of IRF8 modulation on JEV replication remains elusive. Moreover, the pathways regulated by IRF8 to initiate and amplify pathological neuroinflammation are not well understood. Here, we demonstrated the effect of IRF8 modulation on JEV replication, microglial activation, and immune cells infiltration in the brain.

Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

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.ImportanceJEV 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.

Viewpoint of a WHO Advisory Group Tasked to Consider Establishing a Closely-monitored Challenge Model of Coronavirus Disease 2019 (COVID-19) in Healthy Volunteers.

WHO convened an Advisory Group (AG) to consider the feasibility, potential value, and limitations of establishing a closely-monitored challenge model of experimental severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) in healthy adult volunteers. The AG included experts in design, establishment, and performance of challenges. This report summarizes issues that render a COVID-19 model daunting to establish (the potential of SARS-CoV-2 to cause severe/fatal illness, its high transmissibility, and lack of a "rescue treatment" to prevent progression from mild/moderate to severe clinical illness) and it proffers prudent strategies for stepwise model development, challenge virus selection, guidelines for manufacturing challenge doses, and ways to contain SARS-CoV-2 and prevent transmission to household/community contacts. A COVID-19 model could demonstrate protection against virus shedding and/or illness induced by prior SARS-CoV-2 challenge or vaccination. A limitation of the model is that vaccine efficacy in experimentally challenged healthy young adults cannot per se be extrapolated to predict efficacy in elderly/high-risk adults.

Proteomic landscape of Japanese encephalitis virus-infected fibroblasts.

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.

Proteins involved in actin filament organization are key host factors for Japanese encephalitis virus life-cycle in human neuronal cells.

Multiple membrane trafficking networks operate in the eukaryotic cell and are hijacked by viruses to establish infection. Recent studied have highlighted that viruses can exploit distinct pathways depending on the cell type. Japanese encephalitis virus (JEV), a neurotropic flavivirus, can infect neuronal cells through a clathrin-independent endocytic mechanism. To further characterize the membrane trafficking requirements for JEV infection of neuronal cells, we have performed a RNA interference-based study targeting 136 proteins in the human cell line IMR-32. Through quantitative RT-PCR and plaque assays we have validated that JEV infection in neuronal cells was independent of clathrin, and identified host-factors that were crucial for establishment of infection. Several of these proteins were involved in regulation of actin filament organization such as RHOA, RAC1, proteins of the ARP2/3 complex and N-WASP family, LIMK1, PAK1 and ROCK2. The small molecule inhibitors of ARP2/3 complex, CK-548 and of the N-WASP, Wiskostatin inhibited virus replication highlighting the important roles of these proteins in the virus life-cycle. We also identified ATG12, BECN1, VAPA, VAPB and VCP proteins as crucial host-factors for JEV replication across epithelial and neuronal cell lineages.

Japanese Encephalitis Virus-induced let-7a/b interacted with the NOTCH-TLR7 pathway in microglia and facilitated neuronal death via caspase activation.

MicroRNAs (miRNAs) released from the activated microglia upon neurotropic virus infection may exacerbate the neuronal damage. Here, we identified let-7a and let-7b (let-7a/b) as one of the essential miRNAs over-expressed upon Japanese Encephalitis virus (JEV) infection and released in the culture supernatant of the JEV-infected microglial cells through extracellular vesicles. The let-7a/b was previously reported to modulate inflammation in microglial cells through Toll-like receptor 7 (TLR7) pathways; although their role in accelerating JEV pathogenesis remain unexplored. Therefore, we studied the role of let-7a/b in modulating microglia-mediated inflammation during JEV infection and investigated the effect of let-7a/b-containing exosomes on primary neurons. To this end, we examined let-7a/b and NOTCH signaling pathway in TLR7 knockdown (KD) mice. We observed that TLR7 KD or inhibition of let-7a/b suppressed the JEV-induced NOTCH activation possibly via NF-κB dependent manner and subsequently, attenuated JEV-induced TNFα production in microglial cells. Furthermore, exosomes secreted from let-7a/b over-expressed microglia when transferred to uninfected mice brain induced caspase activation. Exosomes secreted from virus-infected or let-7a/b over-expressed microglia when co-incubated with mouse neuronal (Neuro2a) cells or primary cortical neurons also facilitated caspase activation leading to neuronal death. Thus, our results provide evidence for the multifaceted role of let-7a/b miRNAs in JEV pathogenesis. Let-7a/b can interact with TLR7 and NOTCH signaling pathway and enhance TNFα release from microglia. On the other hand, the exosomes secreted by JEV-infected microglia can activate caspases in uninfected neuronal cells which possibly contribute to bystander neuronal death. Cover Image for this issue: doi: 10.1111/jnc.14506.

Membrane trafficking RNA interference screen identifies a crucial role of the clathrin endocytic pathway and ARP2/3 complex for Japanese encephalitis virus infection in HeLa cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is one of the leading global causes of virus-induced encephalitis. The infectious life-cycle of viruses is heavily dependent on the host membrane trafficking network. Here, we have performed a RNA-interference-based screen using a siRNA panel targeting 136 membrane trafficking proteins to identify the key regulators of JEV infection in HeLa cells. We identified 35 proteins whose siRNA depletion restricts JEV replication by over twofold. We observe that JEV infection in HeLa cells is largely dependent on components of the clathrin-mediated endocytic (CME) pathway. Proteins involved in actin-filament-based processes, specifically CDC42 and members of the ARP2/3 complex are crucial for establishment of infection. Pharmacological pertubations of actin polymerization, a small molecule inhibitor of actin nucleation by the ARP2/3 complex - CK-548 - and the inhibitor of neural Wiskott-Aldrich syndrome proteins- Wiskostatin- inhibited JEV replication, highlighting the important role of the dynamic actin network. Other proteins involved in cargo-recognition for CME and endomembrane system organization were also validated as essential host factors for virus replication.

Diphenyleneiodonium enhances oxidative stress and inhibits Japanese encephalitis virus induced autophagy and ER stress pathways.

Diphenyleneiodonium (DPI) and N-acetyl-l-cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fibroblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy induction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of inhibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes.

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

Japanese encephalitis virus activates autophagy through XBP1 and ATF6 ER stress sensors in neuronal cells.

Endoplasmic reticulum (ER) stress and autophagy are key cellular responses to RNA virus infection. Recent studies have shown that Japanese encephalitis virus (JEV)-induced autophagy negatively influences virus replication in mouse neuronal cells and embryonic fibroblasts, and delays virus-induced cell death. Here, we evaluated the role of ER stress pathways in inducing autophagy during JEV infection. We observed that JEV infection of neuronal cells led to activation of all three sensors of ER stress mediated by eIF2α/PERK, IRE1/XBP1 and ATF6. The kinetics of autophagy induction as monitored by levels of SQSTM1 and LC3-II paralleled activation of ER stress. Inhibition of the eIF2α/PERK pathway by siRNA-mediated depletion of proteins and by the PERK inhibitor had no effect on autophagy and JEV replication. However, depletion of XBP1 and ATF6, alone or in combination, prevented autophagy induction and significantly enhanced JEV-induced cell death. JEV-infected cells depleted of XBP1 or ATF6 showed reduced transcription of ER chaperones, ERAD components and autophagy genes, resulting in reduced protein levels of the crucial autophagy effectors ATG3 and BECLIN-1. Conversely, pharmacological induction of ER stress in JEV-infected cells further enhanced autophagy and reduced virus titres. Our study thus demonstrates that a crucial link exists between the ER stress pathways and autophagy in virus-infected cells, and that these processes are highly regulated during virus infection.

Japanese encephalitis virus invasion of cell: allies and alleys.

The mosquito-borne flavivirus, Japanese encephalitis virus (JEV), is the leading cause of virus-induced encephalitis globally and a major public health concern of several countries in Southeast Asia, with the potential to become a global pathogen. The virus is neurotropic, and the disease ranges from mild fever to severe hemorrhagic and encephalitic manifestations and death. The early steps of the virus life cycle, binding, and entry into the cell are crucial determinants of infection and are potential targets for the development of antiviral therapies. JEV can infect multiple cell types; however, the key receptor molecule(s) still remains elusive. JEV also has the capacity to utilize multiple endocytic pathways for entry into cells of different lineages. This review not only gives a comprehensive update on what is known about the virus attachment and receptor system (allies) and the endocytic pathways (alleys) exploited by the virus to gain entry into the cell and establish infection but also discusses crucial unresolved issues. We also highlight common themes and key differences between JEV and other flaviviruses in these contexts.

Unraveling the web of viroinformatics: computational tools and databases in virus research.

The beginning of the second century of research in the field of virology (the first virus was discovered in 1898) was marked by its amalgamation with bioinformatics, resulting in the birth of a new domain--viroinformatics. The availability of more than 100 Web servers and databases embracing all or specific viruses (for example, dengue virus, influenza virus, hepatitis virus, human immunodeficiency virus [HIV], hemorrhagic fever virus [HFV], human papillomavirus [HPV], West Nile virus, etc.) as well as distinct applications (comparative/diversity analysis, viral recombination, small interfering RNA [siRNA]/short hairpin RNA [shRNA]/microRNA [miRNA] studies, RNA folding, protein-protein interaction, structural analysis, and phylotyping and genotyping) will definitely aid the development of effective drugs and vaccines. However, information about their access and utility is not available at any single source or on any single platform. Therefore, a compendium of various computational tools and resources dedicated specifically to virology is presented in this article.

Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Rotavirus is the most common cause of severe dehydrating gastroenteritis in developing countries. Safe, effective, and affordable rotavirus vaccines are needed in these countries. We aimed to assess the efficacy and tolerability of a monovalent human-bovine rotavirus vaccine for severe rotavirus gastroenteritis in low-resource urban and rural settings in India. METHODS: We did a randomised double-blind, placebo-controlled, multicentre trial at three sites in Delhi (urban), Pune (rural), and Vellore (urban and rural) between March 11, 2011, and Nov 5, 2012. Infants aged 6-7 weeks were randomly assigned (2:1), via a central interactive voice or web response system with a block size of 12, to receive either three doses of oral human-bovine natural reassortant vaccine (116E) or placebo at ages 6-7 weeks, 10 weeks, and 14 weeks. Infants' families, study investigators, paediatricians in referral hospitals, laboratory staff, and committee members were all masked to treatment allocation. The primary outcome was incidence of severe rotavirus gastroenteritis (≥11 on the Vesikari scale). Efficacy outcomes and adverse events were ascertained through active surveillance. Analysis was by intention to treat and per protocol. The trial is registered with Clinical Trial Registry-India (CTRI/2010/091/000102) and ClinicalTrials.gov (NCT01305109). FINDINGS: 4532 infants were assigned to receive the 116E vaccine and 2267 to receive placebo, of whom 4354 (96%) and 2187 (96%) infants, respectively, were included in the primary per-protocol efficacy analysis. 71 events of severe rotavirus gastroenteritis were reported in 4752 person-years in infants in the vaccine group compared with 76 events in 2360 person-years in those in the placebo group; vaccine efficacy against severe rotavirus gastroenteritis was 53·6% (95% CI 35·0-66·9; p=0·0013) and 56·4% (36·6-70·1; p<0·0001) in the first year of life. The number of infants needed to be immunised to prevent one severe rotavirus gastroenteritis episode was 55 (95% CI 37-97). The incidence of severe rotavirus gastroenteritis per 100 person-years was 1·5 in the vaccine group and 3·2 in the placebo group, with an incidence rate ratio of 0·46 (95% CI 0·33-0·65). Prevalence of immediate, solicited, and serious adverse events was similar in both groups. One case of urticaria in the vaccine group and one each of acute gastroenteritis and suspected sepsis in the placebo group were regarded as related to the study product. We recorded six cases of intussusception in the vaccine group and two in the placebo group, all of which happened after the third dose. 25 (<1%) infants in the vaccine group and 17 (<1%) in the placebo group died; no death was regarded as related to the study product. INTERPRETATION: Monovalent human-bovine (116E) rotavirus vaccine is effective and well tolerated in Indian infants. FUNDING: Department of Biotechnology and the Biotechnology Industry Research Assistance Council, Government of India; Bill & Melinda Gates Foundation to PATH, USA; Research Council of Norway; UK Department for International Development; National Institutes of Health, Bethesda, USA; and Bharat Biotech International, Hyderabad, India.

Japanese encephalitis virus expands regulatory T cells by increasing the expression of PD-L1 on dendritic cells.

The mechanisms underlying Japanese encephalitis virus (JEV) pathogenesis need to be thoroughly explored to delineate therapeutic approaches. It is believed that JEV manipulates the innate and adaptive compartments of the host's immune system to evade immune response and cross the blood-brain barrier. The present study was thus designed to investigate the functional modulation of DCs after exposure to JEV and to assess the consequences on CD4(+) T-lymphocyte functions. Human monocyte-derived DCs were either infected with 1 MOI of live virus, UV-inactivated virus, or were mock-infected. Replication-competent JEV induced a significant increase in the expression of maturation markers 48 h postinfection, along with that of programmed cell death 1 ligand 1 (PD-L1; also called B7-H1 and CD274). JEV-infected DCs expanded the Treg cells in allogenic mixed lymphocyte reactions. The expansion of Treg cells by JEV-infected DCs was significantly reduced upon blocking PD-L1 using an antagonist. In addition, JEV-infected DCs significantly altered the proliferation and reduced the polarization of Th cells toward the Th1-cell phenotype. The results, for the first time, suggest that JEV evades the host's immune system by modulating the crosstalk between DCs and T lymphocytes via the PD-L1 axis.

Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication.

Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.

MiR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses.

BACKGROUND: Microglial cells, which are resident macrophages of the central nervous system, play important roles in immune responses and pathogenesis. Japanese encephalitis virus (JEV) is a neurotropic virus that infects microglial cells in brain. Several microRNAs including miR-155 and miR-146a play an important role in defining the microglia inflammatory profile. In this study, we have investigated the effect of miR-155 and miR-146a modulation on JEV infection as well as innate immune responses in human microglial cells. METHODS: In vitro studies were performed in JEV-infected human microglial CHME3 cells. miR-155 or miR-146a were overexpressed and total RNA and protein were extracted following JEV-infection. Expression of genes involved in innate immune responses was studied by PCR array, quantitative real-time PCR (qPCR), western blot and Fluorescence activated cell sorter (FACS). JEV replication was monitored by studying the viral RNA by qPCR, protein by western blot, and titres by plaque assay. RESULTS: Overexpression of miR-155 in CHME3 cells resulted in significantly reduced JEV replication whereas miR-146a overexpression had an insignificant effect. Additionally, interferon regulatory factor 8 (IRF8) and complement factor H (CFH) were induced during JEV infection; however, this induction was attenuated in miR-155 overexpressing cells following JEV infection. Further, JEV-induced NF-κB regulated downstream gene expression was attenuated. Interestingly, an increased level of CD45, a negative regulator of microglia activation and a reduced phosphorylated-Signal Transducers and Activators of Transcription (p-STAT1) expression was observed in miR-155 overexpressing cells upon JEV infection. CONCLUSION: Induction of miR-155 in human microglial cells may negatively modulate JEV-induced innate immune gene expression and may have a beneficial role in limiting JEV replication in human microglial cells.