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Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus. [ABSTRACT FROM AUTHOR]

Background: Until recently, arbovirus surveillance is mainly focused on mosquito and tick vectors, resulting in the discovery of several mosquito- and tick-borne arboviruses. However, the role of sandflies in arbovirus transmission and disease has remained largely unexplored. This study sought to isolate and characterize arboviruses from phlebotomine sandflies from selected pastoral ecozones in the North Rift region of Kenya. Methods: Sandflies were collected from selected sites in North Rift Kenya between 2015 and 2018. They were sorted and pooled by sex, site, and collection date. The pools were homogenized and inoculated onto Vero cells for virus isolation. The positive pools were analyzed by polymerase chain reaction targeting different arboviruses. The isolates were further characterized by highthroughput sequencing using Illumina Miseq platform. Results: Approximately 28,226 sandflies translating to 824 pools were sampled from the selected regions. A total of 11 showed reproducible cytopathic effects on Vero cells. We identified five arboviruses: sindbis (n = 4) from Kacheliba and Baringo, Chandipura (n = 4) from Turkana and Baringo, Koutango (n = 1) and Ntepes (n = 1) from Baringo, and Bogoria (n = 1) from Kacheliba. The percent identities of the identified viruses were approximately 80% to 98% compared to known viruses in GenBank, suggesting that some of them could be novel viruses. Conclusion: This study successfully isolated and characterized five arboviruses from sandflies. The findings suggest that sandflies are potential hosts of a wide range of arboviruses and are therefore important vectors to consider in arbovirus surveillance and evaluated for their ability to transmit them. Further studies are needed to determine the public health importance and extent of exposure of these viruses to humans and livestock populations. [ABSTRACT FROM AUTHOR]

BackgroundUntil recently, arbovirus surveillance is mainly focused on mosquito and tick vectors, resulting in the discovery of several mosquito- and tick-borne arboviruses. However, the role of sandflies in arbovirus transmission and disease has remained largely unexplored. This study sought to isolate and characterize arboviruses from phlebotomine sandflies from selected pastoral ecozones in the North Rift region of Kenya.MethodsSandflies were collected from selected sites in North Rift Kenya between 2015 and 2018. They were sorted and pooled by sex, site, and collection date. The pools were homogenized and inoculated onto Vero cells for virus isolation. The positive pools were analyzed by polymerase chain reaction targeting different arboviruses. The isolates were further characterized by high-throughput sequencing using Illumina Miseq platform.ResultsApproximately 28,226 sandflies translating to 824 pools were sampled from the selected regions. A total of 11 showed reproducible cytopathic effects on Vero cells. We identified five arboviruses: sindbis (n = 4) from Kacheliba and Baringo, Chandipura (n = 4) from Turkana and Baringo, Koutango (n = 1) and Ntepes (n = 1) from Baringo, and Bogoria (n = 1) from Kacheliba. The percent identities of the identified viruses were approximately 80% to 98% compared to known viruses in GenBank, suggesting that some of them could be novel viruses.ConclusionThis study successfully isolated and characterized five arboviruses from sandflies. The findings suggest that sandflies are potential hosts of a wide range of arboviruses and are therefore important vectors to consider in arbovirus surveillance and evaluated for their ability to transmit them. Further studies are needed to determine the public health importance and extent of exposure of these viruses to humans and livestock populations.

Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus.

Chandipura virus (CHPV) is an emerging encephalitic virus with outbreak potential in a pediatric population. It causes acute encephalitis, with clinical symptoms leading to death within 48–72 h and an alarmingly high case fatality rate up to 55%–78%. Despite the high mortality rate in children, no vaccines or antivirals are currently available; thus, repurposing licensed drugs seems to be one of the attractive therapeutic approaches. Among the various options available, Favipiravir emerged as a promising candidate, and its unique characteristics and clinical efficacy have garnered significant attention and demonstrated considerable potential in the fight against viral diseases. In the current study, we have evaluated the antiviral effect of Favipiravir against CHPV by Plaque reduction assay and viral growth kinetics assay in Vero cells and in vivo effect of drug treatment against lethal viral challenge was analysed in 10‐day‐old CD1 mice. A dose‐dependent reduction in CHPV plaque size and number was observed in Vero cells treated with Favipiravir, with an EC50 of 92.26 μM. Complete inhibition of CHPV replication was observed at 320 μM drug concentration and 50% cytotoxicity (CC50) at 4774 μM, indicating a high selectivity index 51.24. In vivo, studies in mice showed 100% survival with 300 mg/kg/day of Favipiravir given orally till seventh‐day postinfection. The study provides evidence of the antiviral activity of Favipiravir against CHPV infection, and further clinical evaluation may alleviate the associated mortality. [ABSTRACT FROM AUTHOR]

Background & objectives: Sandflies are implicated as vectors of Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae). The virus is prevalent in central India including Vidarbha region of Maharashtra. CHPV causes encephalitis in children below 15 yr of age with case fatality rates ranging from 56 to 78 per cent. The present study was undertaken to determine the sandfly fauna in the CHPV endemic Vidharba region. Methods: A year round survey of sandflies was conducted at 25 sites in three districts of Vidarbha region. Sandflies were collected from their resting sites using handheld aspirators and identified using taxonomical keys. Results: A total of 6568 sandflies were collected during the study. Approximately 99 per cent of the collection belonged to genus Sergentomyia, which was represented by Ser. babu, Ser. bailyi and Ser. punjabensis. Genus Phlebotomus was represented by Ph. argentipes and Ph. papatasi. Ser. babu was the predominant species (70.7%) collected during the study. Ph. argentipes was detected in four villages with 0.89 per cent, whereas Ph. papatasi was detected in only one village with 0.32 per cent of the total collection. CHPV could not be isolated despite processing all the sandflies for virus isolation in cell culture. Interpretation & conclusions: The present study showed influence of higher temperature and relative humidity on sandfly population dynamics. An important observation during the study was the absence or decline in the population of Ph. papatasi and Ph. argentipes in the study area. Surge in Sergentomyia population and their breeding/resting in close vicinity to humans pose a concern as they are known to harbour CHPV and other viruses of public health importance. [ABSTRACT FROM AUTHOR]

Chandipura virus (CHPV) is an emerging human pathogen of great clinical significance. In this study, we have investigated the susceptibility pattern of both normal and cancer cell lines of human origin to wild-type (wt) CHPV in order to explore the possibility of developing CHPV as an oncolytic vector (OV). Marked cytopathic effect along with enhanced virus output was observed in cancer cell lines (HeLa, A549, U-138, PC-3, and HepG2) in comparison to normal human adult dermal fibroblast (HADF) cells. At an MOI of 0.1, cancer cell lines were differentially susceptible to CHPV, with cells like HeLa and U-138 having pronounced cell death, while the PC-3 were comparatively resistant. All cell lines used in the study except U-138 restricted CHPV infection to varying degrees with IFN-β pre-treatment and supplementation of interferon (IFN) could neither activate the IFN signaling pathway in U-138 cells. Finally, U-138 tumor xenografts established in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice showed significant delay in tumor growth in the CHPV-challenged animals. Thus, targeted cytopathic effect in cancer cells at a very low dose with restricted replication in normal cells offers a rationale to exploit CHPV as an oncolytic vector in the future.

Background: First isolated in 1965 from a case of febrile encephalopathy, the Chandipura virus (CHPV) causes sporadic cases as well as periodic outbreaks of encephalitis in parts of India. Transmitted by sandflies and mosquitoes, CHPV infection has high mortality within 48 hours of hospitalisation, with children bearing the brunt of the illness. The virus garnered global attention in the middle of 2024 as India witnessed its largest outbreak in the last two decades., Objective: This article aims to synthesise the existing knowledge on various aspects of CHPV and outline current actions needed as well as potential directions for future research., Content: Between early June and August 15th 2024, India reported 245 cases of encephalitis from the states of Gujarat and Rajasthan, 64 of which were laboratory-confirmed CHPV infections. The mortality toll of the outbreak was 82, accounting for a case fatality rate of 33%. With this outbreak, the virus has expanded its niche from central and southern to north-western India. Significant advancements in the understanding of the neuropathogenesis of the virus and the development of diagnostic assays have been made in the 21 st century. However, no specific antiviral drugs or vaccines are available. A G-protein-based recombinant vaccine and an inactivated vaccine have shown favourable results in pre-clinical trials. The need of the hour is to fast-track the development of an effective vaccine. A high suspicion for early identification and prompt referral of cases, decentralized diagnostic facilities, sensitization of healthcare workers, integrated vector management and effective reporting and surveillance systems are all needed to curb the menace of this perilous pathogen. The current outbreak should serve as a wake-up call to foster intersectoral collaboration between policymakers, public health experts, epidemiologists, virologists, neurologists, paediatricians, and anthropologists to develop and implement effective strategies against the virus., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Abstract Background Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1β, in human microglial cells during CHPV infection. Methods To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1β in this study. Results The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. Conclusions This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells.

Chandipura virus (CHPV) is a neurotropic virus, known to cause encephalitis in humans. The microRNAs (miRNA/miR) play an important role in the pathogenesis of viral infection. The present study is focused on the role of miRNAs during CHPV (strain 1653514) infection in human microglial cells. The deep sequencing of CHPV‐infected human microglial cells identified a total of 12 differentially expressed miRNA (DEMs). To elucidate the role of DEMs, the target gene prediction, Gene Ontology term (GO Term), pathway enrichment analysis, and miRNA‐messenger RNA (mRNA) interaction network analysis was performed. The GO terms and pathway enrichment analysis provided 146 enriched genes; which were involved in interferon response, cytokine and chemokine signaling. Further, the WGCNA (weighted gene coexpression network analysis) of the enriched genes were discretely categorized into three modules (blue, brown, and turquoise). The hub genes in the blue module may correlate to CHPV induced neuroinflammation. Altogether, the miRNA‐mRNA interaction network and WGCNA study revealed the following pairs, hsa‐miR‐542‐3p and FAF1, hsa‐miR‐92a‐1‐5p and MYD88, and hsa‐miR‐3187‐3p and TNFRSF21, which may contribute to neuroinflammation during CHPV infection in human microglial cells. Highlight: Chandipura virus infects Central Nervous System.Chandipura Virus modulates the microRNA expression pattern in human microglial cells.Chandipura virus affects the cellular gene expression via changes in expression patterns of microRNAs.Chandipura virus infects microglial cells and contributes to the neuroinflammatory events. [ABSTRACT FROM AUTHOR]

Background & objectives: Aedes vittatus (Bigot), an anthropophilic mosquito, plays an important role in the maintenance and transmission of yellow fever (YF), dengue (DEN), chikungunya (CHIKV) and Zika (ZIK) viruses in Africa. In India, though natural isolation of none of these viruses was reported from the mosquito, experimental studies have shown vector competence to DEN and CHIK viruses. Despite wide prevalence in India, their potential in transmitting viruses of public health importance viz., Japanese encephalitis (JEV), West Nile (WNV), Chandipura (CHPV), Chittoor (CHITV) etc., has never been investigated. The objective of the present study is to determine the vector potential of the mosquito to these viruses. Methods: Mosquitoes were infected by intra-thoracic inoculation as well as by oral feeding, and growth kinetics was determined. Virus dissemination to organs was investigated by determining virus in the harvested organs on specified days’ post infection (PI). Vector competence was determined by detecting the virus in saliva. Results: Intra thoracic inoculation has shown vector competence of the mosquito to JEV, WNV, CHIV and CHPV. However, using the oral route of infection, replication was observed with only WNV, JEV and CHITV. High degree of WNV replication (6.7log TCID50/ml) with rapid dissemination to wings, legs and salivary glands was seen from 5th day PI onwards. WNV was detected in saliva with a titer of 0.7log10 TCID50/ml on 5th day PI. JEV and CHITV replicated in the mosquito yielding 3log and 4log10 TCID50/ml on 5th and 10th day PI respectively, but virus was not detected in saliva till 15th day PI. Interpretation & conclusion: From the results it is difficult to indict the mosquito as a vector of the viruses studied. However, presence of WNV in saliva of the mosquito shows its potential as a bridge vector and poses a concern especially when virulent WNV strains are circulating in the country.

Chandipura virus (CHPV) has emerged as a significant cause of acute encephalitis in India, especially affecting pediatric populations. This study aimed to analyze the temporal trends, clinical characteristics, and epidemiological features of CHPV infections reported in outbreaks across India. A comprehensive literature search on CHPV cases was conducted using Google Scholar, PubMed, and the Cochrane Library. Original research studies on laboratory-confirmed clinical cases of CHPV infections, available as full-text articles, were included. Data on outbreak characteristics, clinical presentations, diagnostics, and epidemiological factors were extracted and analyzed. Six studies met the inclusion criteria. The review revealed a geographical expansion of CHPV outbreaks across India over time, with a consistent seasonal pattern (May to September) coinciding with the monsoon season. CHPV predominantly affects children under 15 years of age, presenting with rapid-onset encephalitis characterized by high fever, altered consciousness, and seizures. Case fatality rates were alarmingly high, ranging from 28.6% to 78.3% within the first 48 hours of symptom onset. Diagnostic approaches evolved over the study period, with increasing use of molecular techniques. Entomological investigations consistently identified sandflies, particularly Phlebotomus argentipes, as potential vectors, though their precise role in transmission remains to be fully elucidated. CHPV is an emerging public health threat, especially for children under 15 years. Early diagnosis is crucial, as CHPV is associated with high mortality within the first 24-48 hours of symptom onset. Challenges include limited long-term follow-up data, potential underreporting of mild cases, and gaps in understanding transmission dynamics., Competing Interests: Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Kanabar et al.)

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly., Competing Interests: Declaration of interests The University of Colorado-Boulder currently holds the PCT patents for the use of gectosome technology, with X.Z., Q.X., Z.L., and X.L. as inventors. X.L. is a co-founder and member of the Scientific Advisory Board of OnKure Inc., which has no relationships or competing interests to this study. X.L. owns equity in OnKure Inc. X.L. is also a co-founder and interim president of Vesicle Therapeutics, Inc, a startup company that licensed gectosome technology from the University of Colorado-Boulder. X, L., X.Z., and Q.X. own equity in Vesicle Therapeutics, Inc., (Copyright © 2024. Published by Elsevier Inc.)

Background: Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1β, in human microglial cells during CHPV infection. Methods: To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1β in this study. Results: The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. Conclusions: This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells. [ABSTRACT FROM AUTHOR]

Chandipura virus (CHPV) is a tropical pathogen, suggesting its involvement in childhood encephalitis syndrome in India. No reports are available in adult human beings for its pathogenicity. Similarly, in adult mice, the virus does not develop pathogenesis by parenteral route except for intracranial route of infection. The virus is remarkably nonpathogenic to adult immunocompromised nude mice. In vitro in tissue culture, the CHPV infects and kills many types of cells. All of these properties could qualify the CHPV to be a candidate virus for tumor therapy. To prove this, an experimentally induced tumor in a mouse was infected with live CHPV. The results showed that intra-tumoral injection reduced the volume of tumor and increased the longevity of the mice. The study concludes that the CHPV may be a safe tumor therapy virus. More precisely, the discovery of CHPV protein with oncolytic potential may lead to the development of novel drugs/therapeutics. [ABSTRACT FROM AUTHOR]

Chandipura virus (CHPV) is an emerging tropical pathogen in India. The virus has been reported to be associated with an acute encephalitis syndrome in young children with a case fatality rate of 55% to 75%. Clinical management with symptomatic treatment is the only option available to treat infected patients. No vaccines are available for prophylaxis. In light of the prophylactic limitations, antiviral therapy would play an important role in control of CHPV infection. In the present study, ribavirin (RBV), an antiviral drug widely accepted for human use and having an antiviral effect on many RNA and DNA viruses, was tested against the CHPV. A screening assay that scores for the virus‐mediated plaque formation in the cultured Vero cells was used. RBV exhibited 50% inhibitory concentration (IC50) at 89.84 ± 1.8 µM. The drug was very effective when the cells were treated either within an hour postinfection or 4 to 6 hours before infection. The plaque morphology was different in RBV treated cells; the plaques were smaller in size as compared with the plaques in the virus infected cells. The study reports the antiviral activity of RBV against CHPV, and hence, suggests the possible utility of RBV in CHPV infected patients to mitigate the disease. A further clinical trial is needed before introducing the drug for human use against CHPV infection. Highlights: The 50% inhibitory ribavirin concentration (IC50) for the Chandipura virus is 89.84±1.8 µMRibavirin inhibits replication of Chandipura virus when the Vero cells treated with drug 4 to 6 hr before infection or one hr after infection.In Vero cells, ribavirin acts late replication cycle of Chandipura virus. [ABSTRACT FROM AUTHOR]

Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens, including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae, is a deadly human pathogen known to cause fatal encephalitis, especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and, thus, is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum, we have demonstrated that CHPV neutralization is C3, C4, and C1q dependent and independent of factor B, suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches, we showed (i) a direct association of C1q to CHPV, (ii) deposition of complement proteins C3b, C4b, and C1q on CHPV, and (iii) virus aggregation. Depletion of C8, an important component of the poreforming complex of complement, had no effect on CHPV, further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV. [ABSTRACT FROM AUTHOR]

Chandipura virus (CHPV) belongs to the family Rhabdoviridae and has a single-stranded RNA genome that causes encephalitis among children in India's tropical states. Activation of the antiviral immune response upon viral infection is important for the host's defense. In response to CHPV infection, the brain resident macrophages (microglial cells) control the pathogenic insults. The microRNAs (miRNAs) are 22 nts non-coding RNAs that serve as delicate regulators of their target genes at the post-transcriptional level. In this study, we explored miR-155 mediated antiviral response in CHPV infected human microglial cells. The gene and protein expression patterns were studied through quantitative real-time PCR (qPCR) and immunoblotting, respectively. Additionally, miRNA target validation was done by overexpression and knockdown of miR-155. We observed an increased expression of miR-155 in CHPV infected human microglial cells. The upregulated miR-155 suppresses the Suppressor of Cytokine Signalling 1 (SOCS1). Reduced SOCS1, in turn, led to enhanced phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and induction of Interferon-β (IFN-β), which promoted the expression of IFN-stimulated gene 54 (ISG54) and IFN-stimulated gene 56 (ISG56). In this study, miR-155 positively modulated the cellular antiviral response by enhancing type I IFN signalling through inhibition of SOCS1 in CHPV infected microglial cells. [ABSTRACT FROM AUTHOR]

Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.

Background & objectives: Chandipura virus (CHPV) is an emerging pathogenic rhabdovirus with a high case fatality rate. There are no reports of a minigenome system for CHPV, which could help its study without having to use the infectious agent. This study was, therefore, undertaken for the establishment of T7 polymerase-driven minigenome system for CHPV. Methods: The minigenome rescue system for CHPV consists of three helper plasmids expressing the nucleocapsid protein (N), phosphoprotein (P) and large protein (L) based on a recombinant vaccinia virus expressing bacteriophage T7 polymerase (vTF7-3). The minigenome construct is composed of a reporter gene, flanked by the non-coding regions of CHPV. Two minigenomes were constructed in an antigenome or complimentary sense, expressing luciferase or green fluorescent protein (GFP). The minigenome system was evaluated by co-transfection of the minigenome construct and three helper plasmids into CV-1 cells and analysis of the reporter gene activity. Results: All the helper proteins were expressed from the helper plasmids confirmed by Western blotting. Expression of reporter genes was observed from both the GFP and luciferase-based minigenomes. Green fluorescence could be visualized directly in live CV-1 cells. Luciferase activity was found to be significantly different from control. Interpretation & conclusions: The results showed that the helper plasmids provided all the necessary viral structural proteins required for the production of minigenome mRNA template, which in turn could rescue the expression of reporter genes. Thus, these minigenomes can be applied to mimic the manifestation of CHPV life cycle. [ABSTRACT FROM AUTHOR]

Background: Interaction between immune system and Chandipura virus (CHPV) during different stages of its life cycle remain poorly understood. The exact route of virus entry into the blood and CNS invasion has not been clearly defined. The present study was undertaken to assess the population in PBMC that supports the growth of virus and to detect active virus replication in PBMC as well as its subsets.Methods: PBMC subsets viz.: CD3(+), CD14(+), CD19(+), CD56(+)cells were separated and infected with CHPV. The infected cells were then assessed for transcription (N gene primer) and replication (NP gene primer) of CHPV by PCR. The supernatant collected from infected cells were titrated in Baby Hamster Kidney (BHK) cells to assess virus release. The cytokine and chemokine expression was quantified by flow cytometry.Results: Amplification of N and NP gene was detected in CD14(+) (monocyte) and CD19(+) (B cell), significant increase in virus titre was also observed in these subsets. It was observed that, although the levels of IL-6 and IL-10 were elevated in CD14(+) cells as compared to CD19(+)cells, the differences were not significant. However the levels of TNFα and IL-8 were significantly elevated in CD14(+) cells than in CD19(+)cells. The levels of chemokine (CXCL9, CCL5, CCL2, CXCL10) were significantly elevated in CHPV infected PBMC as compared to uninfected cells. CCL2 and CXCL9 were significantly increased in CHPV infected CD14(+)cells as compared to CD19(+) cells.Conclusion: CD14(+)and CD19(+)cells support active replication of CHPV. High viral load was detected in CD14(+) cells infected with CHPV hence it might be the primary target cells for active replication of CHPV. An elevated levels of cytokines and chemokines observed in CD14(+) cells may help in predicting the pathogenecity of CHPV and possible entry into the central nervous system. [ABSTRACT FROM AUTHOR]

Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae) garnered global attention as an emerging neurotropic pathogen inflicting high mortality in children within 24 h of commencement of symptoms. The 2003-2004 outbreaks in Central India witnessed case fatality rates ranging from 56-75 per cent in Andhra Pradesh and Gujarat with typical encephalitic symptoms. Due to the acute sickness and rapid deterioration, the precise mechanism of action of the virus is still unknown. Recent studies have shown increased expression of CHPV phosphoprotein upto 6 h post infection (PI) demonstrating CHPV replication in neuronal cells and the rapid destruction of the cells by apoptosis shed light on the probable mechanism of rapid death in children. Phlebotomine sandflies are implicated as vectors due to their predominance in endemic areas, repeated virus isolations and their ability to transmit the virus by transovarial and venereal routes. Significant contributions have been made in the development of diagnostics and prophylactics, vaccines and antivirals. Two candidate vaccines, viz. a recombinant vaccine and a killed vaccine and siRNAs targeting P and M proteins have been developed and are awaiting clinical trials. Rhabdomyosarcoma and Phlebotomus papatasi cell lines as well as embryonated chicken eggs have been found useful in virus isolation and propagation. Despite these advancements, CHPV has been a major concern in Central India and warrants immediate attention from virologists, neurologists, paediatricians and the government for containing the virus. [ABSTRACT FROM AUTHOR]

An encephalitis outbreak in 2003 in children from India was attributed to Chandipura virus. Sequence analyses of G, N, and P genes showed 95.6%–97.6% nucleotide identity with the 1965 isolate (G gene, 7–11 amino acid changes); N and P genes were highly conserved.

Chandipura virus ( CHPV; genus Vesiculovirus, family Rhabdoviridae) induces neuronal death through the Fas-mediated extrinsic apoptosis pathway. What propels this apoptosis remains unclear, although oxysterols have been reported to be key players in neurodegeneration. In our study of CHPV-infected brain samples, we observed over-expression of genes such as apolipoprotein E, Cyp46a1, Srebf-1 and Nsdhl. This backs up the hypothesis that CHPV replication demands cholesterol that is supplied by apolipoprotein E through low density lipid receptors, lipid metabolism being pivotal for viral replication. We were able to illustrate this with over-expression of low density lipid receptors in CHPV-infected neurons. An upsurge of cholesterol concentration has been observed in neurons, triggering the expression of Cyp46a1 enzyme and culminating into the conversion of cholesterol to 24( S)-hydroxycholesterol. Increased 24( S)-hydroxycholesterol concentration is toxic to neurons, propelling neuronal apoptosis through the Fas-mediated extrinsic apoptosis pathway. For the first time, perturbation of cholesterol homeostasis in brain is shown to be utilized by the viruses for both maturation and the release of its matured virions outside the cells for continuous neuropathogenesis. [ABSTRACT FROM AUTHOR]

The rhabdovirus matrix (M) protein is a multifunctional virion protein that plays major role in virus assembly and budding, virus-induced inhibition of host gene expression and cytopathic effects observed in infected cells. The myriad roles played by this protein in the virus biology make it a critical player in viral pathogenesis. Therefore, discerning the interactions of this protein with host can greatly facilitate our understanding of virus infections, ultimately leading to both improved therapeutics and insight into cellular processes. Chandipura virus (CHPV; Family Rhabdoviridae , Genus Vesiculovirus ) is an emerging rhabdovirus responsible for several outbreaks of fatal encephalitis among children in India. The present study aims to screen the human fetal brain cDNA library for interactors of CHPV M protein using yeast two-hybrid system. Ten host protein interactors were identified, three of which were further validated by affinity pull down and protein interaction ELISA. The study identified novel human host interactors for CHPV which concurred with previously described associations in other human viruses. [ABSTRACT FROM AUTHOR]

Highlights: [•] Molecular mechanisms of CNS invasion by several neurotropic viruses are compared. [•] Modes by which CHPV could surpass BBB to invade CNS are speculated. [•] Potential CHPV-host protein interactions for experimental research are identified. [ABSTRACT FROM AUTHOR]

Background & objectives: An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra state, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV) were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission. Methods: Twenty nine pools of Sergentomyia spp. comprising 625 specimens were processed for virus isolation in Vero E6 cell line. Diagnostic RT-PCR targeting N-gene was carried out with the sample that showed cytopathic effects (CPE). The PCR product was sequenced, analysed and the sequences were deposited in Genbank database. Results: CPE in Vero E6 cell line infected with three pools was detected at 48 h post infection. However, virus could be isolated only from one pool. RT-PCR studies demonstrated 527 nucleotide product that confirmed the agent as CHPV. Sequence analysis of the new isolate showed difference in 10-12 nucleotides in comparison to earlier isolates. Interpretation & conclusions: This is perhaps the first isolation of CHPV from Sergentomyia spp. in India and virus isolation during transmission season suggests their probable role in CHPV transmission. Further studies need to be done to confirm the precise role of Sargentomyia spp. in CHPV transmission. [ABSTRACT FROM AUTHOR]