Persistence of West Nile Virus circulation in North-East region of India: A prospective facility-based study spanning over a decade.

BACKGROUND OBJECTIVES: To understand the distribution and presence of West Nile (WN) among the acute encephalitis syndrome (AES) patients, a prospective facility-based study was conducted over 13 years (2007-2019). METHODS: During the period, 8957 patients hospitalized with AES in northeastern India were tested for the evidence of WN virus infection by IgM ELISA followed by Plaque reduction neutralization test (PRNT). RESULTS: Of the samples tested 324 (3.62%) were found positive for WN IgM antibodies and 73 paired sera showed a fourfold rise in neutralizing antibody titre by PRNT. The study establishes WN as a noteworthy cause of encephalitis in an erstwhile endemic area for Japanese encephalitis (JE). INTERPRETATION CONCLUSION: Therefore, it is important that WN is recognized as an important acute encephalitis syndrome (AES) causing aetiology in this region and the National centre for vector borne disease control (NCVBDC) guidelines for detection of AES may be modified accordingly. Thus, inclusion of WN in routine diagnosis, along with establishment of an integrative surveillance network with one-health approach will be important.

Global emergence of West Nile virus: Threat & preparedness in special perspective to India.

West Nile virus (WNV) is a mosquito-borne single-stranded RNA neurotropic virus within the family Flaviviridae. The virus was first reported in the West Nile province of Uganda in 1937. Since then, sporadic cases have been reported until the last two decades when it has emerged as a threat to public health. The emergence of WNV with more severity in recent times is intriguing. Considering this phenomenon, the WNV-affected areas of the world were distinguished as old versus new in a depicted world map. The present review showcases the historical and epidemiological perspectives of the virus, genetic diversity of prevailing lineages and clinical spectrum associated with its infection. Emergence of the virus has been discussed in special context to India because of co-circulation of different WNV lineages/strains along with other flaviviruses. Recent laboratory diagnostics, vaccine development and clinical management associated with WNV infection have also been discussed. Further, the research gaps, especially in context to India have been highlighted that may have a pivotal role in combating the spread of WNV.

Impact of the Season on Total Polyphenol and Antioxidant Properties of Tea Cultivars of Industrial Importance in Northeast India.

Tocklai vegetative (TV) cultivars are extensively planted in the tea-growing regions of Northeast India. The present investigation explores the impact of season on the total polyphenol (TP) content and the antioxidant activity of thirty-one TV cultivars (TV1-TV31) and four other commercially popular cultivars, namely, Betjan, Kharijan, S.3A/3, and T.3E/3. The TP content of the cultivars was observed to be highest in the monsoon season, with values ranging from 230.57 to 283.53 mg g-1. In the pre-monsoon season and autumn, the TP content ranged from 197.87 to 256.77 mg g-1 and from 169.97 to 223.50 mg g-1, respectively. Antioxidant activity was measured through DPPH, ABTS, FRAP, and lipid peroxidation inhibition assays. The cultivars showed the highest antioxidant activity in the monsoon in tandem with TP content. A bivariate correlation indicated a highly significant (p ≤ 0.01) positive correlation of antioxidant activity with TP content (R2 = 0.83-0.96).

Black tea bioactives as inhibitors of multiple targets of SARS-CoV-2 (3CLpro, PLpro and RdRp): a virtual screening and molecular dynamic simulation study.

The global pandemic due to the novel Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has taken more than a million lives. Lack of definitive vaccine/drugs against this highly contagious virus has accelerated exploratory research on novel natural and synthetic inhibitors. Tea is a rich source of bioactives and known to have antiviral properties. In this study, an in silico strategy involving ADMET property screening, receptor-ligand docking and molecular dynamic (MD) simulation was employed to screen potential tea bio-active inhibitors against three selected targets (RdRp, 3CLpro and PLpro) of SARS-CoV-2. Among the 70 tea bioactives screened, theaflavin 3,3'-di-gallate (TF3), Procyanidin B2 and Theaflavin 3-gallate (TF2a) exhibited highest binding affinities towards RdRp, 3CLpro/Mpro and PLpro targets of SARS-CoV-2 with low docking scores of -14.92, -11.68 and -10.90 kcal/mol, respectively. All of them showed a substantial number of hydrogen bonds along with other interactions in and around the active sites. Interestingly, the top bioactives in our study showed higher binding affinities compared with known antiviral drugs. Further, the top protein-ligand complexes showed less conformational changes during binding when subjected to MD simulation for 100 nanoseconds. The MMPBSA results revealed that RdRp-TF3, 3CLpro-Procyanidin B2 and PLpro-TF2a complexes were stable with binding free energies of -93.59 ± 43.97, -139.78 ± 16.51 and -96.88 ± 25.39 kJ/mol, respectively. Our results suggest that theaflavin 3,3'-digallate, Theaflavin 3-gallate and Procyanidin B2 found in black tea have the potential to act as inhibitors for selected targets of SARS-CoV-2 and can be considered as drug candidates in future studies against COVID-19.

The Biological Effects of Polystyrene Nanoplastics on Human Peripheral Blood Lymphocytes.

Environmental exposure to microplastics (MPs) and nanoplastics (NPs) is an increasing concern from human health perspectives. Little information on the genotoxic and cytotoxic potential of NP particles in human cells is available. We aimed to assess the cytotoxic and genotoxic potential of polystyrene nanoplastics (PSNPs) at different concentrations (2000µg/mL, 1000µg/mL, and 500µg/mL) by using chromosomal aberration (CA) and cytokinesis-block micronucleus assays (CBMN) on human peripheral lymphocytes. Dose-dependent hemolytic activity and cell viability were observed against the PSNPs exposure. Increased chromosomal aberrations, such as chromosomal breaks and dicentric chromosomes, and an increase in nucleoplasmic bridge (NBP) formation and nuclear budding (NBUD) were observed. The frequency of mitotic index (MI) decreased significantly in the PSNP-exposed groups from lower to higher concentrations. A significant increase in micronuclei (MN) formation and cytostasis% and a dose-dependent reduction in nuclear division index (NDI) in PSNP-exposed groups indicated oxidative stress-mediated cytotoxicity, DNA damage, and genomic instabilities due to PSNP exposure in human lymphocyte cells. This study highlights the importance of understanding the toxic mechanisms and associated chronic and acute health effects on humans due to exposure to this pervasive environmental pollutant.

Tea Bioactive Modulate Innate Immunity: In Perception to COVID-19 Pandemic.

Innate immunity impairment led to disruption in cascade of signaling pathways upregulating pro-inflammatory cytokines, diminish interferons, depleted natural killer cells and activate reactive oxygen species production. These conditions severely affected body's ability to fight against infectious diseases and also plays a pivotal role in disease progression. Here, in emphasis is on nutritional immunity for regulating effective innate immune response for combating against infectious diseases like novel coronavirus disease (COVID 19). Drawing from discoveries on in-vitro experiments, animal models and human trials, tea polyphenols, micronutrients, and vitamins has the potential to modulate and enhance innate immune response. This article provides a comprehensive review on tea (Camellia sinensis L) infusion (a hot water extract of dried processed tea leaves prepared from young shoots of tea plant) as an innate immunity modulator. Tea infusion is rich in polyphenols; epigallocatechin gallate (EGCG) and theaflavin (TF), major green and black tea polyphenols, respectively. Studies showed their immunomodulatory competence. Tea infusions are also rich in alkaloids; caffeine and its intermediates, theophylline and theobromine, which have anti-inflammatory properties. Tea plant being an acidophilic perennial crop can accumulate different micronutrients, viz., copper (Cu), iron (Fe), manganese (Mn), selenium (Se), and zinc (Zn) from growing medium, i.e., from soil, which led to their considerable presence in tea infusion. Micronutrients are integral part of innate immune response. Overall, this review presents tea infusion as an important source of nutritional immunity which can enhance innate immune response in order to mitigate the unprecedented COVID-19 pandemic.

Theaflavins, polyphenols of black tea, inhibit entry of hepatitis C virus in cell culture.

The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3'-monogallate (TF2), and theaflavin-3-3'-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.

Pharmacodynamics of aminoglycosides and tetracycline derivatives against Japanese encephalitis virus.

OBJECTIVE: To explore the antiviral activity of antibiotic compounds, mainly aminoglycosides and tetracyclines against Japanese encephalitis virus (JEV) induced infection in vitro. METHODS: Antiviral activity were evaluated against JEV using cytopathic effect inhibition assay, virus yield reduction assay, caspase 3 level, extracellular viral detection by antigen capture ELISA and viral RNA levels. RESULTS: JEV induced cytopathic effect along with reduction of viral progeny plaque formation indicated antiviral potential of the compounds suggesting that antibiotics had broad spectrum activity. Doxycycline and kanamycin administration in dose dependent manner declined viral RNA replication. CONCLUSIONS: The present study shows kanamycin and doxycycline can affect virion structure and alter replication causing inhibition of JEV induced pathogenesis in vitro.

Comparison of ß-Propiolactone and Formalin Inactivation on Antigenicity and Immune Response of West Nile Virus.

West Nile Virus (WNV) is a pathogenic arbovirus that belongs to genus Flavivirus under family Flaviviridae. Till now there are no approved vaccines against WNV for human use. In this study, the effect of two alkylating agents, formaldehyde and ß-PL, generally used for inactivated vaccine preparation, was assessed on the basis of antigenic and immunogenic potential of the inactivated WNV. Lineage 5 WNV isolates were inactivated by both formalin and ß-PL treatments. Inactivation was confirmed by repeated passage in BHK-21 cell line and infant mice. Viruses inactivated by both the treatments showed higher antigenicity. Immune response in mice model showed serum anti-WNV antibody titre was moderately higher in formalin inactivated antigen compared to ß-PL inactivated antigen. However, no significant differences were observed in neutralization antibody titre. In conclusion, we can state that both formaldehyde and ß-PL inactivation processes were found to be equally efficient for inactivation of WNV. However, they need to be compared with other inactivating agents along with study on cell mediated immune response.

Characterization of Japanese encephalitis virus (JEV) genotype III clinical isolates in northeast India.

BACKGROUND: Japanese encephalitis virus (JEV) is one of the major etiological agents responsible for causing large numbers of acute encephalitis syndrome (AES) cases in the northeastern region of India. This study was carried out to establish and characterize the circulating strain of JEV in the region in order to understand the disease epidemiology. METHODS: Virus isolation was attempted from 121 patients that presented with AES. Phylogenetic analysis was done using the Kimura-2-Parameter model based on envelope and pre-membrane gene sequence. A pathogenecity study was done in the Swiss albino mice model and assessed by Kaplan-Meier survival analysis. RESULTS: The phylogenetic analysis of the two JEV isolates obtained placed them within genotype (G)III, where they form a subclade within the Vellore group of Indian JEV strains. Neutralization assays suggested similarity between the study isolates and prototype Vellore JEV strain P20778. Pathogenesis in mice suggested that the circulating GIII JEV strains were neuroinvasive. CONCLUSIONS: This study showed that a pathogenic GIII JEV strain was circulating in the northeastern region of India. This finding is important as it is contrary to the belief that GI is gradually replacing GIII as the dominant genotype in Asia. GenBank accession numbers: HQ270470, JQ434468, HQ246155, JX018170.

Characterization of West Nile virus (WNV) isolates from Assam, India: insights into the circulating WNV in northeastern India.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes subclinical symptoms, febrile illness with possible kidney infarction and encephalitis. Since WNV was first serologically detected in Assam during 2006, it has become recognized as an important etiological agent that causes acute encephalitis syndrome (AES) in addition to endemic Japanese encephalitis virus (JEV). Therefore, isolating and characterizing the currently circulating strain of WNV is important. The virus was isolated from the cerebrospinal fluid (CSF) of two patients that presented with AES. The genotyping of the isolates HQ246154 (WNIRGC07) and JQ037832 (WNIRTC08) based on the partial sequencing of 921 nucleotides (C-prM-E) of the genome placed them within lineage 5 along with other Indian strains isolated prior to 1982, but the present circulating virus formed a distinct subclade. The derived amino acid sequence alignment indicated substitution in A81T and A84P of the capsid region in HQ246154. A cross-neutralization assay suggested substantial antigenic variation between isolates. The pathogenesis in mice that suggested the circulating WNV was neuroinvasive and comparatively more pathogenic than previous strains from India.

Genetic diversity and gene structure of mitochondrial region of Anopheles minimus (Diptera: Culicidae) - major malaria vector of North east India.

OBJECTIVE: To depict mitochondrial genetic variation for the first time among Anopheles minimus (An.minimus) (Diptera: Culicidae) species from two malaria endemic states of NE India. METHODS: Phylogeographic analysis was carried at 9 out of 12 sites of An.minimus confirmed malaria endemic places. RESULTS: All sequences were Adenine-Thymine rich regions. Transitions were observed in 6 sequences where 5 mutations were synonymous substitutions and in 1 case non synonymous mutation was observed. Three distinct clusters of haplotypes were generated. Haplotype diversity and low nucleotide diversity were studied. Overall negative values obtained from Tajima's D test and Fu'sFS test indicate a recent genetic population expansion. Network analysis has explained sequence diversity that was also shown by mutations in 6 sequences. CONCLUSIONS: High genetic diversity observed within the populations of An.minimus species has several possible implications for vector control in the region.

Immunogenic potential and protective efficacy of formalin inactivated circulating Indian strain of West Nile virus.

OBJECTIVE: To assess the best suitable condition for virus inactivation, and to study the immunogenic potential and protective efficacy of a circulating West Nile virus (WNV) strain in Assam. METHODS: Bulk preparation of circulating WNV: WNIRGC07 (GeneBank ID: HQ246154), was undertaken in a bioreactor using cytodex-1. Virus Inactivation was done in three different conditions; 22 °C, 4 °C and room temperature. The virus preparations were evaluated for antigenicity by ELISA and toxicity by cell proliferation kit. Virus efficacy was done in-vivo on swiss albino mice against standard Indian WNV and Japanese encephalitis virus (JEV) strain. Humoral and cell mediated immune response was evaluated in mice sera by ELISA and neutralization assay. RESULTS: Inactivation at 22 °C was found to be more suitable in terms of less toxicity and high antigenicity. The same was selected to study the immune response and efficacy in mice. It induced neutralizing antibody titre of 1: 625 and high IgG response. In vivo experiment showed 100% protective efficacy against WNV and 20.8% cross protective efficacy against JEV. Further assessment of cellular immunity through immunized mice revealed augmentation of high levels of pro-inflammatory cytokines and moderate levels of anti-cytokines indicating a mixed balance of Th1 and Th2 response. CONCLUSIONS: Findings suggest that formalin inactivated Indian WNV strain has a good immunogenic potential. This is the first study on assessment of immunogenic potential of a lineage 5 strain of WNV. Our study reveals that it would be a promising and effective candidate for vaccine studies which warrants further evaluation.