Genomic Characterization of Mosquito Isolates of Chikungunya Virus (Outbreak Strains 2022) Using Next-Generation Sequencing.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

ArVirInd-a database of arboviral antigenic proteins from the Indian subcontinent.

Background: Studies on antigenic proteins for arboviruses are important for providing diagnostics and vaccine development. India and its neighboring countries have a huge burden of arboviral diseases. Data mining for country-specific sequences from existing bioinformatics databases is cumbersome and time-consuming. This necessitated the development of a database of antigenic proteins from arboviruses isolated from the countries of the Indian subcontinent. Methods: Arboviral antigenic protein sequences were obtained from the NCBI and other databases. In silico antigenic characterization was performed (Epitope predictions) and data was incorporated into the database. The front end was designed and developed using HTML, CSS, and PHP. For the backend of the database, we have used MySQL. Results: A database, named ArVirInd, is created as a repository of information on curated antigenic proteins. This enlists sequences by country and year of outbreak or origin of the viral strain. For each entry, antigenic information is provided along with functional sites, etc. Researchers can search this database by virus/protein name, country, and year of collection (or in combination) as well as peptide search for epitopes. It is available publicly via the Internet at http://www.arvirind.co.in. ArVirInd will be useful in the study of immune informatics, diagnostics, and vaccinology for arboviruses.

Epidemiological findings for the first and second waves of COVID-19 pandemic in Maharashtra, India.

India is one of the worst affected countries during the COVID-19 pandemic. We carried out comparative analyses of the COVID-19 situation in the Maharashtra state, India for the first and second waves. Epidemiological and demographics data were obtained from open sources and the Government of Maharashtra. Mathematical modeling and analyses were conducted to estimate the epidemiological parameters like basic reproduction number (R0) for the first wave at different times. The districts with a higher percentage of the urban population recorded a higher attack rate during the first wave. However, during the second wave, the rural population was more affected. The effective reproduction number (Re) was estimated for the second wave at different times. The second wave affected more individuals than the first wave due to various factors such as strictness of restrictions or the lack of it and the emergence of new strains.

Design of a multi-epitope peptide vaccine candidate against chandipura virus: an immuno-informatics study.

Chandipura virus (CHPV) is an emerging pathogen responsible for acute encephalitic syndrome (AES) in pediatric population in India. Several outbreaks of CHPV have been reported from different states of India since the year 2003. At present there is no vaccine or therapeutic measures available to curtail the disease. In this study, we have identified both T-cell and B-cell epitopes of different antigenic proteins of CHPV like Nucleoprotein (N), Phosphoprotein (P) and Matrix protein (M) along with the immuno-dominant glycoprotein (G) and conducted in silico characterization for the same. The idea is to design a multi-epitope peptide construct using the epitopes, which were found to be non-toxic, non-allergenic and possessing high immunogenicity. The final multi-epitope construct named as: MEC-CHPV, comprised of ß-defensin adjuvant at N-terminal for enhancement of immunogenicity followed by fourteen B-cell epitopes, four Helper T-cell epitopes and six Cytotoxic T-cell epitopes. The characterization of designed construct was carried out in terms of physicochemical parameters, antigenicity and allergenicity. The 3D structure prediction was performed. Molecular docking and molecular-dynamics simulation of MEC-CHPV with Toll like receptors (TLR-3 and TLR-8) showed stable interactions. In silico cloning of MEC-CHPV in pET30a(+) expression vector was also conducted using codon optimization. The in silico immune-simulation indicated a typical immune response against MEC-CHPV when used as a potential vaccine. This study provides a cost-effective and time-saving way to design a peptide vaccine candidate against CHPV using immuno-informatics approach. Development of the MEC-CHPV construct may pave the way for future laboratory experiments.Communicated by Ramaswamy H. Sarma.

Cyclophilin A: a possible host modulator in Chandipura virus infection.

Chandipura virus (CHPV), belonging to the genus Vesiculovirus of the family Rhabdoviridae, has been identified as one of the causes of pediatric encephalitis in India. Currently, neither vaccines nor therapeutic drugs are available against this agent. Considering that the disease progresses very fast with a high mortality rate, working towards the development of potential therapeutics against it will have a public health impact. Although the use of viral inhibitors as antiviral agents is the most common way to curb virus replication, the mutation-prone nature of viruses results in the development of resistance to antiviral agents. The recent development of proteomic platforms for analysis of purified viral agents has allowed certain upregulated host proteins that are involved in the morphogenesis and replication of viruses to be identified. Thus, the alternative approach of inhibition of host proteins involved in the regulation of virus replication could be explored for their therapeutic effectiveness. In the current study, we have evaluated the effect of inhibition of cyclophilin A (CypA), an immunophilin with peptidyl-prolyl cis/trans-isomerase activity, on the replication of CHPV. Treatment with cyclosporin A, used in vitro for the inhibition of CypA, resulted in a 3-log reduction in CHPV titer and an undetectable level of CypA in comparison to an untreated control. An in silico analysis of the interaction of the CHPV nucleoprotein with the human CypA protein showed stable interaction in molecular docking and molecular dynamics simulations. Overall, the results of this study suggest a possible role of CypA in facilitating CHPV replication, thus making it one of the potential host factors to be explored in future antiviral studies.

Differential susceptibility & replication potential of Vero E6, BHK-21, RD, A-549, C6/36 cells & Aedes aegypti mosquitoes to three strains of chikungunya virus.

Background & objectives: Chikungunya virus (CHIKV), a mosquito-borne arthritogenic virus causes infections ranging from febrile illness to debilitating polyarthralgia in humans. Re-emergence of the virus has affected millions of people in Africa and Asia since 2004. During the outbreak, a new lineage of the virus has evolved as an adaptation for enhanced replication and transmission by Aedes albopictus mosquito. A study was designed to compare the susceptibility of four vertebrate cell lines, namely Vero E6 (African green monkey kidney), BHK-21 (Baby hamster kidney), RD (human rhabdomyosarcoma), A-549 (human alveolar basal epithelial cell) and C6/36 (Ae. albopictus) to Asian genotype and two lineages of East, Central and South African (E1:A226 and E1:A226V) of CHIKV. Methods: One-step growth kinetics of different CHIKV strains was carried out in the above five cell lines to determine the growth kinetics and virus yield. Virus titre was determined by 50 per cent tissue culture infectious dose assay and titres were calculated by the Reed and Muench formula. Growth and virus yield of the three strains in Ae. aegypti mosquitoes was studied by intrathoracic inoculation and virus titration in Vero E6 cell line. Results: Virus titration showed Vero E6, C6/36 and BHK-21 cell lines are high virus yielding with all the three lineages while RD and A-549 yielded low virus titres. C6/36 cell line was the most sensitive and yielded the maximum titre. Ae. aegypti mosquitoes, when inoculated with high titre virus, yielded an almost equal growth with the three strains while rapid growth of E1:A226V and Asian strain was observed with 1 log virus. Interpretation & conclusions: C6/36 cell line was found to be the most sensitive and high yielding for CHIKV irrespective of lineages while Vero E6 and BHK-21 cell lines yielded high titres and may find application for vaccine/diagnostic development. Infection of Ae. aegypti mosquitoes with the three CHIKV strains gave almost identical pattern of growth.

Assessment of NS1 protein as an early diagnostic marker for Kyasanur forest disease virus.

BACKGROUND & OBJECTIVES: Due to the emergence of Kyasanur forest disease (KFD) virus to new regions in India, there is an urgent need to develop an early diagnostic system, which is cost-effective and can be efficiently used with minimum paraphernalia. The non-structural-1 (NS1) protein is known to be an early diagnostic marker for flaviviruses. Furthermore, NS1 antigen capture ELISA kits developed using bacterially expressed dengue NS1 protein are commercially available. METHODS: Based on the data available on dengue virus, West Nile virus and other flaviviruses, bacterially expressed Kyasanur forest disease virus (KFDV) NS1 protein and polyclonal serum raised against the NS1 protein in mice and rabbit were used to develop an antigen capture ELISA for early diagnosis of the virus. The feasibility of this ELISA was further tested using in silico predictions. RESULTS: KFDV NS1 gene was cloned, expressed and confirmed by SDS-PAGE and western blotting. An antigen detection ELISA was standardized and sensitivity and specificity was tested with other flaviviruses. KFDV acute phase 43 samples were tested and only two were found to be positive for KFDV NS1 antigen. Superimposition of KFDV NS1 and TBEV NS1 revealed a root mean square distance (RMSD) of ~0.79 Å covering 1220 backbone atoms. This implies that the structures are very similar in terms of 3D fold. The identity of amino acid composition between these proteins was 73.4% and similarity was 92.9%, as revealed from the pairwise comparison. INTERPRETATION & CONCLUSION: The study points out that the half-life, expression and secretion levels of KFDV NS1 protein are not sufficient enough for its use as early diagnostic marker. The protein may have to be expressed in eukaryotic host to counter the lack of glycosylation in bacterial plasmid based expression of proteins. Hence, bacterially expressed KFDV NS1 protein may not be an ideal early diagnostic marker for the virus.

Identification of a conserved neutralizing epitope in the G-protein of Chandipura virus.

Chandipura virus (CHPV), associated with an encephalitic illness in humans, has caused multiple outbreaks with high mortality in central and western India in recent years. The present study compares surface glycoprotein (G-protein) from prototype and recent outbreak strains using in silico tools and in vitro experiments. In silico epitope predictions (B-cell and T-helper cell) for the sequences, 3D structure prediction and comparison of the G-proteins of the strains: I653514 (Year 1965), CIN0327 (Year 2003) and 148974 (Year 2014) revealed that the CHPV G-protein is stable and antigenic determinants are conserved. A monoclonal antibody developed against strain CIN0327 (named NAbC) was found to neutralize prototype I653514 as well as the currently circulating strain 148974. In silico antigen-antibody interaction studies using molecular docking of predicted structures of NAbC and G-proteins of various CHPV strains led to the identification of a conserved neutralizing epitope in the fusion domain of G-protein, which also contained a putative T-helper peptide. The identification of a conserved neutralizing epitope in domain IV (fusion domain amino acids 53 to 172) of CHPV G-protein is an important finding that may have the scope towards the development of protective targets against CHPV infection.

Bioinformatics characterization of envelope glycoprotein from Kyasanur Forest disease virus.

BACKGROUND & OBJECTIVES: Kyasanur Forest disease (KFD) is a febrile illness characterized by haemorrhages and caused by KFD virus (KFDV), which belongs to the Flaviviridae family. It is reported to be an endemic disease in Shimoga district of Karnataka State, India, especially in forested and adjoining areas. Several outbreaks have been reported in newer areas, which raised queries regarding the changing nature of structural proteins if any. The objective of the study was to investigate amino acid composition and antigenic variability if any, among the envelope glycoprotein (E-proteins) from old and new strains of KFDV. METHODS: Bioinformatic tools and techniques were used to predict B-cell epitopes and three-dimensional structures and to compare envelope glycoprotein (E-proteins) between the old strains of KFDV and those from emerging outbreaks till 2015. RESULTS: The strain from recent outbreak in Thirthahalli, Karnataka State (2014), was similar to the older strain of KFDV (99.2%). Although mutations existed in strains from 2015 in Kerala KFD sequences, these did not alter the epitopes. INTERPRETATION & CONCLUSIONS: The study revealed that though mutations existed, there were no drastic changes in the structure or antigenicity of the E-proteins from recent outbreaks. Hence, no correlation could be established between the mutations and detection in new geographical areas. It seems that KFDV must be present earlier also in many States and due to availability of testing system and alertness coming into notice now.

Rainfall and Chikungunya incidences in India during 2010-2014.

Changing climate scenario has resulted in recent emergence and re-emergence of various arboviral diseases including Chikungunya. This disease is caused by Chikungunya virus (CHIKV), which belongs to Togaviridae family of viruses and spread by Aedes mosquitoes. A resurgence of CHIKV and its rapid global spread has been observed since 2004. The disease reemerged in India in 2005, after a gap of 32 years, causing massive outbreaks in some states and circulating thereafter. In the present paper we analyze CHIKV incidence data from India (2010-2014) with a view to understand association with environmental parameters, if any. Data on country-wide occurrences of CHIKV cases were considered from the National Vector Borne Disease Control Board, India. Meteorological data for different climatic subdivisions of India were obtained and processed mathematically. State-wise association of number of cases with rainfall, if any, were studied by statistical analyses. We observe that prevailing temperature range was favorable for CHIKV propagation and the occurrences were modulated by average rainfall. Most affected states were West Bengal, Maharashtra and Karnataka. Overall for India, favorable climatic conditions have contributed to incidences of CHIKV during the study period. There is strong positive association between rainfall variations and occurrence of CHIKV cases.

Differences in Type I interferon response in human lung epithelial cells infected by highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses.

Influenza A virus infection induces type I interferons (IFNs α/ß) which activate host antiviral responses through a cascade of IFN signaling events. Herein, we compared highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses isolated from India, for their replication kinetics and ability to induce IFN-ß and interferon-stimulating genes (ISGs). The H5N1 virus showed a higher replication rate and induced less IFN-ß and ISGs compared to the H11N1 virus when grown in the human lung epithelial A549 cells, reflecting the generation of differential innate immune responses during infection by these viruses. The non-structural 1 (NS1) protein, a major IFN-antagonist, known to help the virus in evading host innate immune response was compared from both the strains using bioinformatics tools. Analyses revealed differences in the composition of the NS1 proteins from the two strains that may have an impact on the modulation of the innate immune response. Intriguingly, H5N1 virus attenuated IFN-ß response in a non-NS1 manner, suggesting the possible involvement of other viral proteins (PB2, PA, PB1/PB1-F2) of H5N1 in synergy with NS1. Preliminary analyses of the above proteins of the two strains by sequence comparison show differences in charged residues. The insight gained will be useful in designing experimental studies to elucidate a probable role of the polymerase protein(s) in association with NS1 in inhibiting the IFN signaling and understanding the molecular mechanism governing the difference.

Zika virus: Indian perspectives.

The emergence of Zika virus (ZiV), a mosquito borne Flavivirus like dengue (DEN) and chikungunya (CHIK), in Brazil in 2014 and its spread to various countries have led to a global health emergency. Aedes aegypti is the major vector for ZiV. Fast dissemination of this virus in different geographical areas posses a major threat especially to regions where the population lacks herd immunity against the ZiV and there is abundance of Aedes mosquitoes. In this review, we focus on current global scenario, epidemiology, biology, diagnostic challenges and remedial measures for ZiVconsidering the Indian perspective.

Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus.

Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection.

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes.

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.

Antigenic variability in Neuraminidase protein of Influenza A/H3N2 vaccine strains (1968 - 2009).

Antigenic drift and shift involving the surface proteins of Influenza virus gave rise to new strains that caused epidemics affecting millions of people worldwide over the last hundred years. Variations in the membrane proteins like Hemagglutinin (HA) and Neuraminidase (NA) necessitates new vaccine strains to be updated frequently and poses challenge to effective vaccine design. Though the HA protein, the primary target of the human immune system, has been well studied, reports on the antigenic variability in the other membrane protein NA are sparse. In this paper we investigate the molecular basis of antigenic drift in the NA protein of the Influenza A/H3N2 vaccine strains between 1968 and 2009 and proceed to establish correlation between antigenic drift and antigen-antibody interactions. Sequence alignments and phylogenetic analyses were carried out and the antigenic variability was evaluated in terms of antigenic distance. To study the effects of antigenic drift on the protein structures, 3D structure of NA from various strains were predicted. Also, rigid body docking protocol has been used to study the interactions between these NA proteins and antibody Mem5, a 1998 antibody.

Molecular basis of antigenic drift in Influenza A/H3N2 strains (1968-2007) in the light of antigenantibody interactions.

The emergence of new strains of Influenza virus have caused several pandemics over the last hundred years with the latest being the H1N1 Swine flu pandemic of 2009. The Hemagglutinin (HA) protein of the Influenza virus is the primary target of human immune system and is responsible for generation of protective antibodies in humans. Mutations in this protein results in change in antigenic regions (antigenic drift) which consequently leads to loss of immunity in hosts even in vaccinated population (herd immunity). This necessitates periodic changes in the Influenza vaccine composition. In this paper, we investigate the molecular basis of the reported loss of herd immunity in vaccinated population (vaccine component: Influenza A/X-31/1968 (H3N2)) which resulted in the outbreak due to strain Influenza A/Port Chalmers/1/1973 (H3N2). Also, the effects of antigenic drift in HA protein (H3N2 vaccine strains 1968-2007) on the 3D structures as well as interactions with BH151, a 1968 antibody, has been studied. Rigid body molecular docking protocol has been used to study the antigen-antibody interactions. We believe that the present study will help in better understanding of host-pathogen interactions at the molecular level.

Antigen-Antibody docking reveals the molecular basis for cross-reactivity of the 1918 and 2009 Influenza A/H1N1 pandemic viruses.

To understand the reported cross-reactivity of the 2009 H1N1 and the 1918 H1N1 pandemic viruses we docked the crystal structure of 2D1, an antibody derived from a survivor of the 1918 pandemic, to the structures of hemaglutinin (HA) of the 2009 strain and seasonal H1 vaccine strains. Our studies revealed that 2D1 binds to the 2009 HA at antigenic site 'Sa', with stabilizing contacts, similar to that in an available co-crystal structure of 2D1-1918 HA. However, 2D1 failed to bind to the known antigenic sites in the HAs of seasonal strains. Our study thus reveals the molecular basis for pre-existing immunity in elderly people to the 2009 pandemic virus.

Delineation of an epitope on domain I of Japanese encephalitis virus Envelope glycoprotein using monoclonal antibodies.

The Envelope glycoprotein (E-protein) of Japanese encephalitis virus (JEV) is the major structural component on the virion surface and is a primary target for the host immune system. Two monoclonal antibodies (MAbs) NHA-I (IgG2b) and NHA-II (IgM) against JEV (Indian strain 733913) were earlier developed in the authors' laboratory and found to be cross-reactive to nuclear histones. However, the epitope specificity of these MAbs has remained unknown. The present study was carried out to delineate the epitopes recognised by these MAbs on the E-protein of JEV strain 733913. The variable regions of the NHA-I and NHA-II were sequenced and the tertiary structures predicted. Molecular docking of the MAbs with the structural model of the JEV E-protein demonstrated that NHA-I binds to a predicted antigenic determinant (residue position 18-33) in domain-I. To understand the epitope specificity and check for possible cross-reactivity of these MAbs, comparative analysis of interactions with the known crystallographic structure of the West Nile virus (WNV) E-protein was also carried out. The studies predicted a differential binding of NHA-I but not of NHA-II between JEV and WNV. Mutagenesis studies could help analyse the specificity of NHA-I. The NHA-II appears to be cross-reactive as it docked in the groove region between domains I and III of both the JEV and WNV E-proteins. In laboratory assays, namely, ELISA and immunofluorescence assay both the MAbs reacted equally with JEV while the NHA-I did not show any reactivity with WNV. In silico results were thus validated by laboratory experiments. The present study would help in better understanding of virus-host interactions at the molecular level, and also be useful for the future design of vaccines as well as peptide based diagnostics.

Electroporation enhances radiation and doxorubicin-induced toxicity in solid tumor in vivo.

Treatment of cancer patients is subject to limitations in radiotherapy and chemotherapy. This necessitates development of new protocols, and the present work reports on the effects of a combination of local electroporation with ionizing radiation and/or anticancer drug doxorubicin hydrochloride (DOX) on subcutaneous solid tumor murine fibrosarcoma. Localized treatment of fibrosarcoma tumor, grown in right hind leg of Swiss mice, has been carried out using DOX (0.6 mg/kg body weight), radiation (Co 60 gamma-rays, dose rate 0.37 Gy/min) and electroporation (1 kV/cm, 200 micros, 8 pulses per burst, 10 bursts) individually or in combinations. Measurements of the tumor growth kinetics after treatment with combinations have revealed significant growth delay. The treatment groups, (i) radiation and electroporation, (ii) DOX and electroporation, and (iii) radiation, DOX and electroporation, have yielded tumor growth delays (TGDs) of 1.22, 1.5, and 1.73 days, respectively, compared to control with the tumor volumes being 53%, 57%, and 49% that of control on the final day of observation. These results suggest that the antitumor effects of a moderate dose of gamma radiation and low concentration of DOX can be significantly enhanced by combination with electroporation.

ISHAN: sequence homology analysis package.

Sequence based homology studies play an important role in evolutionary tracing and classification of proteins. Various methods are available to analyze biological sequence information. However, with the advent of proteomics era, there is a growing demand for analysis of huge amount of biological sequence information, and it has become necessary to have programs that would provide speedy analysis. ISHAN has been developed as a homology analysis package, built on various sequence analysis tools viz FASTA, ALIGN, CLUSTALW, PHYLIP and CODONW (for DNA sequences). This JAVA application offers the user choice of analysis tools. For testing, ISHAN was applied to perform phylogenetic analysis for sets of Caspase 3 DNA sequences and NF-kappaB p105 amino acid sequences. By integrating several tools it has made analysis much faster and reduced manual intervention.