Molecular identification and phylogenetic analysis of chikungunya virus among dengue-negative patients in Kolkata, India.

Dengue and chikungunya are co-circulating vector-borne diseases that share a significant number of clinical symptoms. To identify variables to aid physicians in making rapid and effective diagnostic decisions, we performed molecular diagnosis of the chikungunya virus and examined the clinical manifestations of chikungunya cases to identify the prevalence among dengue-negative individuals in Kolkata. Dengue suspected patients' samples were collected during January 2020-December 2021 and Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) methods have been performed to confirm the prevalence of chikungunya infection among dengue-negative patients. By performing phylogenetic analysis, comparing clinical classifications, identifying disease aetiology using clinical and laboratory factors, and evaluating the time course of several clinical variables, we have evaluated the clinical manifestations linked to dengue and chikungunya virus infections. Chikungunya infection was found in 15.1% and 6.3% of the 635 dengue-negative patients, as determined by ELISA and RT-PCR, respectively. Arthritis and myalgia were more common in chikungunya-infected patients at the time of hospital admission while conjunctivitis, photosensitivity, arthralgia, Anorexia, fatigue, retro-orbital pain, vomiting, dermatitis, or swollen glands were significantly presented as an overlapping symptom. Although dengue and chikungunya infections have significant clinical overlap, basic clinical and laboratory criteria can predict these diseases at presentation for proper management. Effective management enables doctors to treat and care for patients properly and contributes to the development of control measures for these infections in a medical setting.

Emergence of West Nile virus in West Bengal, India: a new report.

Background: The ICMR virus unit in Kolkata functions as an Appex Referral Laboratory for the detection of dengue (DENV) and chikungunya (CHIKV) infections in the eastern part of India. In spite of efforts for confirmatory diagnosis, some samples remain undiagnosed every year. West Nile virus (WNV) infection may mimic either dengue (flavivirus) or chikungunya (alphavirus) like illness. WNV is endemic in the tropical region where its principal/potential vectors are Aedes and Culex. Methods: We explored the existence of WNV within undiagnosed samples to identify the emergence of a new public health problem. Results: Of 1278 sera samples, 574 were negative for DENV and CHIKV either by ELISA or by reverse transcriptase (RT)-PCR. Of these 574 negative samples, 83 (14.5%) and 141 (24.56%) were positive for WNV by ELISA and RT-PCR, respectively; no samples were positive for WNV by both methods. After assembling raw sequencing data, partial envelope genome sequence of West Bengal isolates, WNV was compared through BLAST with other WNV Indian strains and 98% homology detected. Phylogenetic analysis of one West Bengal isolates (Accession No. KY421790) and 28 Indian isolates available in GenBank, indicated close clustering. Conclusions: The serological and molecular approaches have clearly established the emergence of WNV in West Bengal. Hence, for proper case management, detection of WNV in common febrile illness is strongly recommended.

Complete genome sequence of two genotype III Japanese encephalitis virus isolates from West Bengal, India.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE), caused by a mosquito-borne virus JE virus (JEV), is a serious health problem in West Bengal, India. In this study, we report the complete genome sequence of two JEV isolates from West Bengal. The amino acid and nucleotide sequence homology was compared with other Indian strains. METHODS: Two JEV isolates (IND-WB-JE1 and IND-WB-JE2) obtained in 2008 and 2010, respectively, from two districts of the State of West Bengal, respectively were analyzed for genetic variations by sequencing the 10934 bp whole genome of the virus. Of these two districts, one was covered under JE vaccination programme in 2007. RESULTS: Phylogenetic analysis showed that both the isolates belonged to the genotype III. A total of 16 mutations were identified in the two isolates studied with respect to Vellore P20778 strain. One unique mutation A3215S was only found in IND-WB-JE2 isolate, but not in the isolate IND-WB-JE1. These two isolates showed maximum homology with P20778 strain of India. INTERPRETATION & CONCLUSIONS: This study reports on complete gene based phylogenetic analysis of JEV isolates from the State of West Bengal. It was evident from the results that JEV was still under circulation in both vaccine covered and not covered districts of West Bengal.

Japanese encephalitis associated acute encephalitis syndrome cases in West Bengal, India: A sero-molecular evaluation in relation to clinico-pathological spectrum.

Japanese encephalitis (JE) is a major public health problem in Asia and worldwide and it is responsible mainly for viral acute encephalitis syndrome (AES). The sole etiologic agent of JE is Japanese encephalitis virus (JEV). Although JE/AES cases have been regarded traditionally as a disease of children, a growing number of patients with JE/AES cases are also seen in the adult age group every year in the state of West Bengal, India in spite of vaccination. Therefore, a systematic study was performed to differentiate and characterize the clinico-pathological parameters and viral diversity among the patients of different age groups. Viral diversity was also evaluated from the JE/AES cases, depending on their disease severity. A total of 441 JE/AES cases were included in this study. By MAC-ELISA, 111 samples were found JEV IgM positive and among the IgM negative cases, 26 samples were found RT-PCR positive against JEV infection. Neck rigidity, abnormal behavior, convulsion, protein in CSF, WBC in CSF, and aspartate transaminase in blood differed significantly among the patients of pediatric-adolescent and adult group in both IgM positive and RT-PCR positive cases. Viral diversity was increased significantly in the pediatric-adolescent group compared to adult patients. Interestingly, with the rise in disease severity the viral diversity was found to be increased among the patients, irrespective of their age distribution. Based on clinico-pathological parameters and analysis of viral diversity, it can be concluded that viral diversity which occurs naturally is likely to affect disease severity, especially in the patients of pediatric-adolescent group.

Molecular characterization of chikungunya virus circulating in urban and rural areas of West Bengal, India after its re-emergence in 2006.

BACKGROUND: In the state of West Bengal, chikungunya virus (CHIKV) has re-emerged in 2006 after its last occurrence in 1963-1965 in this state. The virus rapidly affected almost every district of this state, with high morbidity. Based on complete sequences of structural region of CHIKV genome, we determined the molecular characterization of the virus circulating in this state from 2006-2012. METHODS: CHIKV was isolated from 20 acute CHIKV RT-PCR positive serum samples in C6/36 mosquito cell line. These samples were collected from 20 patients with a clinical history of ≤2 days of fever and chikungunya-like illness. Those patients were residing in some outbreak areas in the state of West Bengal during 2006-2012. Isolation was confirmed through RT-PCR and sequencing. RESULTS AND CONCLUSIONS: Two sub-lineages of East-central-southern African (ECSA) genotype of CHIKV strains were circulating simultaneously in this state during the study period; one type was circulating in rural areas of the state from 2006 whereas another type was isolated from the metropolitan city of Kolkata in 2011 and 2012. The mutational pattern of those CHIKV strains suggests that the transmission of the viruses might be facilitated by different species of Aedes mosquitoes. Our results represent an important first step towards understanding the circulating strains of CHIKV in the state of West Bengal with the geographical variation.

Envelope protein gene based molecular characterization of Japanese encephalitis virus clinical isolates from West Bengal, India: a comparative approach with respect to SA14-14-2 live attenuated vaccine strain.

BACKGROUND: Increasing virulence of Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen is of grave concern because it causes a neurotrophic killer disease Japanese Encephalitis (JE) which, in turn, is responsible globally for viral acute encephalitis syndrome (AES). Despite the availability of vaccine, JE/AES cases and deaths have become regular features in the different rural districts of West Bengal (WB) state, India, indicating either the partial coverage of vaccine or the emergence of new strain of JEV. Therefore, a study was undertaken to characterize and compare the complete envelope (E) protein gene based molecular changes/patterns of JEVs circulating in WB. METHODS: Total of 98 AES case-patients' samples were tested to detect the presence of JEV specific immunoglobulin M (IgM) antibody by Mac-ELISA method. Only JEV IgM negative samples with a history of ≤3 days' illness were screened for virus isolation and RT-PCR. E gene sequences of JEV isolates were subjected to molecular phylogeny and immunoinformatics analysis. RESULTS: Present study confirmed JEV etiology in 39.7% and 29.1% of patients presenting ≤15 days' febrile illness, as determined by Mac-ELISA and RT-PCR respectively. Phylogenetic analysis based on complete E gene sequences of JEV isolates showed the co-circulation of JEV genotype I (GI) with genotype III (GIII). This study also demonstrated that isolate-specific crucial amino acid substitutions were closely related to neurovirulence/neuroinvasiveness of JE. On the basis of immunoinformatics analysis, some substitutions were predicted to disrupt T-cell epitope immunogenicity/antigenicity that might largely influence the outcome of vaccine derived from JEV GIII SA14-14-2 strain and this has been observed in a previously vaccinated boy with mild JE/AES due to JEV GI infection. CONCLUSIONS: Based on molecular evolutionary and bioinformatic approaches, we report evolution of JEV at a local level. Such naturally occurring evolution is likely to affect the disease profile and the vaccine efficacy to protect against JEV GI may demand careful evaluation.

Molecular evidence for the occurrence of Japanese encephalitis virus genotype I and III infection associated with acute encephalitis in patients of West Bengal, India, 2010.

BACKGROUND: Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is the sole etiologic agent of Japanese Encephalitis (JE); a neurotropic killer disease which is one of the major causes of viral encephalitis worldwide with prime public health concern. JE was first reported in the state of West Bengal, India in 1973. Since then it is being reported every year from different districts of the state, though the vaccination has already been done. Therefore, it indicates that there might be either partial coverage of the vaccine or the emergence of mutated/new strain of JEV. Considering this fact, to understand the JEV genotype distribution, we conducted a molecular epidemiological study on a total of 135 serum/cerebrospinal fluid (CSF) samples referred and/or collected from the clinically suspected patients with Acute encephalitis syndrome (AES), admitted in different district hospitals of West Bengal, India, 2010. FINDINGS: JEV etiology was confirmed in 36/135 (26.6%) and 13/61 (21.3%) 2-15 days' febrile illness samples from AES cases by analyzing Mac-ELISA followed by RT-PCR test respectively. Phylogenetic analysis based on complete envelope gene sequences of 13 isolates showed the emergence of JEV genotype I (GI), co-circulating with genotype III (GIII). CONCLUSION: This study represents the first report of JEV GI with GIII, co-circulating in West Bengal. The efficacy of the vaccine (derived from JEV GIII strain SA-14-14-2) to protect against emerging JEV GI needs careful evaluation. In future, JE outbreak is quite likely in the state, if this vaccine fails to protect sufficiently against GI of JEV.

Molecular typing of dengue virus circulating in kolkata, India in 2010.

Dengue is one of the major public health threats in Kolkata. Every year, blood samples with dengue-like illness are referred to us from different medical colleges and hospitals in Kolkata for the detection of dengue infection in them. In 2010, a total of 378 samples were referred to us for that purpose. All the samples were tested for the detection of IgM antibodies by ELISA method, followed by RT-PCR test for the detection of serotypes. Only 173 samples were ELISA positive. Out of 378 samples, 108 were RT-PCR positive. Out of 108 samples, 74 samples had monotypic infection with different serotypes of DENV and 33 samples had dual infections with DENV-2 and DENV-3. Only one sample had the infection with DENV-1, DENV-2, and DENV-3. DHF was found mainly among the patients, infected with multiple dengue serotypes. Only 3 dengue monotypic infected patients had suffered from DHF.

A comparative study of clinical features between monotypic and dual infection cases with Chikungunya virus and dengue virus in West Bengal, India.

Chikungunya virus (CHIKV) and dengue virus (DENV) are circulating individually in the state of West Bengal, India. However, after 1965 the dual-infection caused by both viruses had not been recorded until 2010. In 2010, an investigation of the febrile cases was carried out to confirm the involvement of both viruses simultaneously. A total of 550 blood samples were tested for the detection of immunoglobulin M (IgM) antibody against both CHIKV and DENV. Serology by the enzyme-linked immunosorbent assay method confirmed that 131 (23.8%) and 104 (18.9%) patients had IgM antibody against CHIKV and DENV, respectively, whereas 68 (12.4%) had IgM antibodies against both CHIKV and DENV. Fever, joint pain, rashes, headache, myalgia, and nausea/vomiting are the common features in the case of both monotypic and dual-infection. Severe arthralgia and swelling of joints were common only in CHIKV-positive cases and abdominal pain was mainly associated with DENV infection. Diarrhea was reported only by the dual-infected patients (16.2%).

Rapid spread of chikungunya virus following its resurgence during 2006 in West Bengal, India.

Re-emergence of chikungunya virus (CHIKV) in West Bengal was detected after almost 40 years when an outbreak of fever occurred in Baduria village (West Bengal, India) in October 2006. The symptoms of CHIKV infection are similar to those of dengue virus (DENV) infection. Serum samples were tested for detection of IgM antibody to CHIKV and DENV and the aetiological agent was detected as CHIKV. RT-PCR was carried out for confirmation of CHIKV infection. By 2009, CHIKV had spread rapidly within ten districts of West Bengal. Middle-aged women (age group 31-40 years) were predominantly affected. Here we report the analysis of 2134 serum samples collected during 2006-2009 from the different districts of West Bengal, among which IgM antibody to CHIKV and DENV was detected in 403 and 199 samples, respectively. This report highlights the gradual dominating activity of CHIKV with dengue-like clinical features in dengue-endemic regions such as West Bengal.

Mass scale screening of common arboviral infections by an affordable, cost effective RT-PCR method.

OBJECTIVE: To develop a rapid, cost effective RT-PCR method for the mass scale diagnosis of such diseases at the viremia stage to find out the actual disease burden in that area. METHODS: For this purpose, cases with the history of only short febrile illness were considered. Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome (AES) were selected. RESULTS: Out of 157 samples, 42 and 74 were detected as dengue and chikungunya, respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method. CONCLUSIONS: This cost effective RT-PCR method can detect the total positive cases that remain undetected by ELISA method. Moreover, this method is capable to detect the viral RNA from patients' sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.