Genotypic characterization of Japanese encephalitis virus circulating in swine population of India: Genotype-III still in dominance.

Swine is considered as a suitable sentinel to predict Japanese encephalitis virus (JEV) outbreaks in humans. The present study was undertaken to determine the circulating genotypes of JEV in swine population of India. A total of 702 swine serum samples from four states of western, northern, northern-temperate, and north-eastern zones of India were screened by real-time RT-PCR targeting envelope gene of JEV, which showed positivity of 35.33%. The viral copy number ranged from 3 copies to 6.3 × 104 copies/reaction. Subsequently, the capsid/prM structural gene region of JEV positive samples was amplified by nested RT-PCR, sequenced, and genetically characterized. The phylogenetic analysis of the partial sequences of the capsid gene of 42 JEV positive samples showed that they all belonged to genotype-III (G-III) of JEV. Notably, JEV positive swine samples showed high nucleotide identity with human isolates from China and Nepal which explains the probable spillover of infection between neighboring countries probably by migratory birds. The novel mutations were observed in JEV positive sample B8 at C54 position (Phe → Ser), and JEV positive sample K50 at C62 (Thr → Ala) and C65 (Leu → Pro) positions which were absent from other JEV isolates reported till now. The mutation at the C66 position (Leu → Ser) observed in live attenuated vaccine SA14-14-2 strain was not found in JEV positive samples of our study. The detection of the G-III JE virus from climatically diverse states of India reinforces the need to continue the ongoing human vaccination program in India by extending vaccine coverage in temperate states.

Sero-molecular epidemiology of Japanese encephalitis virus in swine population of western Uttar Pradesh, India: Unraveling the geographical expansion of the virus.

BACKGROUND & OBJECTIVES: Swine is a good sentinel for forecast of Japanese encephalitis virus (JEV) outbreaks in humans. The present study was envisaged with objectives to know the sero-conversion period of JEV and to assess the prevalence of JEV in swine population of western Uttar Pradesh state of India. METHODS: A total of 252 swine serum samples were screened using IgM ELISA over the period of one year to determine the sero-conversion rate and compared seasonally to check the transmission peak of virus. Further, 321 swine blood and serum samples were collected from all seven divisions of western Uttar Pradesh to determine prevalence of JEV using real time RT-PCR and ELISA. RESULTS: Seasonal sero-conversion rate was high during monsoon and post-monsoon (32%) followed by winter (22.91%) and summer (10.71%) seasons. The sero-conversion was observed in all months indicating viral activity throughout the year in the region. The low degree of correlation was found between meteorological variables (day temperature, rainfall) and sero-conversion rate. A total of 52 samples (16.19%) were found positive by real time RT-PCR while sero-positivity of 29.91% was observed using IgG and IgM ELISA(s). The overall prevalence of JEV was 39.25%. INTERPRETATION & CONCLUSION: The presence of JEV was recorded throughout the year with peak occurrence during monsoon and post-monsoon season indicating that virus has spread its realm to western region of the state. The information generated in the present study will aid in initiating timely vector control measures and human vaccination program to mitigate risk of JEV infection in the region.

First report on the detection of Japanese encephalitis virus in fruit bats from India.

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

Development and evaluation of lateral flow assay for sero-diagnosis of Japanese encephalitis in swine.

Japanese encephalitis (JE) is a re-emerging mosquito-borne zoonotic flaviviral disease. Swine sero-convert 2-3 weeks before infection occurs in humans and thus serves as a suitable sentinel for JE surveillance and outbreak prediction in human population. The present study was conducted with the objective of developing a lateral flow assay (LFA) for detecting JEV antibodies in swine sera. Three different formats were tried using recombinant NS1 protein as antigen in order to select the best format. In format I, gold nanoparticles were conjugated with antigen followed by spotting of antigen on NCM as test line and anti-antigen IgG on NCM as control line. In format II, gold nanoparticles were conjugated with antigen followed by spotting of staphylococcal protein A as test line and anti-antigen IgG as control line. Format III used gold nanoparticles conjugated with goat anti-pig IgG followed by spotting of antigen as test line and pig IgG as control line. Amongst the three formats, format II was found to be superior with 100% relative diagnostic sensitivity and 100% relative diagnostic specificity during monsoon and post-monsoon period. A panel of 500 field swine serum samples was tested using format II which revealed sero-positivity of 15.6%, and the format was found suitable to screen swine serum samples during monsoon and post-monsoon period.

Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population.

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2-3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 µg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 µg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 µl of GNP conjugate and 50 µl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

Draft genome sequences of Brucella melitensis from human and animal sources in India.

We present the draft genome sequences of 23 Brucella melitensis isolates derived from human and animal sources across India with genome size predominantly at 3.207 M and uniform GC content (57.24%) across isolates. The accession numbers and detailed sequencing data enhance the utility of this resource for further genomic studies.

Spatio-temporal epidemiology of Japanese encephalitis virus infection in pig populations of eastern Uttar Pradesh, India, 2013-2022.

AIMS: Japanese encephalitis (JE) is endemic in India. Although pigs are considered important hosts and sentinels for JE outbreaks in people, limited information is available on JE virus (JEV) surveillance in pigs. METHODS AND RESULTS: We investigated the spatio-temporal distribution of JEV seroprevalence and its association with climate variables in 4451 samples from pigs in 10 districts of eastern Uttar Pradesh, India, over 10 years from 2013 to 2022. The mean seroprevalence of IgG (2013-2022) and IgM (2017-2022) was 14% (95% CI 12.8-15.2) and 10.98% (95% CI 9.8-12.2), respectively. Throughout the region, higher seroprevalence from 2013 to 2017 was observed and was highly variable with no predictable spatio-temporal pattern between districts. Seroprevalence of up to 60.8% in Sant Kabir Nagar in 2016 and 69.5% in Gorakhpur district in 2017 for IgG and IgM was observed, respectively. IgG seroprevalence did not increase with age. Monthly time-series decomposition of IgG and IgM seroprevalence demonstrated annual cyclicity (3-4 peaks) with seasonality (higher, broader peaks in the summer and monsoon periods). However, most variance was due to the overall trend and the random components of the time series. Autoregressive time-series modelling of pigs sampled from Gorakhpur was insufficiently predictive for forecasting; however, an inverse association between humidity (but not rainfall or temperature) was observed. CONCLUSIONS: Detection patterns confirm seasonal epidemic periods within year-round endemicity in pigs in eastern Uttar Pradesh. Lack of increasing age-associated seroprevalence indicates that JEV might not be immunizing in pigs which needs further investigation because models that inform public health interventions for JEV could be inaccurate if assuming long-term immunity in pigs. Although pigs are considered sentinels for human outbreaks, sufficient timeliness using sero-surveillance in pigs to inform public health interventions to prevent JEV in people will require more nuanced modelling than seroprevalence and broad climate variables alone.

Exploring genetic determinants of antimicrobial resistance in Brucella melitensis strains of human and animal origin from India.

Introduction: Antimicrobial resistance (AMR) in Brucella melitensis, the causative agent of brucellosis, is of growing concern, particularly in low and middle-income countries. This study aimed to explore the genetic basis of AMR in B. melitensis strains from India. Methods: Twenty-four isolates from humans and animals were subjected to antimicrobial susceptibility testing and whole-genome sequencing. Results: Resistance to doxycycline (20.80%), ciprofloxacin (16.67%), cotrimoxazole (4.17%), and rifampicin (16.67%) was observed. Genome analysis revealed efflux-related genes like mprF, bepG, bepF, bepC, bepE, and bepD across all isolates, however, classical AMR genes were not detected. Mutations in key AMR-associated genes such as rpoB, gyrA, and folP were identified, intriguingly present in both resistant and susceptible isolates, suggesting a complex genotype-phenotype relationship in AMR among Brucella spp. Additionally, mutations in efflux genes were noted in resistant and some susceptible isolates, indicating their potential role in resistance mechanisms. However, mutations in AMR-associated genes did not consistently align with phenotypic resistance, suggesting a multifactorial basis for resistance. Discussion: The study underscores the complexity of AMR in B. melitensis and advocates for a holistic multi-omics approach to fully understand resistance mechanisms. These findings offer valuable insights into genetic markers associated with AMR, guiding future research and treatment strategies.

Development of dipstick enzyme linked immunosorbent assay for on-site sero-diagnosis of Japanese encephalitis in swine.

Japanese encephalitis (JE) is an important viral zoonotic disease in Asia, especially in rural and suburban areas where rice cultivation and pig farming coexist. Pigs serve as a suitable sentinel model, the surveillance of which could predict a potential JE outbreak in human population in the immediate vicinity. However, existing diagnostics like ELISA and VNT require sophisticated laboratory facilities which are more often not available in field conditions. In the present study, we aimed at developing recombinant non-structural (NS1) protein-based dipstick IgG ELISA as an on-site assay for sero-diagnosis of JE in swine. The assay was standardized by optimizing various parameters and the following conditions were found to be ideal including 1 µg of rNS1 protein in carbonate buffer per strip of nitrocellulose membrane comb; bovine serum albumin as blocking agent at 4 °C overnight; serum dilution of 1:10 and conjugate dilution of 1:5000 in skimmed milk powder. Relative diagnostic sensitivity and specificity of dipstick IgG ELISA was 100% and 92.9%, respectively. The dipstick assay was validated in three laboratories as per OIE guidelines. The storage life of dipstick was up to 7 months at 4 °C. The assay is easy to perform and the results can be interpreted with visual observation that precludes the need for absorbance reading equipment. The standardized dipstick assay was found promising for screening swine serum samples in field conditions. Timely detection of JE virus in swine will aid in predicting the outbreak in humans and thus in taking suitable preventive and control measures.

Detection of recent infection of Japanese encephalitis virus in swine population using IgM ELISA: A suitable sentinel to predict infection in humans.

Japanese encephalitis (JE) is a mosquito-borne flaviviral zoonotic disease and is one of the major causes of encephalitis in children. Swine, being an amplifier host of Japanese encephalitis virus (JEV), play an important role in its epidemiology. Therefore, early detection of either JEV or antibodies against JEV in swine is a feasible alternative for initiating necessary measures to prevent the spread of infection to humans. Since IgM antibodies appear early in swine sera, recombinant NS1 protein based indirect IgM ELISA was developed in the present study with the objective to know the recent infection of swine population with JEV. The relative diagnostic sensitivity and specificity of the developed ELISA was 95.34% and 98.6%, respectively. The developed ELISA was found to have excellent reproducibility on inter-laboratory and inter-institutional validation studies. A total of 3,027 field swine sera samples were screened using the developed ELISA and 488 samples were found positive for IgM against JEV with an overall sero-positivity of 16.12% in swine population of India. The highest sero-positivity was observed in swine population of Eastern zone of India which coincided with the maximum number of human JE cases reported from this zone during the same period. Further, antibody kinetics study revealed that the IgM antibodies against NS1 protein of JEV started appearing in swine sera at day 5 and disappeared completely by day 40. The IgG antibodies started appearing at day 7, and remained for more than 365 days indicating the suitability of IgM ELISA to know the recent infection of JEV. The developed IgM ELISA can be readily incorporated into surveillance programs for detection of JEV activity in swine population so that outbreaks in humans can be prevented by taking suitable preventive measures.