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.

Seroprevalence of Japanese encephalitis virus in pig populations of Tamil Nadu, India: Exploring the tropical endemic link of virus.

Japanese encephalitis virus (JEV) is a major cause of encephalitis in Southeast Asia. Tamil Nadu, a state located in the southern part of India, contributes substantially to the national burden of human JE cases every year. However, limited information is available on the epidemiology of JE in pig populations of Tamil Nadu. A cross-sectional study was conducted to assess JEV prevalence in pig populations of Tamil Nadu. A total of 710 pigs reared in 118 farms across 10 districts of Tamil Nadu were sampled using multistage cluster random sampling. Serum samples were analyzed for their JEV status using Immunoglobulin M (IgM) and Immunoglobulin G (IgG) Enzyme-Linked Immunosorbent Assay (ELISA). At the animal-level, the apparent JEV seroprevalence was 60.4% (95% CI: 56.8% - 64.0%) and the true seroprevalence was 50.1% (95% CI: 47.0% - 53.2%). The herd-level apparent seroprevalence was 94.1% (95% CI: 88.1% - 97.5%) and the true seroprevalence was 93.3% (95% CI: 89.5% - 96.2%). The intensity of JEV circulation was high in all the districts, with seroprevalence ranging between 43% and 100%. Pigs across all age categories were seropositive and a high overall seroprevalence of 95.2% (95% CI: 76.2% - 99.9%) was recorded in pigs older than 12 months. JEV seropositivity was recorded in all the seasons but the prevalence peaked in the monsoon (67.9%, 95% CI: 61.1% - 74.2%) followed by winter (65.1%, 95%CI: 57.4% - 72.2%) and summer (53.3%, 95% CI: 47.8% - 58.8%) seasons. The results indicate that JEV is endemic in pigs populations of the state and a one health approach is essential with collaborative actions from animal and public health authorities to control JE in Tamil Nadu, India.

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.

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.

Point-of-care detection of Japanese encephalitis virus biomarker in clinical samples using a portable smartphone-enabled electrochemical "Sensit" device.

Japanese encephalitis (JE), a neglected tropical zoonotic disease prevalent in south-east Asian and western pacific countries, caused by the flavivirus JE virus (JEV), has a dearth of electrochemical point-of-care (PoC) diagnostic tools available to manage endemic breakouts. To overcome this, we have developed a screen-printed carbon electrode (SPCE) immunosensor for rapid PoC detection of JEV nonstructural 1 (NS1) antigen (Ag), found circulating in serum of infected individuals using a smartphone based portable "Sensit" device. The modification of SPCE surface with JEV NS1 antibody (Ab) was confirmed via observation of globular protein structures via scanning electron microscopy (SEM), increase in electrode surface hydrophilicity via contact angle measurement and decrease in current via differential pulse voltammetry (DPV). The fabrication and testing parameters were optimized based on highest current output obtained using DPV. The SPCE was tested for detection limit of target JEV NS1 Ag ranging from 1 fM to 1 µM, which was determined as 0.45 fM in spiked serum. The disposable immunosensor was also found to be highly specific in detecting JEV NS1 Ag over other flaviviral NS1 Ag. Finally, the modified SPCE was clinically validated by testing 62 clinical JEV samples using both a portable miniaturized electrochemical "Sensit" device coupled with a smartphone and a laboratory-based potentiostat. The results were corroborated with gold-standard RT-PCR and showed 96.77% accuracy, 96.15% sensitivity, and 97.22% specificity. Hence, this technique may further be developed into a one-step rapid diagnostic tool for JEV, especially in rural areas.

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.

Prevalence of brucellosis in livestock of African and Asian continents: A systematic review and meta-analysis.

Brucellosis is a highly contagious bacterial disease that mainly affects ruminants, but it may affect equines, canines, and felines. The disease is of utmost significance from an economic standpoint in countries where there is no national brucellosis prevention and eradication policy in operation. A systematic review was done to estimate disease burden, incidences, prevalence, and geographical distribution critical in planning appropriate intervention strategies for the control and prevention of Brucellosis. Research articles that were published during the period 2000-2020 were considered for this study after reinforced scrutiny by two independent authors. Meta-regression was used to examine heterogeneity, and subgroup and sensitivity analyses were used to calculate residual heterogeneity and the pooled prevalence of Brucellosis in livestock. Confounders such as geography, a diagnostic test, and species had the greatest R 2 values of 17.8, 8.8, and 2.3%, respectively, indicating the presence of heterogeneity and necessitating more research into sensitivity and subgroup analysis. The combined pooled prevalence of brucellosis in both Asia and African countries was 8% when compared to 12% in the Indian livestock population. The findings of our systematic review and meta-analysis indicate that brucellosis continues to be an important animal and public health concern in developing countries of Asia and Africa, as evidenced by the prevalence rate of brucellosis in these regions. Our findings suggested that well-planned epidemiological surveillance studies in different geographic settings are needed to generate reliable data on disease burden including the economic loss in Asian and African countries.

Immuno-chromatic probe based lateral flow assay for point-of-care detection of Japanese encephalitis virus NS1 protein biomarker in clinical samples using a smartphone-based approach.

Lateral flow assays (LFAs) are one of the most economical, point-of-care (PoC) diagnostic assays that exploit the colorimetric properties of gold nanoparticles (AuNPs). Up to the best of our knowledge, no rapid antigen-based LFA exists for Japanese Encephalitis Virus (JEV) detection. Herein, we have reported a novel portable sandwich-type LFA for on-site detection of the non-structural 1 (NS1) secretory protein of JEV. In-house JEV NS1 antibodies (Abs) were generated and labelled with AuNPs as immunoprobes. A glass fibre membrane conjugate pad was soaked with AuNPs-Ab solution, while the JEV NS1 Ab and anti-rabbit IgG 2° Ab were coated as the test and control lines, respectively, on a nitrocellulose (NC) membrane. Different layers of the LFA were fabricated and various parameters were standardised for optimum colour intensity development. JEV negative serum samples spiked with JEV NS1 Ags (linear range - 1 pg ml-1 to 1 µg ml-1) were applied onto the sample pad and the intensity of the red colour developed on the test line increased with increasing concentration of Ag. The visual limit of detection (LOD) determined from the LFA was 10 pg ml-1, which corresponded to the LOD determined by the graphical data obtained from ImageJ software and the Colorimeter smartphone application. Furthermore, the colorimetric based immunosensor showed minimal non-specific detection of other closely related flaviviral NS1 Ags in the spiked serum, provided a rapid result within 10 min, showed storage stability up to a month at 4 °C, successfully detected the JEV NS1 protein in clinically infected pig serum samples, and hence, may be developed into a PoC screening diagnostic kit for JEV.

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.

Prevalence of Japanese encephalitis (JE) virus in mosquitoes and animals of the Asian continent: A systematic review and meta-analysis.

BACKGROUND: Japanese encephalitis (JE) is a viral zoonotic disease that has been found in several countries of Asia and is responsible for high mortality and morbidity of men and animals in rural and sub-urban endemic areas due to the virus re-circulation among diverse hosts and vectors. The present study estimates the prevalence of the JE virus in the vector and animal population of the Asian continent using a systematic review and meta-analysis. METHODS: The Cochran collaborators' Preferred Reporting Items for Systematic Reviews and Meta-Analysis [PRISMA] guidelines were used for systematic review and meta-analysis. The heterogeneity was observed in meta-regression analysis due to several factors including region, species, and different diagnostic assays used in various studies. Thus we did sensitivity and subgroup analysis. RESULTS: The prevalence of the JE virus was calculated using a total sample size of 47,391. Subgroup analysis revealed the JE virus prevalence of 39% in the Southeast Asia region, followed by East Asia with 35% and South Asia with 15% prevalence. Hence, the overall pooled prevalence of the JE virus was 26% in the Asian continent. CONCLUSIONS: The highest proportion of infection was found in pigs amongst all animals, reinforcing the fact that they can be used as sentinels to predict outbreaks in humans. The findings of this study will enable researchers and policymakers in better understanding the disease's spatial and temporal distribution, as well as in creating and implementing location-specific JE prevention and control measures.

Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

Japanese encephalitis (JE) is a re-emerging mosquito borne disease, for which equines are most susceptible amongst all animals. Detection of specific immunoglobulin 'M' (IgM) is considered as an ideal way to diagnose recent JE virus infection in equines due to low virus load and short-term viremia. The present study was undertaken to develop a sensitive and specific recombinant NS1 protein based indirect IgM-ELISA and IgM capture (MAC) ELISA to diagnose recent infection of JEV in equines. Indirect IgM ELISA was standardized with relative diagnostic sensitivity and specificity of 100% and 88.5%, respectively. The validation of indirect IgM-ELISA in different laboratories revealed excellent reproducibility with Cohen's kappa value ranging between 0.84 and 1. The standardization of MAC ELISA was attempted using checker board titration method and non-specific binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum raised in swine against the antigen was observed. Hence, the MAC ELISA was standardized with monoclonal capture antibody; however, its diagnostic performance could not meet the satisfactory limit. Due to better sensitivity and less turnaround time, indirect IgM-ELISA was employed to screen 821 equine serum samples revealing 33.73% positivity of IgM antibodies against JEV in equine population of India. The high JEV sero-positivity warrants the need for vaccination in Indian equine population along with the demand for research focused towards anti-viral therapy. The indirect IgM-ELISA developed in the present study could be useful to diagnose acute or recent infection of JEV in equines as well as in sero-epidemiological studies.

Sero-positivity of Japanese Encephalitis Virus in Equine Population of India Using IgG ELISA: Unraveling the Need for Vaccination.

Japanese encephalitis (JE) is a mosquito borne flaviviral zoonoses, causing fatal disease in equines and humans. JE is endemic in most of the states of India with occurrence of human cases every year. The horses are not vaccinated against JE in India and thus they are at more risk of acquiring the disease. Due to nonavailability of indigenously developed ELISA and high cost of imported kits, regular sero-surveillance is not being carried out to assess the true picture of JE virus in equine population of India. Therefore, a recombinant NS1 protein based indirect IgG ELISA was developed with the objective to assess the sero-positivity of JE virus in equine population of India. The diagnostic sensitivity and specificity of developed ELISA was 84.73% and 86.70%, respectively. The validation studies revealed good reproducibility of ELISA with kappa value ranging from 0.75 to 1 between the results of different laboratories. A total of 2,069 horse serum samples were screened using the developed ELISA and 401 samples were positive for IgG against JEV with an overall sero-positivity of 19.38% in equine population of India. A sero-positivity of 25.90% and 12.22% was recorded in Himachal Pradesh and Jammu-Kashmir, both hill states of North zone of India for the first time, revealing the spread of virus to the nonendemic parts of the country. The high sero-positivity of JE virus recorded in equine population warrants the need for initiation of vaccination of horses in India to prevent the morbidity and mortality.

Evaluation of carboxyl beads based latex agglutination test for rapid sero-diagnosis of Japanese encephalitis.

Japanese encephalitis (JE) is a major public health problem in the South Asian countries including India. Pigs serve as a relevant sentinel model, the surveillance of which could predict a potential JE outbreak in human population nearby. However, existing serological detection methods like Enzyme-Linked Immuno Sorbent Assay (ELISA), virus neutralization test (VNT) and Haemagglutination Inhibition (HI) require elaborative laboratory facilities which are invariably not available in field conditions. Recognizing the lacunae, attempts were made to develop recombinant antigen (rNS1) based latex agglutination test (LAT) as a rapid on-site test using covalent coupling method. Four different formats were evaluated using different coupling buffers, blocking buffers and reaction conditions. The format in which borate buffer at alkaline pH (8.5) was used for coupling of antigen with carboxylated beads followed by blocking with skimmed milk powder was found to be the best amongst all. Developed latex based test was used for screening of 207 pig serum samples for JE which revealed relative diagnostic sensitivity and specificity of 80.2% and 95.2%, respectively in comparison with indirect IgG ELISA. Hence, the present study demonstrated that covalently coupled recombinant antigen based LAT could be used as a reliable screening test for surveillance of JE in pigs under field conditions.

Latex agglutination test for rapid on-site serodiagnosis of Japanese encephalitis in pigs using recombinant NS1 antigen.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a mosquitoe-borne viral zoonotic disease and globally around three billion people are at the risk of disease. The occurrence of JE cases has shown a rising trend during last decade in India. Pig is the amplifying host for JE virus and serves as a suitable sentinel model for the prediction of disease outbreak in humans. The development of a diagnostic test that is suitable for surveillance of JE in pigs is the need of the hour. The existing tests require elaborate laboratory facilities which make their application in rural settings difficult. Therefore, realizing the need for a rapid test, efforts were made to standardize a latex agglutination test (LAT) for serodiagnosis of JE in pigs. METHODS: Standardization of LAT by physical adsorption of recombinant NS1 (non-structural) protein of JE virus onto latex beads was done by altering six different variables, namely the antigen concentration, sensitization condition, surface blocking agent, blocking condition, particle concentration and reaction time. The standardized latex-protein complex was used for screening 246 pig serum samples under optimal conditions. RESULTS: The test was standardized with a diagnostic sensitivity and specificity of 82.24 and 87.83%, respectively. Screening of 246 field pig serum samples using standardized LAT showed a seropositivity of 50.4%. The results were available within 5 min after addition of test serum sample to the sensitized beads. INTERPRETATION & CONCLUSION: The findings of the study highlight the potential of LAT as a rapid on-site assay for JE diagnosis in pigs which would aid in predicting JE outbreaks in humans.

Development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus.

Canine parvovirus (CPV) is the leading viral cause of enteritis in dogs and occurs mainly in 6- to 8-week-old pups. Rapid diagnosis of CPV under field conditions is now possible due to commercially available immunochromatographic (IC) assays. However, these commercial kits are somewhat expensive because they utilize a minimum of two monoclonal antibodies (mAbs) targeting different epitopes as capture and detector antibodies. Using only a single mAb for both capture and detection purpose may reduce the sensitivity of the assay. In the present study, efforts were made to develop an economical assay that can be utilized for diagnosis of CPV under Indian conditions with a high level of confidence. Rabbit polyclonal antibodies (pAbs) generated against recombinant truncated VP2 proteins of CPV were used as capture antibodies because they can be produced economically, while a commercial anti-CPV mAb was used as the detector antibody. The detection limit of the test strip was 6.6×105 TCID50/ml, and it specifically detected CPV-2, CPV-2a and CPV-2b while displaying no cross-reactivity with other common canine enteric pathogens. The relative sensitivity/specificity of pAb based strip test was 71%/92% and 71%/100% in relation to the hemagglutination test and a commercial IC kit, respectively, with substantial agreement. In addition, two commercially available mAbs targeting different epitopes were also used for development of another IC assay, which showed sensitivity, and specificity of 82%/87% and 90%/98% in relation to the hemagglutination test and commercial kit. Hence, the present strip test based on a combination of mAb and pAb provides an acceptable alternative for onsite and cost-effective diagnosis of CPV infection.