Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in the Asia-Pacific region. Amplification of JEV in pigs is a potent driver for spillover of the infection to humans, and hence monitoring of virus dynamics in pigs can provide insights into JEV ecology. To study the dynamics of natural JEV infection in a tropical region, two groups of immunologically naïve pigs consisting of six animals per group were kept as sentinels on two different farms in the district of Thanjavur, Tamil Nadu, India. In a longitudinal study conducted from May 2022 to October 2023, nested RT-PCR and indirect ELISA were used to track the dynamics of JEV and the humoral response in pigs. Synchronous and asynchronous seroconversion in pigs was recorded on two different farms with different management practices. Repeated infections with JEV were recorded in all of the sentinel animals throughout the study period, irrespective of the season. Phylogenetic analysis revealed the presence of JEV genotype III in the region. It was observed that the IgG response to natural JEV infection did not last long, which might have been the reason for repeated infections in the sentinel animals. The longest period during which IgG was present at detectable levels in this study was two months, after which the pigs could once again amplify the virus. A significant positive correlation was found between wind speed and JEV incidence in sentinel animals. Our results offer a different perspective on the relationship between JEV and its amplifying host that contradicts the assumption that pre-immune pigs are resistant to JEV amplification. Our findings could have a major impact on our understanding of the ecology of JEV in tropical regions, where there is a high burden of JE despite coordinated prevention efforts that have relied on achieving a long-lasting immune response.

Detection and quantification of a virus play a crucial role in diagnosis, determination of infection stage, and decision on treatment strategies. Further, it plays a crucial role in monitoring the disease propagation during the pandemic. A wide range of analytical methods are used to detect and quantify viruses. The currently available methods are, however, laborious, costly, and time-consuming. Also, the accuracy of many of these methods is debatable. We propose a novel method for the detection and quantification of coronavirus based on the clay-virus-electrolyte interaction using the sedimentation method. Newcastle disease virus (NDV), a Coronavirus surrogate (CS), and Infectious bronchitis virus (IBV), an avian coronavirus, were used in this study. Due to changes in inter-particle forces of clay in the presence of the virus, the sedimentation rate of the clay-electrolyte system changed. A protocol was thus developed based on the changes in the sedimentation rate to predict the presence and quantity of the given virus. The sedimentation rates were recorded by mixing the bentonite clay with the CS or IBV and 0.1 M NaCl electrolyte solution at a solid-to-liquid ratio of 1:50. The sedimentation rates were varied with different CS concentrations due to different particle-particle associations due to the sorption of virus particles on the clay surface. The proposed method was successfully validated for using the plaque assay techniques for CS virus and RT-PCR for the IBV. The proposed method is simple, cost-effective, and very useful for the detection of coronavirus and other viruses' presence and quantification. Moreover, the methodology is useful to diagnose the Newcastle disease virus (NDV), which causes infection among several avian species during avian outbreaks.

Japanese encephalitis (JE) is widely prevalent in the Asia-Pacific region. There is limited information on JE virus (JEV) seroprevalence as well as the associated epidemiologic and ecological factors in India. This study aimed to determine the seroprevalence of JEV and associated risk factors in swine populations from climatically diverse regions of the country. A total of 1205 swine serum samples were collected from the backyard/established/slaughterhouse settings of the western (Maharashtra state), northern (Uttar Pradesh state), northern temperate (Uttarakhand state), and northeastern (Assam state) zones of India. Information on demographic (swine population), geographic (paddy cultivation area) and weather (annual rainfall)-related variables was collected and analysed to understand their association with JE seropositivity. A JEV seroprevalence of 20.3% (95% CI: 17.9-22.7) using IgM and 16.6% (95% CI: 14.3-19.1) using IgG ELISA(s) was recorded, with an overall seroprevalence of 17.7% (95% CI: 15.8-19.6). The highest seroprevalence was observed in Assam state (30.2%; 95% CI: 25.9-34.7) followed by Maharashtra (17.2%; 95% CI: 13.6-21.1), Uttarakhand (13.4%; 95% CI: 10.1-17.3) and Uttar Pradesh (11%; 95% CI: 8.2-14.3). JEV seroprevalence was highest during the post-monsoon season (40.3%, 95% CI: 34.7-46.0, n = 205), followed by the monsoon (18.9%, 95% CI: 15.7-22.2, n = 410), winter (17.0%, 95% CI: 13.5-20.9, n = 290), and summer (3.7%, 95% CI: 1.9-6.2, n = 300) seasons. The final multivariable analysis indicated that odds of infection were 2.59 (95% CI: 1.77-3.81, p = 0.001) times greater in the dense swine population area (population > 7616-81872 v/s 313-2006) and 5.77 (95% CI: 3.80-8.76, p = < 0.001) times greater in the small per capita paddy area (0-0.011 v/s > 0.046-0.091 hectares). This is the first study reporting JEV seroprevalence in the swine population of the northern temperate Indian zone, and it is recommended that sentinel surveillance and JE vaccine coverage be extended to the newly reported areas of virus activity.

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.

Viral quasispecies are distinct but closely related mutants formed by the disparity in viral genomes due to recombination, mutations, competition, and selection pressure. Theoretical derivation for the origin of a quasispecies is owed to the error-prone replication by polymerase and mutants of RNA replicators. Here, we briefly addressed the theoretical and mathematical origin of quasispecies and their dynamics. The impact of quasispecies for major salient human pathogens is reviewed. In the current global scenario, rapid changes in geographical landscapes favor the origin and selection of mutants. It comes as no surprise that a cauldron of mutants poses a significant risk to public health, capable of causing pandemics. Mutation rates in RNA viruses are magnitudes higher than in DNA organisms, explaining their enhanced virulence and evolvability. RNA viruses cause the most devastating pandemics; for example, members of the Orthomyxoviridae family caused the great influenza pandemic (1918 flu or Spanish flu), the SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) outbreak, and the human immunodeficiency viruses (HIV), lentiviruses of the Retroviridae family, caused worldwide devastation. Rapidly evolving RNA virus populations are a daunting challenge for the designing of effective control measures like vaccines. Developing awareness of the evolutionary dispositions of RNA viral mutant spectra and what influences their adaptation and virulence will help curtail outbreaks of past and future pathogens.

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.

Objective: To assess the prevalence of Japanese encephalitis virus (JEV) and associated risk factors in the swine population of Assam. Methods: A total of 432 swine serum and blood samples were collected from Barpeta and Sonitpur districts of Assam and were screened for the presence of JEV antibodies. Information related to risk factors was collected using a self-designed questionnaire from 120 swine-rearing farmers. Linear-mixed models were used for prevalence estimation. Univariate and multivariate regression models were constructed to evaluate the association of demography, season and management practices with JEV positive status. Results: Overall, the JEV infection prevalence was 51.6% at farm and 47.1% at slaughter premises. Phylogenetic analysis of partial sequence of envelope gene of two positive field samples revealed that both isolates belonged to genotype III JEV. Isolate 1 shared a common clade with human isolates while isolate 2 belonged to the same clade as that of other JEV swine strain isolated from India. The final multivariate model showed that two factors including monsoon season (Adjusted OR 5.6; 95% CI 2.1-14.9; P<0.001) and water logging in the area near the pig shelter (Adjusted OR 16.9; 95% CI 6.1-47.3; P<0.001) were associated with greater odds of swine being infected with JEV. Conclusions: High prevalence of JEV in swine population of Assam state indicates a significant risk of virus transmission to humans while risk factor study underlines the urgent need for awareness campaigns in the Assam.

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.

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.

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.