Increased IL-6 Levels and the Upregulation of Iron Regulatory Biomarkers Contribute to the Progression of Japanese Encephalitis Virus Infection's Pathogenesis.

Integrated analysis of iron regulatory biomarkers and inflammatory response could be an important strategy for Japanese encephalitis viral (JEV) infection disease management. In the present study, the inflammatory response was assessed by measuring serum Interleukin-6 (IL-6) levels using ELISA, and the transcription levels of iron homeostasis regulators were analyzed via RT-PCR. Furthermore, inter-individual variation in the transferrin gene was analyzed by PCR-RFLP and their association with clinical symptoms, susceptibility, severity, and outcomes was assessed through binary logistic regression and classification and regression tree (CART) analysis. Our findings revealed elevated levels of IL-6 in serum as well as increased expression of hepcidin (HAMP), transferrin (TF), and transferrin receptor-1 (TFR1) mRNA in JEV infection cases. Moreover, we found a genetic variation in TF (rs4481157) associated with clinical symptoms of meningoencephalitis. CART analysis indicates that individuals with the wild-type TF genotype are more susceptible to moderate JEV infection, while those with the homozygous type are in the high-risk group to develop a severe JEV condition. In summary, the study highlights that JEV infection induces alteration in both IL-6 levels and iron regulatory processes, which play pivotal roles in the development of JEV disease pathologies.

The relationship between MMP-2 rs243865, MMP-9 rs398242 and CXCL-12 rs1801157 gene polymorphisms with Japanese encephalitis disease and disease outcome in North Indian population.

BACKGROUND & OBJECTIVES: Japanese encephalitis virus (JEV) is one of the most important causes of acute and uncontrolled inflammatory disease in Asia. Matrix metalloproteinases (MMPs) and chemokines play a detrimental role in the host response to JE disease, aetiology, and disease outcome. Evidently, MMPs are widely circulated in the brain and regulate various process including microglial activation, inflammation, blood-brain barrier disruption as well as affects central nervous system (CNS). The present study was to assess the association of single nucleotide polymorphisms of MMP-2, MMP-9 and chemokine (CXCL-12/SDF1-3') in the north Indian population. METHODS: We performed case-control study comprising of 125 patients and 125 healthy controls in north Indian population. Genomic DNA was extracted from whole blood and gene polymorphism have been determined by PCR-RFLP method. RESULTS: MMP-2, MMP-9 and CXCL-12 gene was not significantly associated with JE disease, but homozygous (T/T) genotype of MMP-2 was statically associated with disease outcome (p=0.05, OR=0.110). A/G and G/G genotype of CXCL-12 was significantly associated with severity of disease. (p=0.032, OR=5.500, p=0.037, OR= 9.167). The serum level of MMP-2 was observed significantly increased in JE patients with homozygous (T/T) genotype whereas increased MMP-9 level was associated with heterozygous genotype. INTERPRETATION & CONCLUSION: MMP-2, MMP-9 and CXCL-12 gene polymorphism were not associated with JE susceptibility, but MMP-2 may be contributed to disease protection. CXCL-12 was associated with disease severity. In our concern this is the first report from northern India.

Differential expression of circulating microRNAs in serum: Potential biomarkers to track Japanese encephalitis virus infection.

Japanese encephalitis is one of the serious vector-borne viral encephalitis diseases found worldwide and poses a major threat to public health. Most Japanese encephalitis virus (JEV) infections are subclinical; only 1: 250 to 1:1000 infected persons develop clinical presentations. Delay in proper diagnosis of JE affects the timeliness of treatment initiation and increases the mortality rate in patients. Therefore, there is an extreme need to develop potential biomarkers, which might improve the diagnosis and can become the basis for development of new therapeutics. The microRNAs (miRNAs/or miRs) are small noncoding RNAs of 17-24 nucleotides that are known to regulate about 60% of human genes. Although miRNAs have been found to regulate various aspects of innate and adaptive immune responses, less information on circulating miRNAs in JE is known. The study of JEV infected human serum miRNAs will provide novel information for the diagnosis of JE as well as for the improvement of disease outcome. Total RNA, including miRNA, was extracted from serum followed by the complementary DNA (cDNA) synthesis by using sequence-specific primers. cDNA was amplified using target-specific TaqMan MicroRNA Assay. Real-time polymerase chain reaction data was normalized using both exogenous (cel-miR-39) and endogenous (hsa-miR-93) controls. We have found significantly altered expression of miR-155 and miR-21 in serum of JEV infected patients as compared to healthy controls, revealing their role as a a noninvasive biomarker in JE. A significant correlation between miRNAs and JE was observed that offers the basis for miRNAs to serve as a new component to develop possible therapeutic strategies for JE in near future.

Pro-inflammatory and anti-inflamatory cytokine genes polymorphisms and susceptibility to Japanese encephalitis disease in the North Indian population.

BACKGROUND: Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in many regions of Asia. Cytokines, including pro-inflammatory and anti-inflammatory are key regulators playing a detrimental role in the host response to JE infection, pathogenesis and disease outcome. Evidently, the host's cytokine response is genetically determined, representing the complexity of interindividual differences regarding immune response to viral infection. The current study assesses the association of single nucleotide polymorphisms of classical interleukin IL-1ß and IL-10 with JEV susceptibility and disease severity in north Indian population. METHODS: We performed a case-control study using 85 JE patients and 85 healthy controls. Polymorphisms in the IL-1ß (-511 C/T) and IL-10 (-1082 A/G) genes were genotyped using PCR-RFLP. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed in percentage. RESULTS: The mRNA level of IL-1ß and IL-10 were found significantly increased in JE patients. In severe JE patients, IL-1ß mRNA level was significantly higher with heterozygous (C/T) and homozygous (C/C) genotype compared to wild (T/T) genotype and mRNA level of IL-10 was higher in heterozygous genotype (A/G) compared to wild genotype (A/A). The C/T and C/C genotypes of IL-1ß were significantly associated with higher risk of JE infection (p < 0.05, OR = 7.25 and 4.40) whereas, the A/G genotype of IL-10 was associated with a reduced risk of JEV infection (p < 0.05, OR = 0.30). The C allele of IL-1ß was associated with fever and neck stiffness (p < 0.05) and CT genotype was associated with disease severity and worse outcomes in JE patients. Along with this, IL-10 polymorphism was found associated with fever, and AG genotype was found to be associated with worse disease outcomes such as neurological sequelae (p < 0.05). CONCLUSION: Mutant allele and genotype at IL-1ß (-511 C/T) and IL-10 (-1082 A/G) gene polymorphism show increased expression of IL-1ß and IL-10 in JE patients which contribute to disease severity as well as adverse outcomes of disease. Overall this is the first report from northern India, which shows the association of IL-1ß and IL-10 polymorphisms with JEV infection.

Association of interleukin-6 (174 G/C) and interleukin-12B (1188 A/C) gene polymorphism with expression and risk of Japanese encephalitis disease in North Indian population.

BACKGROUND: Japanese encephalitis is an acute inflammatory disease caused by Japanese encephalitis virus (JEV). In this study we aim to determine the association of IL-6 (174) and IL-12B (1188A/C) gene polymorphisms with JEV susceptibility, disease severity and outcomes in north Indian population. METHODS: This study was performed an equal number of cases and control individuals (125). Gene polymorphism has been analyzed by PCR-RFLP and expression by ELISA. RESULTS: Homozygous(C/C) genotypes of IL-12B were significantly associated with protection in JE infection (p = 0.008, OR = 0.368) whereas IL-6 was not associated with JEV infection (p = 0.269, OR = 1.245). The C allele of IL-6 was associated with protection in JE disease and G/C genotype was associated with outcomes with recovered individuals. CONCLUSION: IL-12B gene polymorphism leads to increase level of IL-12B in JE patients, which can contribute to JE susceptibility and disease severity. IL-6 polymorphism has not been associated with susceptibility of JE. Overall, this is the first information from northern India shows association of IL-6 and IL-12B polymorphisms with JE disease.

Increased serum microRNA-29b expression and bad recovery in Japanese encephalitis virus infected patients; A new component to improve the disease recovery.

BACKGROUND: Japanese encephalitis virus (JEV) is a neurotropic mosquito-borne Flavivirus, mainly prevalent in Asia. It is the most important causative agent of acute viral encephalitis in humans. Recently, micro RNAs are discovered as a key regulator of inflammatory and immune responses in various diseases including neurological and viral infections. Thus, this study was proposed to check whether changes in cellular miRNA expression due to JE virus infection, can be detected in circulation which would be helpful in diagnosis and treatment. METHODS: miRNAs (miR-29b and miR-146a) were analyzed in the serum of JEV infected patients using quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: miR-146a was found significantly decreased (p = 0.0008) in JEV infected patients as compared to healthy controls whereas miR-29b was significantly increased (p = 0.001) in JEV patients recovered with neurological sequelae when compared to those recovered without sequelae. CONCLUSION: In conclusion, miRNA can be measured in serum. Studying microRNAs will provide novel information and help us to identify the components that can serve as biomarkers and can lead to new discovery in controlling disease recovery.

Cytomegalovirus infection in pregnant women and its association with bad obstetric outcomes in Northern India.

BACKGROUND: Cytomegalovirus (CMV) infection during pregnancy is far more complex than other infections, due to ability of the virus to be frequently reactivated during the child bearing age and may vertically transmitted to the developing fetus in spite of maternal immunity. Therefore, in the current study we determined the prevalence of CMV infection in pregnant women and tried to identify the role of maternal CMV infection in adverse pregnancy outcomes in Northern India. In this case-control study, 517 pregnant women, out of them 200 in case group and 317 in the control group. The overall 31.72% (164/517) cases were found with active CMV infection. CMV positivity (p=0.026) was significantly associated with bad obstetric history (75/200, 37.50%) compared to normal pregnancy (89/317, 28.07%). CMV infection was predominantly observed in age group 21-25 years. CMV positivity have been found to be significantly higher in women from rural area as compare to those from urban area (p=0.028). However, no significant difference has been observed in case of occupation, income, and haemoglobin level.

Detection of long term cellular immune response to Japanese encephalitis vaccination using IFN-γ ELIspot assay.

Vaccine is the most effective preventive measure against Japanese Encephalitis infection. Role of IFN-γ expressing T cells for JE virus clearance has been described as a part of cellular immunity. Vaccine induced immunity also involve the cellular immune response, therefore the study was aimed to observe induction and persistence of IFN-γ expressing T cells by IFN-γ ELISpot assay. The cell count increased significantly after 28 (P < 0.0001) days post vaccination, and remained higher at all time points (day 28, day 180, day 360) when compared with prevaccination. This study will be helpful for designing future vaccination strategy and improving vaccine efficacy.

An outbreak of encephalitis associated with echovirus 19 in Uttar Pradesh, India, in 2011.

A sequence-independent single-primer amplification method and a modified enterovirus VP1 gene typing primer were used for identification of echovirus 19 and enterovirus 101, which remained undiagnosed by standard enterovirus molecular typing methods. Six different serotypes were identified during this study, with the predominance of ECV 19 (n = 20) followed by echovirus 21 (n = 3), EV 69 and EV 101 (n = 2 each), coxsackievirus B5 and ECV 27 (n = 1 each). To our knowledge, this is the first report of enteroviruses 69 and 101 in encephalitis cases in India.