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 ICAM-1 (K469E) and MCP-1-2518 A > G polymorphism with risk of Japanese encephalitis in North Indian population.

BACKGROUND: Japanese encephalitis virus (JEV) is most important cause of viral encephalitis worldwide. The pathogenesis of this is probably attributed to the host genetic makeup. Intercellular adhesion molecule-1 (ICAM-1) and monocytes chemoattractant protein-1 (MCP-1) play a vital role in host defense mechanism against flavivirus causing encephalitis. We assessed the possible genetic association between ICAM-1 (K469E) and MCP-1-2518 A > G polymorphisms and Japanese Encephalitis in North Indian population. METHODS: We studied ICAM-1(K469E) and MCP-1-2518 A > G polymorphisms with the help of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Expression of ICAM-1 and MCP-1 were determined at mRNA and protein levels in JE patients and healthy controls by real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). RESULTS: Homozygous (E/E) genotype of ICAM-1 was associated with clinical severity (p = 0.015) and outcome (p = 0.04) of JE, whereas, heterozygous (A/G) genotype of MCP-1-2518 A > G was associated with outcome in JE patients (p = 0.01). Among severe cases of JE, a higher level of ICAM-1 was observed in patients with E allele (E/K + E/E) of ICAM-1 (K469E) than non-E allele (K/K). The level of MCP-1 was found significantly increased in JE patients with homozygous (G/G) genotype when compared to wild (A/A) genotype of MCP-1-2518 A > G (p = 0.03). CONCLUSION: ICAM-1 (K469E) and MCP-1-2518 A > G polymorphisms lead to increased level of ICAM-1 and MCP-1 in Japanese Encephalitis which may be associated with severity as well as an adverse outcome of the disease. ICAM-1 (K469E) polymorphism may affect host susceptibility to Japanese encephalitis in North Indian population.

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.

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.

Comparative transcriptome profiling of high and low oil yielding Santalum album L.

East Indian Sandalwood (Santalum album L.) is highly valued for its heartwood and its oil. There have been no efforts to comparative study of high and low oil yielding genetically identical sandalwood trees grown in similar climatic condition. Thus we intend to study a genome wide transcriptome analysis to identify the corresponding genes involved in high oil biosynthesis in S. album. In this study, 15 years old S. album (SaSHc and SaSLc) genotypes were targeted for analysis to understand the contribution of genetic background on high oil biosynthesis in S. album. A total of 28,959187 and 25,598869 raw PE reads were generated by the Illumina sequencing. 2.12 million and 1.811 million coding sequences were obtained in respective accessions. Based on the GO terms, functional classification of the CDS 21262, & 18113 were assigned into 26 functional groups of three GO categories; (4,168; 3,641) for biological process (5,758;4,971) cellular component and (5,108;4,441) for molecular functions. Total 41,900 and 36,571 genes were functionally annotated and KEGG pathways of the DEGs resulted 213 metabolic pathways. In this, 14 pathways were involved in secondary metabolites biosynthesis pathway in S. album. Among 237 cytochrome families, nine groups of cytochromes were participated in high oil biosynthesis. 16,665 differentially expressed genes were commonly detected in both the accessions (SaHc and SaSLc). The results showed that 784 genes were upregulated and 339 genes were downregulated in SaHc whilst 635 upregulated 299 downregulated in SaSLc S. album. RNA-Seq results were further validated by quantitative RT-PCR. Maximum Blast hits were found to be against Vitis vinifera. From this study, we have identified additional number of cytochrome family in high oil yielding sandalwood accessions (SaHc). The accessibility of a RNA-Seq for high oil yielding sandalwood accessions will have broader associations for the conservation and selection of superior elite samples/populations for further genetic improvement program.

Assessment of morphological and genetic variability through genic microsatellite markers for essential oil in Sandalwood (Santalum album L.).

Sandalwood (Santalum album L; family Santalaceae) is a highly significant aromatic oil yielding tree. It is valued for two important traits, heartwood and essential oil obtained from the heartwood. This study was proposed to assess the morphological and genetic variability of sandalwood accessions. For this, genotypes were randomly selected (n = 177) from the 14 populations from three states in southern India. The total heartwood oil content and quality was estimated by UV method and GC-MS. Total 14 oil-specific genic SSR markers were procured to evaluate the genetic diversity among the sandalwood accessions. Total core size, heartwood content, and oil of S. album ranged from 4.4 to 19.1 cm; 0.0 to 17.3 cm; and 0.0 to 5.96% with covariance 27.61, 85.25, and 73.12% followed by mean 9.74, 3.77, and 2.71, respectively. Genetic diversity estimates were highly polymorphic in terms of Na 7.28, Ne 5.89, He 8.0 PIC 0.891, with little Ho, and F-0.922. AMOVA revealed that minimal genetic variation among populations and highest variation was found among individuals with Nm (58.4). The UPGMA reveals the cluster favored the grouping pattern by the PCA analysis. Structure and PCA analysis clustered the entire populations into two major groups with F ST 0.046 in which population of Kerala and Karnataka were pure and Telangana accessions were found admixtures. No significant correlation (r 2 = 0.23, P = 0.00) was observed between heartwood oil and genetic structures. A high degree of transferability of genic markers would facilitate the assessment of novel genotypes for future tree improvement and conservation of Sandalwood populations.