Tapan N. Dhole

Japanese encephalitis: a review of the Indian perspective

Japanese encephalitis virus (JEV) causes Japanese encephalitis, which is a leading form of viral encephalitis in Asia, with around 50,000 cases and 10,000 deaths per year in children below 15 years of age. The JEV has shown a tendency to extend to other geographic regions. Case fatality averages 30% and a high percentage of the survivors are left with permanent neuropsychiatric sequelae. Currently, there is no cure for JEV, and treatment is mainly supportive. Patients are not infectious, but should avoid further mosquito bites. A number of antiviral agents have been investigated; however, none of these have convincingly been shown to improve the outcome of JEV. In this review, the current knowledge of the epidemiology and the pathogenesis of this deadly disease have been summarized.

Preventive strategies for frequent outbreaks of Japanese encephalitis in Northern India

Japanese encephalitis (JE) remains the most important cause of acute viral encephalitis and continues to spread to hitherto unaffected regions like Indonesia, Pakistan and Australia. Approximately 60% of the world population inhabits JE endemic areas. Despite its restricted range mostly in the developing countries, a high annual incidence of 50,000 cases and about 10,000 deaths has been reported. Disease can be fatal in 25% cases. Magnitude of the problem is even more alarming since the survivors are left with serious long-term neuropsychiatric sequelae. Almost every two years, epidemics of JE occur in Indian subcontinent with a high mortality. JE virus infection results in different disease manifestations in host from mild subclinical febrile illness to clinical infections leading to encephalitis. No antiviral treatment is so far available for JE. The prevention of JE can be achieved by controlling the vector or by immunization regime. The vector control in the rural areas, which are the worst affected ones, is practically almost impossible. Three vaccines that have been implicated against JE include inactivated mouse brain derived, inactivated cell culture derived and cell culture derived live attenuated JE vaccine. But each has its own limitation. Currently, attempts to synthesize recombinant DNA vaccine are being made. New therapeutics are on the way of development like use of minocycline, short interfering RNA, arctigenin, rosmarinic acid, DNAzymes etc. However, the immune mechanisms that lead to JE are complex and need to be elucidated further for the development of therapeutics as well as safe and efficacious JE vaccines.

Non‐polio enteroviruses in acute flaccid paralysis children of India: Vital assessment before polio eradication

Aim:  This study is an overview of non‐polio enterovirus (NPEV) circulating in North India studied from the perspective of poliomyelitis eradication. Wild polio cases declined because of intensive oral polio vaccine immunization. As we approach global eradication of poliovirus (PV), NPEV causing acute flaccid paralysis (AFP) are equal cause of concern.

Molecular Epidemiological Study of Enteroviruses Associated with Encephalitis in Children from India

ABSTRACT Enteroviruses have been reported in encephalitis cases. However, clinical and epidemiological characteristics of enteroviruses in encephalitis are not fully established. We prospectively investigated 204 children with encephalitis over a period of 2 years (2009 to 2010) for enterovirus. Enterovirus was detected in 45 specimens (22.1%); of these, 40 were typed by seminested reverse transcription-PCR (RT-PCR) and sequencing of the VP1 gene. Molecular typing of enterovirus revealed the predominance of echovirus 21 associated with an epidemic during the rainy seasons of 2010 and the circulation of echovirus 1, coxsackievirus B1, enterovirus 75, enterovirus 76, coxsackievirus B5, and echovirus 19. The nucleotide divergence among echovirus 21 strains was 0 to 2% at the nucleotide level. This study suggests that enterovirus is an important cause of encephalitis in children from India. To our knowledge, this is the first report of echovirus 21 in encephalitis cases worldwide.

Role of Homozygous DC-SIGNR 5/5 Tandem Repeat Polymorphism in HIV-1 Exposed Seronegative North Indian Individuals

Despite multiple sexual exposures to HIV-1 virus, some individuals remain HIV-1 seronegative. Although several genetic factors have been related to HIV-1 resistance, the homozygosity for a mutation in CCR5 gene (the 32-bp deletion, i.e., CCR5-Delta32 allele) is presently considered the most relevant one. The C-type lectins, DC-SIGN (present on dendritic cells and macrophages) and DC-SIGNR (present on endothelial cells in liver and lymph nodes) efficiently bind and transmit HIV-1 to susceptible cell in trans, thereby augmenting the infection. A potential association of the DC-SIGN and DC-SIGNR neck domain repeat polymorphism and risk of HIV-1 infection is currently under debate. To determine the influence of host genetic factors on HIV-1 resistance, we conducted genetic risk association study in HIV-1-exposed seronegative (n = 47) individuals, HIV-1 seronegative (n = 262) healthy control, and HIV-1-infected seropositive patients (n = 168) for polymorphism in neck domain of DC-SIGN and DC-SIGNR genes. The DC-SIGN and DC-SIGNR genotypes were identified by polymerase chain reaction method in DNA extracted from peripheral blood and confirmed by sequencing. Fisher exact or χ2 test was used for static analysis. DC-SIGN genotype and allele distribution was fairly similar in HIV-1-exposed seronegative, HIV-1 seropositive, and HIV-1 seronegative control. There was no statistical significance in the differences in the distribution of DC-SIGN genotypes. A total of 13 genotypes were found in DC-SIGNR neck repeat region polymorphism. Among all the genotypes, only 5/5 homozygous showed significant reduced risk of HIV-1 infection in HIV-1-exposed seronegative individuals (p = 0.009). A unique genotype 8/5 heterozygous was also found in HIV-1 seropositive individual, which is not reported elsewhere.

Kinetics of cytokine profile during intraperitoneal inoculation of Japanese encephalitis virus in BALB/c mice model.

Infection with Japanese encephalitis virus (JEV) is mostly asymptomatic/subclinical in 90% of the individuals. Host immune response during subclinical JEV infection is poorly understood. We assessed iNOS, IFN-gamma, TNF-alpha, IL-10 and IL-4 production in spleen, brain and sera of intraperitoneally challenged BALB/c mice by RT-PCR and ELISA along with brain histopathology at different days post inoculation (d.p.i.). In spleen of virus infected mice, expression of all cytokines including iNOS mRNA were upregulated till 5d.p.i. followed by decline. At 5d.p.i., IL-10 expression outcompeted TNF-alpha, IFN-gamma and IL-4. However, in the virus infected mice sera, IL-4 production predominated over TNF-alpha and IL-10 at 5d.p.i. Conversely, cytokines expression and iNOS mRNA remained unchanged in the brain of virus infected mice from 1 to 7d.p.i. A significant increase in the cytokine expression was observed at 11d.p.i. (P<0.05) in virus infected mice brain, with the predominance of IL-10 along with the presence of meningeal inflammation and viral RNA by histology and RT-PCR, respectively. We report a biased pattern of cytokine production in sera, brain and spleen of mice intraperitoneally challenged with JEV. IL-10 exerts neuroprotective function during JEV and regulates deleterious effects of proinflammatory cytokines; however, its mechanism needs further investigation.

Emb nucleotide polymorphisms and the role of embB306 mutations in Mycobacterium tuberculosis resistance to ethambutol

The emb locus has been considered a target for ethambutol (EMB). Substitutions of codon 306 in Mycobacterium tuberculosis embB have been shown to be the most frequent and predictive mutations for EMB resistance; however, recent reports question the biological role of this mutation. We sequenced embB, embC and embR of 44 EMB-resistant M. tuberculosis strains and found that 30/44 (68.1%) strains had a resistance-associated mutation in one of the three genes sequenced. The majority of these mutations resulted in amino acid replacements at codon 306, 368, 378, and 406 of EmbB. The most common mutation reported in EmbC was at codon 270, followed by mutation at codon 297. Novel mutations were also reported in EmbR. Mutations in embC and embR were usually present together with mutations in embB. We found 41/44 EMB-resistant isolates to be resistant to other antituberculosis drugs as well. Our data confirm that mutation at emb306 does not confer resistance to EMB but is a rather common polymorphism in clinical strains of M. tuberculosis predisposing them to the development of any type of drug resistance.

Molecular epidemiology of Chikungunya virus: Mutation in E1 gene region

Chikungunya virus is a mosquito-transmitted RNA virus and emerging as a pathogen that has a major public health impact because of the high morbidity including high fever, headache, rash, nausea, vomiting, myalgia, arthralgia with or without neurological manifestation or fulminant hepatitis. One hundred fifty-one patient samples were analyzed during the years 2006-2008, and compared conventional tests and CCRT-PCR (cell culture RT PCR). The conventional tests included ELISA, inoculation into C6/36 cell line and CPE were examined by PCR after RNA extraction. A total of 20/151 (13.2%), 8/151 (5.29%) and 7/151 (4.6%) samples were found to be positive by ELISA, cell culture and PCR, respectively. While 7/20 (35%) of the samples were positive by CCRT_PCR when ELISA 20 positive samples were detected. A total of 5/7 positive strains were sequenced in the E1 gene region. Remarkable changes (M269V, D284E, P294L, S295F, A316V, V322A, and C328W) were observed in the membrane fusion glycoprotein E1. These unique molecular features of the isolates with the continuing epidemic demonstrated high evolutionary potential and thereby indicating higher virulence.

Association of RANTES −403 G/A, −28 C/G and In1.1 T/C polymorphism with HIV‐1 transmission and progression among North Indians

The relationships between host immune factors and HIV‐1 disease progression are still in dispute. The RANTES SNPs exhibit distinct ethnic distribution and are associated with different effects on the course of HIV infection. Therefore, impact of RANTES gene polymorphism on HIV‐1 transmission and progression needs to be evaluated. The RANTES genotypes were identified by PCR‐RFLP method and confirmed by sequencing in HIV‐1 seronegative (HSN; n = 315), HIV‐1 exposed seronegative (HES; n = 47) and HIV‐1 seropositive (HSP; n = 196) patients classified into different clinical stages (i.e. Stages I, II, III). Fisher exact test was used for statistical analysis and Arlequin software for haplotype analysis. RANTES allele −403G, −28C and In1.1 T were the predominant allele in the subject studied. HSP group have higher frequency of RANTES In1.1 T allele compared with HSN (91.32% vs. 86.19%; P = 0.013) and HES (91.32% vs. 78.72%; P = 0.001). Higher frequency of RANTES In1.1 C allele in Stage III was observed, compared with Stage I (14.28% vs. 6.39%) and was significantly associated with high risk (P = 0.047, OR = 2.439, C.I. = 1.061–5.609). Haplotype II (ACT) was significantly higher in HSP compared with HSN (9.69% vs. 1.58%) and associated with high risk (P < 0.001, OR = 6.655, C.I. = 2.443–18.132). There were no significant differences in RANTES −403 A/G and −28 C/G genotype and allele distribution in all the groups compared. Our results implicate that RANTES In1.1 T allele and haplotype II (ACT) may be a risk factor for HIV‐1 transmission while RANTES In1.1 C allele may be risk factor for disease progression among North Indians. J. Med. Virol. 80: 1133–1141, 2008.

Genetic Association of IL-10 Gene Promoter Polymorphism and HIV-1 Infection in North Indians

IntroductionCytokines play a significant role in host immune defense. IL-10 is an anti-inflammatory, immunomodulatory cytokine that can both stimulate and suppress the immune response and inhibits HIV-1 replication in vivo. Interindividual variations in IL-10 production were genetically contributed to polymorphisms within IL-10 promoter region.AimsThe aim of this study was to investigate the association of IL-10 gene promoter −1082 G/A, −819 C/T, and 592 C/A polymorphism on HIV-1 transmission /progression in North Indian individuals.Patients and MethodsA total of 180 HIV-1 seropositive (HSP) stratified on the basis of disease severity (stage I, II, and III), 50 HIV-1 exposed seronegative (HES) and 305 HIV-1 seronegative (HSN) individuals were genotyped for IL-10 gene promoter by polymerase chain reaction-restriction fragment length polymorphism. A suggestive evidence of association was obtained for IL-10 592 C/A promoter polymorphism at the level of allele and genotype distribution. The frequency of IL-10 592 A allele and genotype was significantly increased in HSP compared to HSN (p = 0.013; OR = 1.412 and p = 0.034; OR = 1.685 respectively). Further comparison in between different clinical stages of HIV-1 infected patients of IL-10 592 A allele and genotype revealed a significant increase in its frequency in the stage III compared with those together in stage I (p = 0.004, OR = 2.181 and p = 0.002, OR = 4.156, respectively). This study reports for the first time that IL-10 gene promoter 592 C/A polymorphism may be a risk factor for HIV-1 transmission/progression in HIV-1 infected North Indian individuals.