Gajanan N. Sapkal

Enteroviruses in Patients with Acute Encephalitis, Uttar Pradesh, India

An outbreak of viral encephalitis occurred in northern India in 2006. Attempts to identify an etiologic agent in cerebrospinal fluid by using reverse transcription–PCR showed positivity to enterovirus (EV) in 66 (21.6%) of 306 patients. Sequencing and phylogenetic analyses of PCR products from 59 (89.3%) of 66 specimens showed similarity with EV-89 and EV-76 sequences.

Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients

During investigations into the outbreak of encephalitis in 1996 in the Kerala state in India, an arbovirus was isolated from a Culex tritaeniorhynchus mosquito pool. It was characterized as a Japanese encephalitis and West Nile virus cross-reactive arbovirus by complement fixation test. A plaque reduction-neutralization test was performed using hyperimmune sera raised against the plaque-purified arbovirus isolate. The sera did not show reactivity with Japanese encephalitis virus and were weakly reactive with West Nile virus. Complete open reading frame sequence analysis characterized the arbovirus as Bagaza virus (BAGV), with 94.80 % nucleotide identity with African BAGV strain DakAr B209. Sera collected from the encephalitic patients during the acute phase of illness showed 15 % (8/53) positivity for anti-BAGV neutralizing antibodies. This is the first report of the isolation of BAGV from India. The presence of anti-BAGV neutralizing antibodies suggests that the human population has been exposed to BAGV.

Japanese encephalitis virus produces a CD4+ Th2 response and associated immunoprotection in an adoptive-transfer murine model

Japanese encephalitis is an acute infection of the central nervous system caused by Japanese encephalitis virus (JEV). The importance of an effective humoral response in preventing JEV infection has already been established, although the contribution of cellular immunity remains unclear. This study used an experimental murine model to understand the protective effects of cell-mediated immunity in JEV infection. Fourteen-day-old mice adoptively transferred with JEV-immune splenocytes were found to be protected from peripheral JEV challenge. The survival rate was reduced when transferred cells were depleted of their CD4(+) T-cell population. Correspondingly, increased protection was observed when JEV-primed isolated CD4(+) T cells were transferred compared with isolated CD8(+) T cells. Mice protected from JEV infection by the adoptive transfer of JEV-immune splenocytes had higher levels of immunomodulatory cytokines and decreased expression of pro-inflammatory cytokines. Concurrent with the increase in Th2 cytokines, JEV-specific IgM and IgG1 antibody titres were found to be elevated in protected mice. Taken together, these data indicate a definite role for CD4(+) T cells in protection from lethal JEV infection in naïve 14-day-old mice. Induction of a Th2 cytokine response and IgG1 antibody probably achieves an immunomodulatory effect that results in the enhanced survival of these animals.

West Nile encephalitis outbreak in Kerala, India, 2011

BACKGROUND An outbreak of acute encephalitis syndrome (AES) was reported in Kerala in India in May 2011. The outbreak features were unusual in terms of seasonality, geographical distribution, age group, and clinical manifestations in comparison to the epidemiological features of Japanese Encephalitis. OBJECTIVE To detect the etiology of the acute encephalitis syndrome outbreak. STUDY DESIGN Investigation of outbreak was undertaken by collection of brief clinical history and epidemiological details along with the specimens for viral diagnosis. The serum/CSF samples (patients=208) received from the sentinel hospitals were subjected to IgM capture ELISA and RT-PCR specific for Japanese encephalitis (JE) virus and West Nile virus (WNV). The JE/WN IgM positive samples were further tested by serum neutralization assay for the presence of JE and WNV specific neutralizing antibody. RESULT Most of the affected patients were aged above 15 years. No spatial clustering of the disease was noticed. Cases were observed in premonsoon and early monsoon season and in JE non-endemic area of Kerala. A total of 47 patient samples were positive for in-house JE IgM capture ELISA and WNV IgM capture ELISA. Serum neutralization assay result revealed that 32 of 42 (76.19%) sera were positive for WNV neutralization antibodies. WNV was isolated from a clinical specimen. Phylogenetic analysis of WNV envelope gene revealed 99% homology with Russian Lineage 1 WNV. CONCLUSION West Nile virus (WNV) etiology was confirmed by virus isolation and detection of virus specific antibody from clinical specimen. Phylogenetic analysis grouped the current strain in lineage I West Nile virus.

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes.

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.

Pathogenic and vaccine strains of Japanese encephalitis virus elicit different levels of human macrophage effector functions

In India, Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Macrophages support various neurotropic viruses and influence the immune response. However, the functional status of human macrophages during JEV infection remains unidentified. In this study, we examined the cytokine response and co-stimulatory marker levels in primary human monocyte derived macrophages (MDMs) infected with JE057434 (neurovirulent, primary clinical isolate) or SA14-14-2 (non-neurovirulent, live-attenuated vaccine) JEV strains. We also examined the differential susceptibility of these JEV strains to antiviral effects of interferon and nitric oxide. The results indicate that both JEV strains are capable of inducing various cytokines (type-I IFN, TNFα, IL6 and IL8) and co-stimulatory molecules (CD86 and CD80) in MDMs. However, they varied in replication potential and corresponding interferon sensitivity. SA14-14-2 was highly susceptible to interferon and nitric oxide when compared to JE057434. Thus, reduction in infectious virion production and increased sensitivity of SA14-14-2 towards interferon in MDMs could potentially play a role in limiting viral spread to additional target tissues.

Zika virus: Indian perspectives.

The emergence of Zika virus (ZiV), a mosquito borne Flavivirus like dengue (DEN) and chikungunya (CHIK), in Brazil in 2014 and its spread to various countries have led to a global health emergency. Aedes aegypti is the major vector for ZiV. Fast dissemination of this virus in different geographical areas posses a major threat especially to regions where the population lacks herd immunity against the ZiV and there is abundance of Aedes mosquitoes. In this review, we focus on current global scenario, epidemiology, biology, diagnostic challenges and remedial measures for ZiVconsidering the Indian perspective.

Neutralization escape variant of West Nile virus associated with altered peripheral pathogenicity and differential cytokine profile

In order to understand the factors influencing pathogenicity of a virus, two neutralization escape (NE) variants were selected from wild type lineage 1 West Nile virus (WNV) 68856 strain pathogenic by intra-peritoneal (i.p.) route using monoclonal antibodies (MAbs) against envelope (E) protein. Both NE IF1A7 1.1 and NE IVC3F10 1.2 were resistant to neutralization and were neurovirulent by intra-cranial (i.c.) inoculation. Growth kinetics in porcine stable (PS) kidney and baby hamster kidney (BHK) cells was unchanged. In contrast to parent WNV only NE IF1A7 1.1 failed to cause lethal encephalitis on i.p. inoculation and was non pathogenic. NE IF1A7 1.1 variant showed delayed replication kinetics in murine peritoneal exudate cells (PEC) and Neuro 346 cells in vitro. In comparison with parent WNV and NE IVC3F10 1.2 variant, non pathogenic variant exhibited significantly reduced tumour necrosis factor α (TNF-α) induction in infected animals and PEC. Other cytokines like Interleukin (IL)-10, IL-6 and Interferon (IFN)-β remained unchanged. However, IL-1β did not follow the pattern and was higher only in parent WNV-infected PEC. The E gene sequences of these NE variants showed three common amino acid substitutions at residues E50, E89 and E242. A unique E156 (ser→pro) substitution in NE IF1A7 1.1, was absent in NE IVC3F10 1.2 variant suggested probable virulence marker. Our data indicates possible role of WNV E protein in induction of TNF-α and IL-1β and its association with WNV pathogenesis.

Evaluation of Japanese encephalitis virus polytope DNA vaccine candidate in BALB/c mice

Vaccination is the most important measure available to control the spread of Japanese encephalitis virus. We have previously described the in silico design, in vitro and primary in vivo analyses of polytope construct (P-JEV). Present study indicates that, P-JEV induces significant specific humoral and cellular immune responses in BALB/c mice. Mice were immunized intra-dermally with 1μg plasmid DNA using gene gun and boosted twice. After the second booster, all the mice seroconverted and developed JEV neutralizing antibodies. Enhancement of post-challenge neutralizing antibody titres indicates the dominant role of anamnestic antibody-mediated protection in the mice JEV challenge model. Our study provides an insight demonstrating, intra-dermal DNA administration led to higher seroconversion rates and potentiated T(H)2 type of immune responses. The P-JEV construct is thus capable of generating protective neutralizing antibodies in mice and prime the immune system effectively against subsequent exposure to the virus.

Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus.

Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection.