Evidence of hepatitis A virus infection in the patients with acute encephalitis syndrome in Gorakhpur region, North India.

The etiological agent remained unidentified in a large number of patients hospitalized for acute encephalitis syndrome (AES) in 2008-2009 in Uttar Pradesh and Bihar, north India. All patients were found to present with fever and altered sensorium, while 28%, 19% and 13% showed hepatomegaly, splenomegaly and meningeal signs, respectively. Involvement mostly of children with abnormal hepatic features prompted us to undertake an exploratory study on viral hepatitis A to determine its association, if any, with hepatic derangements. AES patients (n = 2515) and healthy children (n = 167) were investigated for the presence of serum anti-hepatitis A virus (anti-HAV) IgM and anti-Japanese encephalitis (anti-JE) virus IgM by ELISA. Cerebrospinal fluids (CSFs, n = 595) and rectal swabs (n = 182) were examined for anti-HAV IgM and/or HAV RNA. Anti-HAV IgM was detected in the sera of 14.6% patients as against 6.6% of healthy children (p = 0.0042). Anti-JE virus IgM positivity was Keywords: acute encephalitis syndrome; cerebrospinal fluid; hepatitis A virus; anti-HAV IgM; non-Japanese encephalitis.

Changing clinico-laboratory profile of encephalitis patients in the eastern Uttar Pradesh region of India.

A cross-sectional study was done on 100 consecutive paediatric patients presenting with acute encephalitis syndrome. The clinico-laboratory features of all patients were recorded in a prestructured performa. Cerebrospinal fluid and serum samples were tested for: Japanese encephalitis (JE) virus; Chandipura virus; coxsackie virus; dengue virus; enterovirus 76; and West Nile virus. Twenty-two (22.0%) patients were confirmed JE cases and 17% had parasitic or bacteriological aetiology. The remaining 61 cases (61.0%) in which no viral aetiological agent was found were grouped as non-JE cases. Peripheral vascular failure, splenomegaly and hypotonia were distinguishing clinical features found in the non-JE patients. A high mortality of 26.5% was seen in patients with confirmed or presumptive viral encephalitis (22/83). A fatal outcome was independently associated with peripheral vascular failure and pallor at the time of admission. Early recognition of these signs may help clinicians to manage these cases.

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)-ß remained unchanged. However, IL-1ß 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-1ß and its association with WNV pathogenesis.

Immunomodulatory cytokines determine the outcome of Japanese encephalitis virus infection in mice.

Japanese encephalitis virus (JEV) induces an acute infection of the central nervous system, the pathogenic mechanism of which is not fully understood. To investigate host response to JEV infection, 14-day-old mice were infected via the extraneural route, which resulted in encephalitis and death. Mice that received JEV immune splenocyte transfer were protected from extraneural JEV infection. Pathology and gene expression profiles were then compared in brains of mice that either succumbed to JEV infection or were protected from infection by JEV immune cell transfer. Mice undergoing progressive JEV infection had increased expression of proinflammatory cytokines, chemokines, and signal transducers associated with the interferon (IFN) pathway. In contrast, mice receiving immune cell transfer had increased production of the Th2 cytokine IL-4, and of IL-10, with subdued expression of IFN-gamma. We observed IL-10 to be an important factor in determining clinical outcome in JEV infection. Data obtained by microarray analysis were further confirmed by quantitative RT-PCR. Together, these data suggest that JEV infection causes an unregulated inflammatory response that can be countered by the expression of immunomodulatory cytokines in mice that survive lethal infection.

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.

Horizontal and vertical transmission of dengue virus type 2 in highly and lowly susceptible strains of Aedes aegypti mosquitoes.

Isofemale lines of Aedes aegypti mosquitoes highly and lowly susceptible to dengue type 2 (DEN-2) virus (DEN(h) and DEN(l), respectively) were established by oral feeding and individual rearing. The susceptibility at F13 generation was found to be 61% and 25% for the DEN(h) and DEN(l) line, respectively. The virus-infected mosquito females were allowed to probe on bovine albumin phosphate saline pH 7.2 (BAPS) through membrane feeders. The presence of virus in the probed BAPS was determined either by ELISA or by intrathoracic (i.t.) inoculation of mosquitoes or by both methods. The rate of oral transmission of virus was found to be 2 times higher in the DEN(h) isofemale line than in the DEN(l) one. Similarly, vertical transmission rate of the virus was found to be 7 times higher in the DEN(h) line. When batches of eggs obtained from infected female mosquitoes were allowed to hatch after two months the vertical transmission rate of the virus was very high. It is possible that, at room temperature, the virus gets an opportunity to multiply and increase its copy number in the quiescent embryos. The progeny obtained from the infected mosquitoes was found to be capable of transmitting the virus horizontally when allowed to probe on BAPS through the membrane feeder. This is the first report demonstrating horizontal transmission of DEN-2 virus by mosquitoes infected through vertical transmission. The higher vertical transmission rate of the virus in the progeny obtained from the eggs dessicated for a longer time and the horizontal transmission of the virus from the progeny is of very high epidemiological significance.

Monoclonal antibody raised against envelope glycoprotein peptide neutralizes Japanese encephalitis virus.

Epitopes on envelope glycoprotein of Indian strain of Japanese encephalitis virus were delineated by prediction methods. Monoclonal antibodies (MAb) raised against a putative B cell epitope peptide, reacted with the virion in ELISA and immunofluorescence assays. One MAb was also able to neutralize the virus. The reactivity of this MAb against a Sri Lankan strain was checked, since this strain had a substitution within the B cell epitope at position Egp 153 (G-->W). The MAb was able to bind to, but was not able to neutralize the Sri Lankan isolate. The data indicated that the predicted B cell epitope is a neutralizing epitope and may be included in a peptide-based vaccine against the virus.

Recovery from stress in two different postures and in Shavasana--a yogic relaxation posture.

The recovery from induced physiological stress in Shavasana (a yogic relaxation posture) and two other postures (resting in chair and resting supine posture) was compared. Twenty one males and 6 females (age 21-30 yrs) were allowed to take rest in one of the above postures immediately after completing the scheduled treadmill running. The recovery was assessed in terms of Heart Rate (HR) and Blood pressure (BP). HR and BP were measured before and every two minutes after the treadmill running till they returned to the initial level. The results revealed that the effects of stress was reversed in significantly (P < 0.01) shorter time in Shavasana, compared to the resting posture in chair and a supine posture.

Incorporation of T cell counterparts in the fusion partners for generation of human monoclonal antibodies from Staphylococcus aureus stimulated B lymphocytes.

B cell growth and differentiation into immunoglobulin secreting cells is controlled by various cytokines and cell to cell contact with T cells. Fusion partner for human hybridoma therefore should accommodate all or some of these signaling systems to overcome the unique situation of MHC incompatibility, need for specific growth factors simultaneously taking into consideration the downstream processing of the product for the clinical use. We have thus directed our efforts towards the development of a fusion partner which would not need Epstein-Barr virus transformation of B cells prior to fusion. A nontransforming mitogen, formalinized Staphylococcus aureus (FSTA) was used for stimulating human B cells. Successful production of human IgM monoclonal antibody was achieved by incorporating Jurkat-4 cells in existing mouse human heterohybrid through fusion of these cells followed by fusion with human B cells. To accommodate chromosomes of both T and B cells after fusion, human myeloid precursor cells KG1a, and to incorporate T cell, HuT78 cells were fused. CD34+ and CD4+ hybrid of KG1a and HuT 78 cells-434 AM-when used as fusion partner could allow secretion of MAbs, however growth potential was low. SP2/0 cells were then incorporated in 434 AM cells to give myeloma environment to fused human B cells. Rabies virus neutralizing human IgG MAb secreting clone was generated by fusing FSTA stimulated human B cells with this fusion partner.

Indigenous anti-hepatitis A virus IgM capture ELISA for the diagnosis of hepatitis A.

Anti-hepatitis A virus IgM capture ELISA was developed by using the reagents produced in the NIV laboratory. The major reagents of the assay were anti-human IgM antibody, hepatitis A virus (HAV) and anti-HAV IgG-horse radish peroxidase (HRP) conjugate. Of these, anti-human IgM antibodies were generated in rabbit against IgM secreted by human hybridoma clone(G3). HAV was derived from buffalo green money kidney cell line infected with HM-175 strain. Virus purified from the cell lysates was used for immunization of rabbits and guinea-pigs. There was very low anti-HAV response. A seropositive rhesus monkey was inoculated with monkey adapted strain of HAV to boost the anti-HAV antibody titre. Anti-HAV IgGs derived from hyperimmune sera of monkey and hepatitis A patient were conjugated with HRP. The preparations of conjugate--particularly human antibody--HRP conjugate yielded highly satisfactory results in anti-HAV capture ELISA. The assay appears to be specific, sensitive and quick and is useful in differentiating acute HAV infection from other acute infections caused by B, E and non-A non-B hepatitis viruses.

Xenogenic transfer of human lymphocytes in tolerized mice.

Female nu/+ or BALB/c mice were immunized with human peripheral blood lymphocytes (PBL) before and during pregnancy. Pups born to these mothers were inoculated with human PBL or human fetal bone marrow and thymus cells. Tolerization of the pups to human PBL was observed without graft-versus-host reaction. Presence of human immunoglobulins was observed in the pups for 3-4 weeks. Human T cells also could be detected for a period of 3-4 months in these mice.

Analysis of computer-predicted antibody inducing epitope on Japanese encephalitis virus.

Theoretical methods to delineate antibody inducing epitopes have been employed to predict antigenic determinants on envelope glycoprotein (gpE) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV viruses. A predicted region on JE virus gpE 74CPTTGEAHNEKRAD87 was synthesized, conjugated to KLH (KLH-peptide) and used in immunization of mice. A mouse monoclonal antibody (MoAb IVB4) reactive to the peptide was also found to react with native JE virus gpE. Characterization of the idiotypic (ID) determinants with the help of polyclonal domain-specific anti-ID antibodies revealed that polyclonal anti-KLH-peptide antibodies and MoAb IVB4 are flavivirus-cross-reactive to Hx and NHx domains, respectively. The region 74-87 in JE virus gpE has been mapped as a linking area between Hx and NHx domains. Reactivity of the peptide with sera from JE patients and vaccinees also indicated the feasibility of using predicted peptides for diagnostic and prophylastic purposes.

Detection of virus specific IgG subclasses in Japanese encephalitis patients.

During the Japanese encephalitis (JE) epidemic in 1988 at Gorakhpur, Uttar Pradesh, 34 cerebrospinal fluid (CSF) samples with 16 matching sera from 34 anti JEV IgM positive (confirmed JE) and 24 CSF samples with 4 matching sera from 24 anti JEV IgM negative (clinical encephalitis) patients were collected and tested for presence of JEV specific IgG by ELISA. Eighteen CSF samples and 8 matching sera from confirmed JE and 5 CSF samples and one matching serum from clinical encephalitis patients positive for JEV specific IgG were further assayed for subclass specificity using specific murine monoclonal antibodies. Almost all the samples exhibited IgG1 as the virus specific subclass. In addition to IgG1, one serum and one CSF sample each from two different confirmed JE patients showed the presence of virus specific IgG4 and IgG3 respectively. Half of the confirmed JE and clinical encephalitis patients exhibited intrathecal synthesis as evident from either elevated IgG index or CSF IgG/CSF albumin ratio. Most of the patients who recovered had predominantly virus specific IgG1 in CSF. It seems likely that IgG1 might have a protective role in clearance of virus from the central nervous system.

Nuclear immunofluorescence in porcine kidney cells infected with Japanese encephalitis virus.

Acetone-fixed porcine stable kidney (PS) cells infected with Japanese encephalitis (JE) virus were stained in indirect fluorescent antibody (FA) assay with anti-JE virus monoclonal (MoAb) and polyclonal (immune PF) antibodies. First positive immunofluorescence (IF) occurred in the cytoplasm with MoAb Hs-1 (anti-envelope, JE-specific) and immune PF after 7 hr post-infection (p. i.); it became prominent by 15 hr to 48 hr (maximum) when cells reacted strongly also with MoAb Hx-3 (flavivirus crossreactive epitope). In addition, 15 to 20% of the infected cells, which revealed positive cytoplasmic IF, showed intranuclear IF with Hs-1, Hx-3, and immune PF by 20 to 24 hr p.i. By 48 hr, the intranuclear IF was not observed or became diminished. These observations indicate that the JE virus specific epitope Hs-1 appeared first followed by the flavivirus cross-reactive epitope Hx-3. Nuclei of the infected cells seem to play some role in the replication of JE virus.

Recognition of helper T cell epitopes in envelope (E) glycoprotein of Japanese encephalitis, west Nile and Dengue viruses.

Helper T (Th) cell antigenic sites were predicted from the primary amino acid sequence (approximately 500 in length) of the envelope (E) glycoprotein (gp) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV flaviviruses. Prediction of Th epitopes was done by analyzing the occurrence of amphipathic segments, Rothbard-Taylor tetra/pentamer motifs and presence of alpha helix-preferring amino acids. The simultaneous occurrence of all these parameters in segments of E gp were used as criteria for prediction as Th epitopes. Only one cross reactive epitope was predicted in the C-terminal region of the E gp predicted segments of all flaviviruses analyzed. This region is one of the longest amphipathic stretch (approximately from 420 to 455) and also has a fairly large amphipathic score. Based on the predicted findings three selected peptides were synthesized and analyzed for their ability to induce in vitro T cell proliferative response in different inbred strains of mice (Balb/c, C57BL6, C3H/HeJ). Synthetic peptide I and II prepared from C-terminal region gave a cross reactive response to JE, WN and Den-II in Balb/c and C3H/HeJ mice. Synthetic peptide III prepared from N-terminal region gave a proliferative response to DEN-II in Balb/c strain only, indicating differential antigen presentation.

Selection of a neutralization-escape variant strain of Japanese encephalitis virus using monoclonal antibody.

An Indian strain of Japanese encephalitis virus (JEV), 733913, a human isolate from Bankura, West Bengal in 1973, with all the functional epitopes designated by a panel of murine monoclonal antibodies (MAbs), was treated with one of the JEV specific HI reactive MAb(Hs-I). This led to selection of a neutralization-escape variant which showed loss of reaction to three different MAbs belonging to the same domain (Hs) and assumed similar characteristics to another JEV strain (755468) also isolated from Bankura in 1975 from mosquitoes. It is possible that selection of such variant might occur in presence of pre-existing JE antibody (Hs-I type) in pigs which are amplifying hosts of JEV. Subsequent dissemination of such variant virus could occur through mosquitoes.

Interferon producing capacity (IPCA) of peripheral mononuclear cell in oral cancer patients.

Interferon producing capacity (IPCA) of peripheral blood mononuclear cells is ability of these cells to produce IFN with suitable IFN inducer. In Vitro IPCA of cryopreserved mononuclear cells (MNC) from peripheral blood of 46 oral cancer patients was studied and was compared to that of healthy, age matched donors. New castle disease virus (NDV) and staphylococcal enterotoxin A (SEA) were used as inducers for evaluating Type alpha IPCA (AIPCA) and Type gamma IPCA (GIPCA) respectively. Age of healthy donors did not influence the AIPCA or GIPCA. Oral cancer patients demonstrated significant low AIPCA (P less than 0.05) (Range Healthy donors 3.5 to 4.6 log 10Iu/ml Oral Cancer 2.0 to 4.6 log 10Iu/ml GIPCA was found to be further depressed (P less than 0.005) (Range Healthy donors 2.87 to 3.6 Log 10 U/ml, Oral cancer 1.7 to 3.6 log 10 U/ml. The depression in IPCA was found to be more pronounced in advanced stage of disease.