Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: Requirement for L3T4(+) T cells

The protective ability of cytotoxic T cells (CTL) raised in vitro against Japanese encephalitis virus (JEV) was examined by adoptive transfer experiments. Adoptive transfer of anti-JEV effecters by intracerebral (i.c.) but not by intraperitoneal (i.p.) or intravenous (i.v.) routes protected adult BALB/c mice against lethal i.c. JEV challenge. In contrast to adult mice, adoptive transfer of anti-JEV effecters into newborn (4-day-old) and suckling (8-14-day-old) mice did not confer protection. However, virus-induced death was delayed in suckling mice compared to newborn mice upon adoptive transfer. The specific reasons for lack of protection in newborn mice are not clear but virus load was found to be higher in newborn mice brains compared to those of adults and virus clearance was observed only in adult mice brains but not in newborn mice brains upon adoptive transfer. Specific depletion of Lyt 2.2(+), L3T4(+) or Thy-1(+) T cell populations before adoptive transfer abrogated the protective ability of transferred effecters. However, when Lyt 2.2(+) cell-depleted and L3T4(+) cell-depleted effecters were mixed and transferred into adult mice the protective activity was retained, demonstrating that both Lyt 2.2(+) and L3T4(+) T cells are necessary to confer protection. Although the presence of L3T4(+) T cells in adoptively transferred effector populations enhanced virus-specific serum neutralizing antibodies, the presence of neutralizing antibodies alone without Lyt 2.2(+) cells was not sufficient to confer protection.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

Structure of sesbania mosaic virus at 3 å resolution

Background: Sobemoviruses are a group of RNA plant viruses that have a narrow host range. They are characterized in vitro by their stability, high thermal inactivation point and longevity. The three-dimensional structure of only one virus belonging to this group, southern bean mosaic virus (SBMV), is known. Structural studies on sesbania mosaic virus (SMV), which is closely related to SBMV, will provide details of the molecular interactions that are likely to be important in the stability and assembly of sobemoviruses. Results: We have determined the three-dimensional structure of SMV at 3 Angstrom resolution. The polypeptide fold and quaternary organization are very similar to those of SBMV. The capsid consists of sixty icosahedral asymmetric units, each comprising three copies of a chemically identical coat protein subunit, which are designated as A, B and C and are in structurally different environments. Four cation-binding sites have been located in the icosahedral asymmetric unit. Of these, the site at the quasi-threefold axis is not found in SBMV. Structural differences are observed in loops and regions close to this cation-binding site. Preliminary studies on ethylene diamine tetra acetic acid (EDTA) treated crystals suggest asymmetry in removal of the quasi-equivalent cations at the AB, BC, and AC subunit interfaces. Conclusions: Despite the overall similarity between SMV and SBMV in the nature of the polypeptide fold, these viruses show a number of differences in intermolecular interactions. The polar interactions at the quasi-threefold axis are substantially less in SMV and positively charged residues on the RNA-facing side of the protein and in the N-terminal arm are not particularly well conserved. This suggests that protein-RNA interactions are likely to be different between the two viruses.

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group, JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis, H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV, Virus replication was found to be inhibited in the brains of animals that mere adoptively transferred with JEV specific CTL as revealed by immunohistological staining as,veil as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.

Inhibition of hepatitis C virus replication by herbal extract: Phyllanthus amarus as potent natural source

Hepatitis C virus infection is a major health problem worldwide. Developing effective antiviral therapy for HCV is the need of the hour. The viral enzymes NS3 protease and NS5B RNA dependent RNA polymerase are essential enzymes for polyprotein processing and viral RNA replication and thus can be potential targets for screening anti-HCV compounds. A large number of phytochemicals are present in plants, which are found to be promising antiviral agents. In this study, we have screened inhibitory effect of different plant extracts against the NS3 and NS5B enzymes of hepatitis C virus. Methanolic extracts were prepared from various plant materials and their inhibitory effects on the viral enzymes were determined by in vitro enzyme assays. Effect on viral RNA replication was investigated by using TaqMan Real time RT-PCR. Interestingly, Phyllanthus amarus root (PAR) extract showed significant inhibition of HCV-NS3 protease enzyme; whereas P. amarus leaf (PAL) extract showed considerable inhibition of NS5B in the in vitro assays. Further, the PAR and PAL extracts significantly inhibited replication of HCV monocistronic replicon RNA and HCV H77S viral RNA in HCV cell culture system. However, both PAR and PAL extracts did not show cytotoxicity in Huh7 cells in the MTT assay. Furthermore, addition of PAR together with IFN-alpha showed additive effect in the inhibition of HCV RNA replication. Results suggest the possible molecular basis of the inhibitory activity of PA extract against HCV which would help in optimization and subsequent development of specific antiviral agent using P. amarus as potent natural source. (C) 2011 Elsevier B.V. All rights reserved.

Mapping of B-cell epitopes of hemagglutinin protein of rinderpest virus

Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus FIN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination. (C) 2002 Elsevier Science (USA).

Interplay between NS3 protease and human La protein regulates translation-replication switch of Hepatitis C virus

HCV NS3 protein plays a central role in viral polyprotein processing and RNA replication. We demonstrate that the NS3 protease (NS3(pro)) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding. More importantly, NS3(pro) binding to the SLIV hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, resulting in inhibition of HCV-IRES activity. Although overexpression of both NS3(pro) as well as the full length NS3 protein decreased the level of HCV IRES mediated translation, replication of HCV replicon RNA was enhanced significantly. These observations suggest that the NS3(pro) binding to HCV IRES reduces translation in favor of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might regulate the molecular switch from translation to replication of HCV.

Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Advances in the Mathematical Modelling of Hepatitis C Virus Dynamics

Mathematical models have provided key insights into the pathogenesis of hepatitis C virus (HCV) in vivo, suggested predominant mechanism(s) of drug action, explained confounding patterns of viral load changes in HCV infected patients undergoing therapy, and presented a framework for therapy optimization. In this article, I present an overview of the major advances in the mathematical modeling of HCV dynamics.