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Background: Concerns of immune cross-reactivity, between epitopes of the group A streptococcal (GAS) M-proteins and host proteins have hindered the progress of an effective GAS vaccine. An ideal M-protein based subunit vaccine should not elicit heart tissue crossreactive antibody responses and should not activate M-protein specific $CD4^+$ T-cells. In the current study we used a bioinformatic and immunoinformatic approach to assess the safety of J8 and J14, chimeric vaccine constructs containing a GAS derived M-protein epitope embedded in flanking GCN4 region. We demonstrate that at the primary amino acid level J8 and J14 show very little homology to human proteins. ProPred, RANKPEP and HLABIND algorithms failed to predict significant binding between the M-protein specific regions of J8 and J14 and class II binding alleles. A single peptide was predicted to bind to HLA class I allele B_2705. This data was supported by cellular proliferation assays demonstrating few periferal blood mononuclear cells (PBMCs) from donors respond to J8 and J14. Reassuringly, there was no correlation between proliferation to these peptides, and proliferation to host proteins. This data suggests that J8 and J14 are unlikely to induce cross-reactive immune responses, and will be safe for use in humans.

The ability of activated T cells to present foreign antigens through the MHC class II pathway has been shown in the case of human, rat and mouse T cells. In the present study, the ability of activated T cells to present their endogenous TCR in association with MHC class II molecules to CD4 + T cells was shown. Upon activation mouse T cells downregulate their surface TCR, which are degraded into peptides in endosomal/lysosomal compartments. The idiopeptides (peptides derived from the variable region of the TCR) are presented to cognate anti-idiotypic CD4 + T cells, resulting in activation and proliferation of these cells. Interaction of idiotypic and anti-idiotypic T cells brought about by presentation of TCR idiopeptide may have important implications for T-cell vaccination and perpetuation of T-cell memory not requiring persisting antigen or long-lived memory cells.

Immunological memory is characterized by quick and enhanced immune response after re-exposure to the same antigen. In the present work, we have shown that anti-idiotypic T cells are generated in mice after immunization with idiotypic T cell clone or polyclonal T cell specific for nucleocapsid protein of Rinderpest virus. Further, we have shown that N specific idiotypic T cell receptors from apoptotic idiotypic T cells are processed by bone marrow derived dendritic cells and presented to cognate anti-idiotypic T cells. Evidence has been provided for the existence of antigen specific T cell idiotypic network in the body. Boosting with antigen specific anti-idiotypic T cells generates memory response in the antigen-primed mice.

$CD8^+ T$ cells are activated by the presentation of antigenic peptide through MHC class I molecules. Newly synthesized proteins formed as defective ribosomal products (DRiPs) can act as a major source of antigenic peptides for MHC class I presentation pathway. Majority of these peptides are generated from the intracellular degradation of self antigens. In the present study, we have shown that newly synthesized T cell receptor (TCR) beta chains formed as DRiPs in T cells are ubiquitinated and degraded by the proteasomes. These TCR-DRiPs are processed and presented by activated T cells to cognate anti-idiotypic $CD8^+ T$ cells. Presentation of TCR idiopeptide (peptide derived from the variable region of idiotypic TCR) by activated T cells leads to Bcl-2 expression and cytokine secretion by anti-idiotypic $CD8^+ T$ cells. Presentation of intracellular antigen by T cells may have important implications in immunoregulation, control of lymphotropic virus infection and autoimmune diseases.

Vaccination remains the most cost-effective means of preventing infectious diseases. Success of vaccination depends on generation of effective memory response. Understanding the mechanism of generation and maintenance of immunological memory would help in the design of rational vaccines. T lymphocytes play a central role in the generation of protective immune response against many microbial infections. A hypothesis known as relay hypothesis was earlier proposed, which explains the maintenance of immunological memory through interaction of idiotypic and anti-idiotypic lymphocytes. In the present study, we have shown that immunization with a model antigen, chicken ovalbumin specific T cell receptor beta chain (idiotypic TCR) generates TCR specific antibody and anti-idiotypic T cell responses as well as ovalbumin specific T cell response. We further show that boosting of ovalbumin primed mice with ovalbumin specific idiotypic $TCR\beta$ DNA or $TCR\beta$ protein gives memory response for ovalbumin. This study provides experimental evidence for perpetuation of immunological memory through idiotypic network interactions.

The role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. They are also shown to activate different cell types, which subsequently present mycobacterial antigens to T cells. Therefore identification of T cell epitopes from this set of proteins may serve to define candidate antigens with vaccine potential. Fifty-two secretory proteins of M. tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nonameric peptides. All possible overlapping nonameric peptide sequences from 52 secretory proteins were generated in silico and analyzed for their ability to bind to 33 alleles belonging to A, B and C loci of HLA class I. Fifteen percent of generated peptides are predicted to bind to HLA with halftime of dissociation T(1/2) >or=100 min and 73% of the peptides predicted to bind are mono-allelic in their binding. The structural basis for recognition of no-namers by different HLA molecules was studied employing structural modeling of HLA class I-peptide complexes and there exists a good correlation between structural analysis and binding prediction. Pathogen peptides that could behave as self- or partially self-peptides in the host were eliminated using a comparative study with the human proteome, thus reducing the number of peptides for analysis. The implications of the finding for vaccine development are discussed vis-a-vis the limitations of the use of subunit vaccine and DNA vaccine.

A pivotal step in the initiation of T-cell immunity is the presentation of antigenic peptides by major histocompatibility complex (MHC) expressed on antigen presenting cells. The expression of MHC class II molecules by mouse T-cells has not been shown unequivocally. In the present work, we demonstrate that activated mouse T-cells synthesize MHC class II molecules de novo and express them on their surface. Further, we have demonstrated that in vitro activated T-cells take up extra-cellular soluble nucleocapsid protein of a morbillivirus. The internalized antigen goes to antigen processing compartment as shown by co-localization of antigen and LAMP-1 using confocal microscopy. We show that activated T-cells express H2M, a chaperone molecule known to have a role in antigen presentation. Further, we demonstrate that activated T-cells process and present internalized extra-cellular antigen to primed T-cells as shown by IL-2 secretion and in vitro proliferation. The presentation of antigen by T-cells may have implications in immuno-regulation, control of infection by lymphotropic viruses and maintenance of immunological memory.

A self-sustaining mechanism for perpetuation of immunological memory is proposed which requires the involvement of idiotypic-anti-idiotypic antibody immune-complexes, antigen-presenting cells and follicular dendritic cells, but not long-lived memory cells or persisting antigens.

The surface glycoproteins of rinderpest virus (RPV) confer protective immunity in cattle. We demonstrated that cattle immunized with a recombinant extracellular baculovirus expressing the hemagglutinin (H) protein of RPV (rECV-H) generate virus neutralizing antibody responses, bovine leukocyte antigen (BoLA) class II restricted helper T cell responses and BoLA class I restricted cytotoxic T cell (CTL) responses against RPV-H and hemagglutinin-neuraminidase (HN) glycoprotein of closely related Peste des petits ruminants virus (PPRV). In this study, employing autologous skin fibroblasts transiently expressing truncations of H and HN in a BoLA class I restricted lympho-proliferation assay, we have mapped a highly homologous domain (amino acids 400-423) on these proteins harboring a CTL epitope. Subsequently, based on sequence comparison with available BoLA class I binding motifs, we have identified a BoLA-A11 binding motif (amino acids 408-416) in the stimulatory domain. Autologous cells pulsed with a synthetic peptide corresponding to this sequence stimulated CTLs from rECV-H immunized as well as tissue culture attenuated RPV vaccinated cattle of different breeds and parentage. This is the first epitope identified in cattle on the attachment glycoproteins of RPV and PPRV.

The negative sense genome RNA of Rinderpest virus, a Paramyxoviridae, is encapsidated with the nucleocapsid protein N and serves as a template for the viral RNA dependent RNA polymerase for transcription and replication. The viral RNA polymerase consists of the large protein L and the phosphoprotein P functioning as the P–L complex. We provide in this report, evidences for specific binding of P protein of Rinderpest virus to the plus sense leader RNA depending on its phosphorylation status. We have also demonstrated that P protein is released from the le RNA:P protein complex upon phosphorylation in vitro. Finally, we have identified that the C-terminal 358–389 amino acid residues of P protein is involved in le RNA binding. The leader RNA binding may signify a hitherto unidentified role for P protein in the viral RNA synthesis. Moreover, our results indicate a possible role for P protein in the transcription-replication switch through leader RNA binding.

The nucleocapsid protein N of Chandipura virus is prone to aggregation in vitro. We have shown that this aggregation occurs in two phases in a nucleation-dependent manner. Electron microscopy suggests that the aggregated state may have a ring-like structure. Using a GFP fusion, we have shown that the N-protein also aggregates in vivo. The P-protein suppresses the N-protein aggregation efficiently, both in vitro and in vivo. Increased lag phase in the presence of the P-protein suggests that chaperone-like action of the P-protein occurs before the nucleation event. The P-protein, however, does not exert any chaperone-like action against other proteins, suggesting that it binds to the N-protein specifically. Surface plasmon resonance and fluorescence enhancement indeed suggest that the P-protein binds tightly to the native N-protein. The P-protein is thus an N-protein-specific chaperone which inhibits the nucleation phase of N-protein aggregation, thus keeping a pool of encapsidation-competent N-protein for viral maturation.

Phosphoprotein P of rinderpest virus (RPV), when expressed in E. coli, is present in the unphosphorylated form. Bacterially expressed P protein was phosphorylated by a eukaryotic cellular extract, and casein kinase II (CK II) was identified as the cellular kinase involved in phosphorylation. In vitro phosphorylation of P-deletion mutants identified the N terminus as a phosphorylation domain. In vivo phosphorylation of single or multiple serine mutants of P protein identified serine residues at 49, 88 and 151 as phospho-acceptor residues. The role of P protein phosphorylation in virus replication/transcription was evaluated using the RPV minigenome system and replication/transcription of a reporter gene in vivo. P protein phosphorylation was shown to be essential for in vivo replication/transcription since phosphorylation-null mutants do not support expression of a reporter gene. Transfection of increased amounts of phosphorylation-null mutant did not support minigenome replication/transcription in vivo.

The RNA dependant RNA polymerase of negative sense RNA viruses is composed of two subunits - the Large protein ( L) and the Phosphoprotein ( P). These two proteins have to form a complex in order to carry out genome transcription and replication. Employing the baculovirus expression system, we demonstrate here, the specific in vivo interaction between the L and P proteins of Rinderpest virus and also the stabilization of L protein when it is present as L+P complex. The regions on either protein involved in such interaction has been studied using the yeast two-hybrid system which indicates that the P binding region resides within the amino terminal 380 amino acid residues of L protein. The L binding region on P protein has been mapped to lie within 347 - 490 amino acids.

The RNA dependent RNA polymerase of Rinderpest virus consists of two subunits—the large protein (L) and the phosphoprotein (P), where L is thought to be responsible for the catalytic activities in association with P protein which plays multiple roles in transcription and replication. The nucleocapsid protein (N) is necessary for encapsidation of genomic RNA, which is required as N–P complex. To understand the different steps of transcription and replication as well as the roles played by the three proteins, an in vitro reconstitution system for RNA synthesis is necessary which is not available for any morbillivirus. We describe here, an in vitro reconstitution system for transcription and replication of Rinderpest virus utilizing a synthetic, positive sense N-RNA minigenome template, free of endogenous viral polymerase proteins and recombinant viral proteins (P + L and P + N) expressed in insect cells by recombinant baculoviruses. We show that although L–P complex is sufficient to synthesize negative sense minigenome RNA, soluble N protein is necessary for encapsidation of RNA as well as synthesis of (+) sense leader RNA and (+) sense minigenome RNA.

The paramyxovirus RNA-dependent RNA polymerase consists of two subunits, the transcription co-factor phosphoprotein P and the large protein L, which possesses all the catalytic functions such as RNA synthesis (both transcription replication), methylation, capping and polyadenylation. The L protein has high sequence homology among the negative sense RNA viruses. The domains and residues on the L protein involved in the above-mentioned activities are not well defined, although the role of conserved GDNQ motif of the putative catalytic centre of L protein of few related viruses have been examined. In order to gain insight into the role played by the GDNQ motif of the L protein of Rinderpest virus (RPV), we have examined mutations at each amino acid in this motif of the L protein of Rinderpest virus and tested the biological activity in vivo and in vitro. Site directed mutants were generated and transiently expressed in mammalian cells and were shown to interact with P protein similar to wild type L. The biological activity of mutant L proteins has been tested in an in vitro reconstituted system capable of carrying out cell-free RNA synthesis on synthetic Rinderpest N-RNA template. Further, the role played by individual amino acids has also been defined in vivo using an in vivo minigenome replication/transcription system which indicated the importance of this conserved sequence in viral RNA synthesis.

In recent times, newly emerging infectious diseases and their causative agents have become the focus of intense investigations by medical researchers and microbiologists, particularly virologists, since many of the causative organisms are viruses. One such disease which has created so much fear world wide is severe acute respiratory syndrome (SARS). Infection with a novel corona virus has been implicated as a possible cause of SARS.Epidemiological and laboratory investigations of SARS are ongoing. WHO estimates that SARS is fatal in around 4% of cases, usually where the person has an underlying condition such as diabetes or heart disease or has a weakened immune system. In 90% of the cases people seem to recover around after a week after being infected. This is about the same mortality rate as in other diseases such as West Nile virus infection. There are currently no antiviral drugs shown to be consistently successful in treating SARS or any corona virus infection, nor anyvaccine against SARS.

The molecular events associated with the transcriptive and replicative cycle of negative-stranded RNA viruses are still an enigma. We took Chandipura virus, a member of the Rhabdoviridae family, as our model system to demonstrate that Phosphoprotein P, besides Nucleocapsid protein N, also acts as a leader RNA-binding protein in its unphosphorylated form, whereas CKII-mediated phosphorylation totally abrogates its RNA-binding ability. However, interaction between P protein and leader RNA can be distinguished from N-mediated encapsidation of viral sequences. Furthermore, P protein bound to leader chain can successively recruit N protein on RNA while itself being replaced. We also observed that the accumulation of phosphorylation null mutant of P protein in cells results in enhanced genome RNA replication with concurrent increase in the viral yield. All these results led us to propose a model explaining viral transcription-replication switch where Phosphoprotein P acts as a modulator of genome transcription and replication by its ability to bind to the nascent leader RNA in its unphosphorylated form, promoting read-through of the transcription termination signals and initiating nucleocapsid assembly on the nascent RNA chain.

The use of transgenic plants as a production system for recombinant subunit vaccines has been considered safe and economical compared to cell culture methods. We have exploited this approach to produce rinderpest virus hemagglutinin (H) protein in transgenic tobacco as a model plant for testing the immunogenicity of plant-derived hemagglutinin protein. The transgenic nature of the plants was confirmed by molecular analysis such as gene specific PCR and Southern hybridization using full-length H gene as a probe. The Mendelian pattern of inheritance of the transgene has been demonstrated in T1 generation. The transgenic plants express the H protein of molecular weight 72 kDa. The plant derived H protein is antigenically authentic as revealed by reactivity with H-specific antibodies as well as convalescent sera. The induction of immune response was tested in mice after intraperitoneal immunization with plant-derived H. High titers of antibodies were induced which were H-specific and they neutralized the infectivity of rinderpest virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects antibodies unique to rinderpest virus (RPV) has been developed. This test can differentiate antibodies against RPV and those against peste des petits ruminants virus. The recombinant RPV hemagglutinin (H)-protein C-ELISA (recH C-ELISA) is based on the ability of a well-characterized monoclonal antibody (MAb) produced with the soluble, secreted form of the H protein (Sec H protein) of RPV made in a baculovirus expression system to compete with the binding of RPV antibodies in the serum of vaccinated or infected, recovered animals to the Sec H protein. The B-cell epitope recognized by the MAb corresponds to amino acids 575 to 583 on the H protein, which is not present on the antigenically closely related peste des petits ruminants virus hemagglutinin-neuraminidase protein. Initially, a positive-negative threshold cutoff value for percent inhibition of 34 was established with 500 known RPV-negative serum samples. The recH C-ELISA was developed with the enzyme immunoassay software of a commercial RPV C-ELISA kit. Comparative analysis of the test results for 700 serum samples obtained with the commercial kit gave a sensitivity of 112.4% and a specificity of 72.4%. Variations in percent inhibition values were observed for the two assay systems. These variations may have been due to the undefined amount of antigen present in the commercial kit as well as the use of a different MAb. The recH C-ELISA detected more positive serum samples compared to the number detected by the commercial kit, with the results confirmed by a virus neutralization test. Thus, recH C-ELISA is a sensitive tool for RPV serosurveillance in disease eradication programs.

We describe the properties of the two heptad repeats (HR1 and HR2) of the Peste des petits ruminants virus (PPRV) fusion protein (F) to obtain insights into the mechanism by which these repeats influence PPRV-mediated cell fusion. Both HR1 and HR2 inhibit PPRV-mediated syncytia formation in Vero cells in vitro. Of these, HR2 was found to be more effective than HR1. We studied the mechanism of fusion inhibition by these two repeats by using various biophysical and biochemical methods either separately or together. CD spectral analysis of these repeats revealed that the alpha-helical content of HR1 and HR2 when used together is higher than that of their simulated spectrum in the mixture, suggesting the formation of a highly structured complex by these repeats. Protease protection assays confirmed that such a complex is highly stable. Electrospray mass spectrometry of protease-digested products of the HR1-HR2 complex showed protection of fragments corresponding to both HR1 and HR2 sequences involved in complex formation. By employing size-exclusion chromatography and chemical cross-linking experiments, we show that three units each of HR1 and HR2 form a complex in which HR1 is a trimer and HR2 is a monomer. Homology-based three-dimensional modeling of this complex showed that HR1 and HR2 together form a six-helix and trimeric coiled-coil bundle. In this model, the HRI trimer forms the core whereas HR2, while interacting with HR1 in an antiparallel orientation, forms a two-stranded coiled-coil structure and lies at the periphery of the structure. These results are discussed in the context of a common fusion mechanism among paramyxoviruses.

Rinderpest causes a devastating disease, often fatal, in wild and domestic ruminants. It has been eradicated successfully using a live, attenuated vaccine from most part of the world leaving a few foci of disease in parts of Africa, the Middle East, and South Asia. We have developed transgenic peanut (Arachis hypogaea L.) plants expressing hemagglutinin (H) protein of rinderpest virus (RPV), which is antigenically authentic. In this work, we have evaluated the immunogenicity of peanut-expressed H protein using mouse model, administered parenterally as well as orally. Intraperitoneal immunization of mice with the transgenic peanut extract elicited antibody response specific to H. These antibodies neutralized virus infectivity in vitro. Oral immunization of mice with transgenic peanut induced H-specific serum IgG and IgA antibodies. The systemic and oral immunogenicity of plant-derived H in absence of any adjuvant indicates the potential of edible vaccine for rinderpest.

A recombinant baculovirus expressing membrane bound form of hemagglutinin-neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein. Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells. The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263-368 and 538-609 and hence the epitopes identified are conformation-dependent. The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera. Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity. Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%). Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties. All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay.

Rinderpest virus (RPV), a member of the genus Morbillivirus within the Paramyxoviridae family, causes a highly contagious and often fatal disease known as rinderpest in wild and domestic ruminants. The envelope of the virus contains two surface glycoproteins, namely the hemagglutinin (H) and the fusion (F) proteins, both of which have been shown to confer protective immunity in animals. In this paper, we demonstrate that single administration of low doses of recombinant H protein of RPV expressed in insect cells in the form of extracellular virus induces long lasting bovine leukocyte antigen class I restricted cytotoxic T-cell (CTL) responses in cattle in the absence of adjuvant. This is the first report of CTL responses in cattle against one of the protective antigens of RPV.

B cell hybridomas expressing class I and II MHC molecules and producing antibodies directed against hemagglutinin protein of Rinderpest virus and human Mucin-1 have been used as surrogate B cells to study T cell responses against the antigens. The observed CTL and lymphoproliferative response indicates that anti-idiotypic B cells termed Jerne cells stimulate both T helper and T cytotoxic cells by virtue of their ability to present recycled or regurgitated peptido-mimics of antigen to T helper cells through class II MHC and de novo synthesized peptido-mimics of antigens to CTLs. Thus, T cell memory response can be perpetuated by anti-idiotypic Jerne B cells and these findings lend support to the earlier proposed relay hypothesis for perpetuation of immunological memory (IM).

Rinderpest virus is the causative agent of a devastating, often fatal disease in wild and domestic bovids that is endemic in Africa, the Middle East and South Asia. The existing live attenuated vaccine is heat-labile, and thus there is a need for the development of new strategies for vaccination. This paper reports the development of transgenic pigeon pea [Cajanus cajun (L.) Millsp.] expressing one of the protective antigens, the hemagglutinin (H) protein of Rinderpest virus. A 2-kb fragment containing the coding region of the H protein was cloned into pBI121 and mobilized into Agrobacterium tumefaciens strain EHA105. Embryonic axes and cotyledonary nodes from germinated seeds of pigeon pea were used for transformation. The presence of the transgene in transgenic plants was confirmed by Southern blots, and the specific transcription of the marker gene in the plants was demonstrated by reverse transcription-polymerase chain reaction. Integration of the H gene into the pigeon pea genome was confirmed by Southern hybridization. The expression of the H protein in the transgenic lines was confirmed by Western blot analysis using a polyclonal monospecific antibody to the H protein. The highest level of expression of the hemagglutinin protein in leaves of pigeon pea was 0.49% of the total soluble protein. The transgenic plants were fertile and the transgene expressed in the progeny.

To study the process of membrane fusion in Morbilliviruses, the fusion (F) glycoproteins of Peste des petits ruminants virus (PPRV) and Rinderpest virus (RPV) were expressed transiently in mammalian cells. The recombinant F proteins were found to be localized at the surface of transfected cells. The fusion activity, as evident from cell fusion assays and lysis of chicken erythrocytes, documented that transiently expressed PPRV F glycoprotein induces cell fusion in the absence of homotypic hemagglutinin–neuraminidase (HN) attachment glycoprotein. The coexpression of homotypic HN increased the extent of fusion by twofold, while the efficiency of fusion was found to be substantially enhanced. In contrast, in RPV F-expressing cells, fusion was detected only when homotypic hemagglutinin (H) or heterotypic HN protein was coexpressed. This differs from the strict type-specific requirement for the attachment protein as in the fusion process of most of the paramyxoviruses. Further, we demonstrate by fluorescence transfer experiments that while PPRV F brings about both hemifusion and complete fusion on its own, RPV F induces only hemifusion while it brings about complete fusion in the presence of homotypic or heterotypic attachment protein.

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus within the family Paramyxoviridae, causes a fatal disease ‘peste des petits ruminants’ in goats and sheep. This enveloped virus is antigenically closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants. PPRV harbors two major surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. The surface glycoproteins of morbilliviruses are highly immunogenic and confer protective immunity. In this study, we investigated the immune responses generated in goats immunized with low doses of purified recombinant extracellular baculovirus carrying a membrane bound form of the HN protein of PPRV without any adjuvant. We report that the immunized goats develop both humoral and cell-mediated immune responses. Antibodies generated in the immunized animals could neutralize both PPRV and RPV in vitro. Further, using a combination of Escherichia coli expressed deletion mutants of PPRV-HN and RPV-H proteins, and synthetic peptides corresponding to the highly conserved N-terminal sequences of MV-H protein, we have mapped an N-terminal T cell determinant (amino acids 123–137) and a C-terminal domain (amino acids 242–609) harboring potential T cell determinant(s) in goats.

Idiotypic determinants of immunoglobulin molecules can evoke both $CD4^+$ and $CD8^+$ T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell (1). The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.

The genes coding for the surface glycoproteins hemagglutinin–neuraminidase (HN) of the peste des petits ruminants virus (PPRV) and hemagglutinin (H) of rinderpest virus (RPV) were cloned in a cytomagalovirus promoter driven expression vector and expressed transiently in mammalian cells. The protein expression was apparent 24 h after transfection and the expressed proteins were detected at the cell surface. The transiently expressed PPRV HN protein was found to be biologically active in possessing hemadsorption and neuraminidase activities. On the other hand, RPV H protein exhibited neuraminidase activity but was deficient in hemadsorption activity. The substrate specificity of the neuraminidase activity of these two proteins differed distinctly. The presence of neuraminidase activity in both PPRV HN and RPV H proteins is unusual among members of the morbillivirus genus.

A mechanism is proposed which explains the perpetuation of B-cell immunological memory indefinitely without requiring the presence of long-living memory cells or persisting antigen. The salient feature of this model is that immunological memory can be perpetuated indefinitely through the mutual interaction of idiotypic and anti-idiotypic B cells. These cells mutually stimulate and clonally expand with either specific or bystander T-cell help. Because B cells can present antigen, they present 'apparently foreign' idiopeptides to T cells. The idiopeptides of de novo synthesized antibody is presented to CD8+ T cells that recognize the idiopeptide-presenting cell as targets and regulate their population. The recycling of immunoglobulins from surface to endosomal compartment of B cells leads to the presentation of idiopeptides by major histocompatibility complex (MHC) class II to CD4+ T cells. Even if the majority of the clonally expanded cells die because of lack of stimulation, cytotoxic T lymphocyte (CTL) lysis or for other reasons, the surviving cells will be able to carry forward the memory. This mechanism also provides a means for affinity maturation through idiotypic selection of somatically mutated high affinity cells or those from the naïve pool. We have termed these two types of complementary B cells as Burnet B cells: those which recognize the antigen or antigen mimic, and Jerne B cells, which can recognize the idiotypes of antibody and carry antigen mimics. The proposed hypothesis can explain differential duration of memory for different antigens, the shelf space paradox, affinity maturation, repertoire shift, etc.

The fusion glycoprotein (F protein) of paramyxoviruses plays a vital role in virus-induced cytopathology. To explore the role of the F protein in peste des petits ruminants virus (PPRV)- induced cytopathology, the F protein of PPRV was purified by immunoaffinity chromatography. The purified F protein, when incubated with chicken erythrocytes, caused lysis suggesting that PPRV F protein is a hemolysin. Furthermore, the hemolysis can be inhibited by hyperimmune serum against F protein. The virus-induced cell fusion (syncytia) was also inhibited by the hyperimmune serum against the F protein. In summary, these results indicate that the purified PPRV F protein is biologically active and is involved in virus-induced hemolysis, cell-fusion and the initiation of infection.

Peste des petits ruminants (PPR) is an important viral disease of goats and sheep prevalent in West Africa and the Middle East. In recent years, PPR has emerged in India, first in the South India and later in North India. To study the genetic relationships between viruses of distinct geographical origin we have sequenced a 322 nucleotide cDNA fragment of the fusion protein gene generated using reverse transcription followed by polymerase chain reaction (PCR) amplification. Viruses from nineteen independent PPR outbreaks were compared; these included the prototype African strain from Senegal and viruses from disease outbreaks which have occurred at different times and locations across Africa, Arabia, the Near East and the Indian subcontinent. Four separate lineages of the virus were identified and the virus isolates from Asia over the past 2 years were all of one lineage which had not previously been identified in Africa or Asia.

Background Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. Results RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3’ end to 5’ end of the genome similar to that exhibited by the virus in infected cells. Conclusion This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

An in vitro transcription system for rinderpest virus (RPV) is described. Ribonucleoprotein complexes isolated from RPV-infected Vero cells, human lung carcinoma cells, or detergent-disrupted purified virions synthesized authentic RPV mRNAs for the N, P, M, F and H genes as identified by dot blot hybridization analysis with individual cDNA clones. The relative abundance of the mRNAs synthesized in vitro decreased from the 3? end of the genome to the 5? end, very similar to that observed with measles virus transcription in vitro. The transcription by purified virions was stimulated three-fold by the addition of infected human lung carcinoma cell lysate, demonstrating the involvement of host factor(s) in mRNA synthesis.

Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.

We have cloned several cDNAs derived from the P gene of rinderpest virus. One of these, derived from a bicistronic N-P mRNA, has been sequenced in its entirety. Sequencing of a section of the others, and comparison with the genome sequence, showed that P gene transcripts, as for other morbilliviruses, were variable; non-templated Gs could be added at a site resembling the normal stop transcription site. Primer extension analysis showed that about half the transcripts were edited. Sequences of the P, C and V proteins encoded by the normal and edited transcripts were compared with those of other morbilliviruses and with those of the more distantly related paramyxoviruses.

The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.

Treatment ofTrigonella foenumgraeceum (fenugreek) seedlings with naphthalene acetic acid plus gibberellic acid enhanced the RNA synthesising capacity of nuclei isolated from the hypocotyl and cotyledonary regions. This increase was more pronounced in the nuclei from the hypocotyl region than from the cotyledonary region.In vitro addition of these phytohormones did not stimulate RNA synthesis by nuclei. The RNA synthesis by mitochondria was not affected by preincubating the seedlings with the hormones. The nuclei isolated from callus cultures of fenugreek hypocotyl treated with the hormone also showed increased RNA synthesis.

A lectin has been purified from the seeds ofMimosa invisa L. by gel filtration and preparative Polyacrylamide gel electrophoresis. The purified lectin was homogeneous as judged by analytical Polyacrylamide gel electrophoresis, immunodiffusion and Immunoelectrophoresis. The apparent molecular weight is 100,000; the protein is a tetramer with two types of subunits (molecular weight 35,000 and 15,000). The lectin is a glycoprotein with approximately 21% carbohydrate and interacts with Sephadex and concanavalin A-Sepharose. It agglutinates erthrocytes non-specifically, does not agglutinate leucocytes and is not mitogenic, agglutinates Mimosa-nodulatingRhizobium and is a panagglutinin; the agglutination is not inhibited by several simple sugars. It is thermo-stable and has no metal ions.

Isolated nuclei from differentiating cultures ofNicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg2+ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH4)2 SO4 and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.

An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of 14 C-amino acids into protein. The effects of various antitubercular drugs and “known inhibitors” of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of “membrane-active” agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of 14 C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains. In vitro, streptomycin was an effective inhibitor of susceptible strains, whereas in streptomycin-resistant strains the concentration of streptomycin at which half-maximal inhibition was produced varied according to the resistance of whole cells, and there was a correlation between the two. In one low-level streptomycin-resistant mutant, the in vitro amino acid-incorporating system was as sensitive to various concentrations of streptomycin as the parental type, and a possible involvement of a membrane site in the development of low-level resistance was indicated. Streptomycin susceptibility and high-level resistance were shown to be ribosomal in nature.

The assimilatory nitrite reductase catalyses the conversion of nitrite to ammonia. The enzyme from Candida utilis has been previously purified to homogeneity and shown to be a heterodimer consisting of 58 kDa and 66 kDa subunits. The enzyme has also been shown to be induced by nitrate and repressed by ammonium ions. The levels of nitrite reductase mRNA, its protein and the enzyme activity were modulated together indicating that the primary level of regulation of this enzyme existed at the transcriptional level. Here we report that the 58 kDa and 66 kDa subunits of the enzyme were differentially phosphorylated under the induced and repressed conditions, indicating a second level of regulation. The highly phosphorylated 66 kDa subunit was shown to be dephosphorylated by calf intestinal alkaline phosphatase. The enzymatic activity associated with the native enzyme also decreased due to the dephosphorylation. Each of the subunits could undergo autophosphorylation at serine/threonine residues as demonstrated by thin layer chromatography and recognition by antibodies to phosphoamino acids. The presence of similar phosphorylated subunits under in vivo conditions has also been demonstrated. A model has been proposed to explain the post-translational regulation of the enzyme.

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).

Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I–III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.

Peroxidase from Mycobacterium tuberculosis H37Rv was purified to homogeneity. The homogeneous protein exhibits catalase and Y (Youatt's)-enzyme activities in addition to peroxidase activity. Further confirmation that the three activities are due to a single enzyme was accomplished by other criteria, such as differential thermal inactivation, sensitivity to different inhibitors, and co-purification. The Y enzyme (peroxidase) was separated from NADase (NAD+ glycohydrolase) inhibitor by gel filtration on Sephadex G-200. The molecular weights of peroxidase and NADase inhibitor, as determined by gel filtration, are 240000 and 98000 respectively. The Y enzyme shows two Km values for both isoniazid (isonicotinic acid hydrazide) and NAD at low and high concentrations. Analysis of the data by Hill plots revealed that the enzyme has one binding site at lower substrate concentrations and more than one at higher substrate concentration. The enzyme contains 6g-atoms of iron/mol. Highly purified preparations of peroxidases from different sources catalyse the Y-enzyme reaction, suggesting that the nature of the reaction may be a peroxidatic oxidation of isoniazid. Moreover, the Y-enzyme reaction is enhanced by O2. Isoniazid-resistant mutants do not exhibit Y-enzyme, peroxidase or catalase activities, and do not take up isoniazid. The Y-enzyme reaction is therefore implicated in the uptake of the drug.