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Peste-des-petits-ruminants is a highly contagious and fatal disease of goats and sheep caused by non-segmented, negative strand RNA virus belonging to the Morbillivirus genus-Peste-des-petits-ruminants virus (PPRV) which is evolutionarily closely related to Rinderpest virus (RPV). The large protein 'L' of the members of this genus is a multifunctional catalytic protein, which transcribes and replicates the viral genomic RNA as well as possesses mRNA capping, methylation and polyadenylation activities; however, the detailed mechanism of mRNA capping by PPRV L protein has not been studied. We have found earlier that the L protein of RPV has RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase activities, and unlike vesicular stomatitis virus (VSV), follows the conventional pathway of mRNA capping. In the present work, using a 5'-end labelled viral RNA as substrate, we demonstrate that PPRV L protein has RTPase activity when present in the ribonucleoprotein complex of purified virus as well as recombinant L-P complex expressed in insect cells. Further, a minimal domain in the C-terminal region (aa1640-1840) of the L protein has been expressed in E. coli and shown to exhibit RTPase activity. The RTPase activity of PPRV L protein is metal-dependent and functions with a divalent cation, either magnesium or manganese. In addition, RTPase associated nucleotide triphosphatase activity (NTPase) of PPRV L protein is also demonstrated. This work provides the first detailed study of RTPase activity and identifies the RTPase domain of PPRV L protein.

The present study envisages expression of immunodominant ectodomain of peste des petits ruminants virus (PPRV) fusion (F) protein in Escherichia coli BL21 (DE3) and its characterization to assess its immunoreactivity. The ectodomain gene sequences corresponding to 222 amino acids, was amplified from PPR vaccine virus, cloned into pET33b vector and expressed in E. coli at an optimal temperature of 37 degrees C with 1 mM IPTG for 5 h. The expressed and Ni-NTA purified PPRV F protein (31 kDa) was characterized by SDS-PAGE and Western blot using anti-his-tagged-conjugate, anti-serum raised against recombinant PPRV F protein, hyper immune serum against whole PPRV and convalescent sera from sheep and goats. The expressed protein was assessed for its immunoreactivity by ELISA and immunoblotting. The antibody response mounted against the recombinant PPRV F protein in immunized rabbits was detected by recombinant PPRV F antigen based indirect ELISA, and whole virus antigen based indirect ELISA, which indicating the native confirmation of the expressed protein in E. coli. Indirect ELISA was optimized using known true positive and negative sera with respect to PPRV antibodies in order to assess the reactivity of the PPRV F protein in detecting PPRV F antibodies in small ruminants. The E. coli expressed recombinant ectodomain of PPRV F protein exhibits immunoreactivity and was able to specifically detect PPRV antibodies in response to both vaccination and disease in natural host. (C) 2016 Elsevier B.V. All rights reserved.

A peptide (J14) containing conformationally restricted epitopes from the M protein of group A streptococcus (GAS) is capable of eliciting protective immune response against GAS infection. However, the protective response may be lost possibly due to its weak secondary-structure when the antigen is fused with other antigens in a recombinant polyepitope vaccine construct. We previously showed that JJo, a conformationally stabilized derivative of dimeric J14, overcomes this problem. We now show that anti JJo antibodies react with diverse GAS isolates found in the Indian sub-continent and that these antibodies are opsonic for GAS. The GAS strains used in this study were isolated from throat and skin swabs from Mumbai, Chennai and Vellore. Sera from mice immunized with recombinant JJo peptide were tested by ELISA, immunofluorescence, flow-cytometry, indirect bactericidal assay and mouse challenge assays to determine specific immunogenicity, opsonic functions and protection against an Indian isolate. We propose that JJo is a robust antigen suitable for inclusion in recombinant multi-epitope vaccines which are potentially affordable option for the pediatric population of developing countries.

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAsin vitroand the abundance of mRNAs follows a gradient of polarity, similar to the occurrencein vivo. In the present work, we demonstrate that the viral mRNAs synthesizedin vitroby the recombinant L or purified RNP are capped and methylated at the N7guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal ofγ-phosphate from triphosphate-ended 25 nt RNA generatedin vitrorepresenting the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

Background Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.Methods As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. Results The sFLT-1 produced by the baculovirus system showed potent antiangiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Conclusions The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.

The PE and PPE proteins ofMycobacterium tuberculosisform a source of antigenic variation among different strains of this bacterium. Two of the PE_PGRS protein-encoding genes,rv3812andrv3018c, are expressed in pathogenic mycobacteria and are implicated, respectively, in the persistence of the organism in macrophages and in virulence. Peptides derived from these proteins have been predicted to bind major histocompatibility complex (MHC) class I with high affinity on the basis of immunoinformatics analysis, suggesting a possible role for these proteins in antimycobacterial immunity. In the present work, using DNA constructs containing therv3812andrv3018cgenes ofM. tuberculosis, the immunogenicity of these proteins was demonstrated in BALB/c mice. Immunization with either DNA construct induced a significant number of CD8+-type T cells and a strong Th1-type response, with high gamma interferon (IFN-γ) and low interleukin-4 responses. Three nonameric peptides of Rv3812 and two of Rv3018c elicited a strong T-cell response in an MHC-restricted manner. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of perforin and IFN-γproduction. Experimentally, these peptides bound with high affinity to MHC H-2Kdand showed low dissociation rates of peptide–MHC complexes. This study suggests that the identified T-cell epitopes may contribute to immunity against tuberculosis if included in a vaccine.

The PE and PPE family of proteins of Mycobacterium tuberculosis have been hypothesized to play important roles in the biology of the organism and some proteins have been shown to be involved in eliciting T-cell responses. Earlier, we had identified putative HLA class I binding epitopes of the PE and PPE proteins of Mycobacterium tuberculosis employing computational and molecular modeling approaches. In the present work, three of the PE/PPE family proteins, coded by Rv1818c, Rv3812 and Rv3018c genes, were selected based on the computational analysis for testing human immune responses. PBMCs from patients with active tuberculosis and healthy, BCG vaccinated, PPD-positive individuals were tested for in vitro proliferative response and gamma-interferon production using synthetic peptides derived from the chosen proteins. Significant differences were seen in the responsiveness between healthy controls and patients. Antigen-specific T-cell lines were established from the PBMCs of healthy controls and their responses to peptide-specific CD8+ T-cell effectors were shown to be present at high frequency in the PBMCs of PPD+ controls. The T-cell lines also showed cytotoxic activity against the peptide pulsed monocytes.

The existing vaccines against influenza are based on the generation of neutralizing antibody primarily directed against surface proteins-hernagglutinin and neuraminidase. In this work, we have computationally defined conserved T cell epitopes of proteins of influenza virus H5N1 to help in the design of a vaccine with haplotype specificity for a target population. The peptides from the proteome of H5NI irus which are predicted to bind to different HLAs, do not show similarity with peptides of human proteorne and are also identified to be generated by proteolytic cleavage. These peptides could be made use of in the design of either a DNA vaccine or a subunit vaccine against V influenza. (c) 2007 Elsevier Ltd. All rights reserved.

Understanding the mechanisms of generation and maintenance of immunological memory is crucial for rational vaccine design.Ahypothesis 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 rinderpest virus hemagglutinin protein specific anti-idiotypic antibody $(Ab2v_\beta)$ DNAs coding for heavy and light chains generates antigen-specific antibody and T cell responses as well as Ab1 specific T cell response. We further show that boosting with the recombinant $Ab_2-v_\beta$ proteins generates B and T cell memory response specific for antigen in anti-id DNA primed mice. This study provides experimental evidence for perpetuation of immunological memory through idiotypic network interactions.

Post-transcriptional modification of viral mRNA is essential for the translation of viral proteins by cellular translation machinery. Due to the cytoplasmic replication of Paramyxoviruses, the viral-encoded RNA-dependent RNA polymerase (RdRP) is thought to possess all activities required for mRNA capping and methylation. In the present work, using partially purified recombinant RNA polymerase complex of rinderpest virus expressed in insect cells, we demonstrate the in vitro methylation of capped mRNA. Further, we show that a recombinant C-terminal fragment (1717-2183 aa) of L protein is capable of methylating capped mRNA, suggesting that the various post-transcriptional activities of the L protein are located in independently folding domains.

Peste des Petits Ruminants (PPR) is a highly contagious transboundary animal disease of wild and domestic small ruminants and notifiable byworld organization for animal health (OIE). It constitutes a serious threat to livestock production in many developing countries, in western Africa and southern Asia. The disease is caused by peste des petits ruminants virus (PPRV), a negative sense, single stranded RNAvirus belonging to the genus Morbillivirus of the family Paramyxoviridae (Gibbs et al. 1979). PPRV causes huge disease burden on agriculture across developing world, affecting small ruminant production and in turn increases poverty in some of the poorest parts of the world (Banyard et al. 2010). In PPR endemic regions of the world, conventional live attenuated vaccines are being used to vaccinate small ruminants (Diallo et al. 1989). Currently, the original Nigerian strain of PPRV (Nig 75/1) is used in the African countries (Diallo 2003). In India, three Indian strains of PPRV have been attenuated and shown to provide protective immunity (Saravanan et al. 2010). Although the live attenuated vaccines are efficacious, they suffer from the same draw back as that of live attenuated Rinderpest vaccine in terms of heat stability. This puts severe constraints on the coverage of small ruminant’s population during vaccination campaigns. Although there is one report of a method for making a heat stable live attenuated vaccine (Worrwall et al. 2000), such a thermo stable vaccine has not been put to use or field tested. Another major disadvantage of using live attenuated vaccine is that the antibody responses they induce in animals cannot be distinguished from those following a natural infection. This makes sero-epidemio surveillance of the disease impossible in endemic areas where a vaccination programme has been or is being implemented. The immune response to a given antigen can be regulated by a number of idiotypic determinants (Ids), and its counterpart, an anti-idiotypic antibody (anti-Id) or Ab2 (Jerne 1974). An idiotype is located on the variable regions of antibody molecules. An internal image anti-Id, Ab2 β recognizes an idiotypic determinant within the antigen combining site and bears a structural resemblance to the original antigen (Bona and Kohler 1984). The internal image Ab2 β antibodies have been shown to induce specific immune responses to hepatitis B surface antigen in chimpanzees (Kennedy et al. 1986) and as a vaccine candidate for the control of bluetongue infection by functionally mimicking VP7 antigen of bluetongue virus (Zhou and Lin 1997). We had earlier demonstrated that an internal image Ab2 against Rinderpest virus/Peste des petits Ruminants virus H/HN protein elicits virus neutralizing antibodies as well as T cell responses in the mouse model (Vani et al. 2007a). Further, using the mouse model, DNA encoding VH region of Ab2 elicited both neutralizing antibody and T cell responses, in the complete absence of viral antigen and these responses were long lasting (Vani et al. 2007b). * M. S. Shaila shaila@mcbl.iisc.ernet.in

Rinderpest virus (RPV) is an important member of the Morbillivirus genus in the family Paramyxoviridae and employs a similar strategy for transcription and replication of its genome as that of other negative sense RNA viruses. Cellular proteins have earlier been shown to stimulate viral RNA synthesis by isolated nucleocapsids from purified virus or from virus-infected cells. In the present work, we show that plus sense leader RNA of RPV, transcribed from 3' end of genomic RNA, specifically interacts with cellular La protein employing gel mobility shift assay as well as UV cross-linking of leader RNA with La protein. The leader RNA synthesized in virus-infected cells was shown to interact with La protein by immunoprecipitation of leader RNA bound to La protein and detecting the leader RNA in the immunoprecipitate by Northern hybridization with labeled antisense leader RNA. Employing a minireplicon system, we demonstrate that transiently expressed La protein enhances the replication/transcription of the RPV minigenome in cells. Sub-cellular immunolocalization shows that La protein is redistributed from nucleus to the cytoplasm upon infection. Our results strongly suggest that La protein may be involved in regulation of Rinderpest virus replication.

Phosphoprotein (P) of negative sense RNA viruses functions as a transcriptional transactivator of the viral polymerase (L). We report here the characterization of oligomeric P protein of rinderpest virus (RPV) and provide a structural basis for its multimerization. By size exclusion chromatography and dynamic light scattering analyses we show that bacterially expressed P protein exists as an oligomer, thus excluding the role of phosphorylation in P protein oligomerization. Gel filtration analyses of various parts of the P protein, also expressed in Escherichia coli, revealed that the predicted coiled coil region in the C-terminal domain is responsible for P protein oligomerization. Dynamic light scattering analysis confirmed the oligomeric nature of the coiled coil region of P. Chemical cross-linking analysis suggested that the C-terminal coiled coil region exists as a tetramer. The tetramer is formed by coiled coil interaction as shown by circular dichroism spectral analysis. Based on sequence homology, we propose a three-dimensional structure of the multimerization domain of RPV P using the crystal structure for multimerization domain of sendai virus (SeV) P as a template. Four-stranded coiled coil structure of the model is stabilized by a series of interactions predominantly between short nonpolar side chains emerging from different strands. In an in vivo replication/transcription system using a synthetic minigenome of RPV, we show that multimerization is essential for P protein function(s), and the multimerization domain is highly conserved between two morbilliviruses namely RPV and peste de petits ruminants virus. These results are discussed in the context of biological functions of P protein among various negative- stranded RNA viruses.

Glycodelin A, also known as placental protein-14, is a multifunctional glycosylated protein secreted by the uterine endometrium during the early phases of pregnancy.It is a known suppressor of T cell proliferation, inducer of T cell apoptosis, and inhibitor of sperm zona binding. Unlike in contraceptive activity, where the glycans on the molecule have been shown to play a crucial role, mutagenesis of the asparagines at sites of N-linked glycosylation $(Asn^{28}$ and $Asn^{63})$ to glutamine shows that the apoptogenic activity of glycodelin A is executed by the protein backbone. Glycosylation at $Asn^{28}$ appears to play a role in the extracellular secretion of the molecule, as mutation of $Asn^{28}$ resulted in a significant decrease in the amount of secreted protein, and loss of both glycosylation sites reduced the secretion drastically. Our results also suggest that the loss of glycosylation does not affect the dimerization status of the molecule.

Hemagglutinin-neuraminidase (HN) gene of Peste des petits ruminants virus (PPRV) has been expressed in pigeonpea for the development of an edible vaccine for Peste des Petits ruminant (PPR). PPRV, causes PPR disease in sheep and goats with high mortality rate. The two surface glycoproteins of PPRV Hemagglutinin-neuraminidase and fusion protein (F) confer protective immunity. We report the successful generation of transgenic pigeonpea (Cajanus cajan (L.) Millsp.) plants and expression of HN protein having biological activity. A 2 fragment containing the coding region of the HN gene from an Indian isolate was cloned into the binary vector pBI121 and mobilized into Agrobacterium tumefaciens strain GV3 101. Cotyledonary nodes from germinated seeds of pigeonpea were used for transformation. The presence of transgenes, NPTII and HN in the plants was confirmed by PCR. The expression of HN protein in the transgenic lines was further confirmed by Western blot analysis using polyclonal monospecific antibody to HN and more importantly plant-derived HN protein was shown to be biologically active as demonstrated by neuraminidase activity. Transgenic plants were fertile and PCR of $T_1$ plants confirmed the inheritance of the transgene.

Development of transgenic plants as an expression system for the delivery of recombinant subunit vaccine through fodder would represent the most efficient and economical means of mass immunization of domestic ruminants as well as susceptible wild life. Rinderpest virus causes a highly contagious disease in cattle, buffaloes, sheep, goats and wild ruminants with a high mortality rate. We have developed transgenic peanut (Arachis hypogea L.) plants expressing the hemagglutinin (H) protein of rinderpest virus. The transgenic peanut plants were produced by Agrobacterium-mediated transformation of shoot apices. The presence and integration of the transgene has been verified by PCR and Southern hybridization. The expression of the hemagglutinin protein was confirmed by immunoblotting analysis employing polyclonal H antibody. The expression of hemagglutinin protein in T1 generation demonstrates the stable integration of H gene into the plant genome.

Rinderpest is an acute, highly contagious often fatal disease of large and small ruminants, both domestic and wild. Global eradication of rinderpest needs a robust, safe and cost-effective vaccine. The causative agent, rinderpest virus (RPV) is an important member of the genus Morbillivirus in the Paramyxoviridae family. We have generated transgenic peanut (Arachis hypogea L.) plants expressing hemagglutinin protein of RPV and report here, the induction of immune responses in cattle following oral feeding with transgenic leaves expressing hemagglutinin protein without oral adjuvant. Hemagglutinin-specific antibody was detected in the serum as confirmed by immunohistochemical staining of virus-infected cells, and in vitro neutralization of virus infectivity. Oral delivery also resulted in cell-mediated immune responses.

Rinderpest virus causes a highly contagious and often fatal disease in domestic and wild ruminants. The surface glycoproteins, hemagglutinin (H) and fusion (F) proteins of this enveloped virus are known to confer protective immunity in cattle. We have reported the generation of a recombinant baculovirus expressing H protein and studied its protective properties in cattle. In this report, we demonstrate that the recombinant baculovirus encoded H protein expressed in insect cells gets incorporated into extracellular baculovirus. Single administration of low doses of purified recombinant extracellular virus with or without adjuvant induces virus neutralizing antibody responses and bovine leukocyte antigen (BoLA) class II restricted helper T cell responses in cattle.

The idiotypic network theory (N. K. Jerne, Ann. Immunol. 125, 373–389, 1974) predicts that any antibody that can be made by an individual would have its preexisting specific complementary B cells in its germline repertoire. We transplanted syngeneic BALB/c mice with live hybridoma cells and demonstrated the simultaneous presence of interacting idiotypic and anti-idiotypic B cells in an individual animal by immuno-cytoadherence assays. Furthermore, we demonstrate that interacting B cells displaying idiotypic and anti-idiotypic antibodies are subjected to lysis by complement. It is therefore tempting to speculate that this complement-sensitive interaction between idiotypic and complementary anti-idiotypic B cells in vivo may provide a mechanism for the regulation of B cell populations.

Rinderpest virus (RPV) is a highly contagious and often fatal disease of domestic and wild ruminants, caused by rinderpest virus of the genus Morbillivirus under the family Paramyxoviridae. Hemagglutinin (H) and fusion (F) proteins of this enveloped virus confer protective immunity against experimental challenge with virulent rinderpest virus. We have earlier demonstrated that immunization with a single dose of recombinant extracellular baculovirus expressing H protein elicits H-specific humoral and lymphoproliferative responses in cattle. The lymphoproliferative responses are predominantly BoLA class II restricted. In this work, we have analyzed lymphoproliferative responses of peripheral lymphocytes from immunized cattle to truncated H protein fragments expressed in E. coli for locating domains harboring T epitopes. One region (aa 113-182) recognized by immune $T_h$ cells is conserved in the H protein of measles virus, which was earlier shown to contain a dominant $T_h$ epitope in mouse. Synthetic peptides within this region of measles virus H protein were used to identify a $T_h$ epitope conserved in the H protein of RPV virus (aa 123-137) in cattle. A second $T_h$ epitope located at the C-terminus of RPV-H was mapped to the region corresponding to aa 512-609 using truncated protein fragments expressed in E. coli. The C-terminal epitope (aa 575-583) was mapped using synthetic peptides corresponding to measles virus H as well as RPV-H protein.

Rinderpest virus (RPV), a member of genus Morbillivirus in the family Paramyxoviridae, causes an acute and often fatal disease in cattle and other large ruminants. A subunit rinderpest vaccine consisting of an immune-stimulating complex (ISCOM) incorporating the RPV haemaggulutinin (H) protein, was examined for its ability to induce protective immunity in cattle, the natural host of RPV. All of four cattle vaccinated with the ISCOM vaccine survived challenge with virulent virus. Three were solidly protected, showing no clinical signs of infection, while the fourth animal developed only mild and transient symptoms. Virus neutralizing antibodies were produced at a significant level in all vaccinated cattle. These results indicate that this ISCOM vaccine is effective in producing protective immunity in cattle and should be a suitable means of delivering glycoprotein antigens from other morbilliviruses

The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells. We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E. coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells. Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c). A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response. The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted. Based on the prediction for H-2d-restricted T-cell motifs we tested the lysis of transfected P815 (H-2d) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins. We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins. Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope. This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides.

The yeast two-hybrid system was used to identify domains involved in specific in vivo interactions between the Rinderpest virus (RPV) phosphoprotein (P) and nucleocapsid protein (N). N and P genes were cloned in both the yeast GAL4 DNA-binding and GAL4 activation domain vectors, which enabled analysis of self and interprotein interactions. Mapping of the domain of P protein involved in its association with itself revealed that the COOH-terminal 32 amino acids (316–347) that forms a part of the highly conserved coiled coil region is important for interaction. In addition, just the coiled coil region of RPV P protein fused to the DNA-binding domain and activation domain of GAL4 was found to be sufficient to bring about activation of the $\beta-$galactosidase reporter. Similarly, mapping of the domains of P protein involved in its interaction with N protein revealed that $NH_2-$terminal 59 amino acids and COOH-terminal 32 amino acids (316–347) involved in P–P interaction are simultaneously required for association with N protein. Interestingly, a P protein mutant with just the $NH_2-$terminal 59 amino acids and the coiled coil domain with all other P protein regions deleted retained its ability to interact with N protein. Furthermore, we were able to show N and P protein interaction in vitro using recombinant N and P proteins expressed in Escherichia coli, demonstrating the existence of direct physical interaction between the two proteins.

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.

The hemagglutinin (H) protein of Rinderpest virus expressed by a recombinant baculo-virus used as a vaccine produced high titres of neutralizing antibody to Rinderpest virus in the vaccinated cattle, comparable to the levels produced by live attenuated vaccine. The immunized cattle were protected against a vaccine-virus challenge, as demonstrated by the failure of development of antibodies to N protein of the vaccine virus. The lack of replication of vaccine virus in the immunized cattle indicated that they are capable of showing a protective response if challenged with a virulent virus.

5-Fluorouracil (5FU), an analogue of uracil, was found to inhibit the production of infectious particles of rinderpest virus (RPV) in Vero cells (African green monkey kidney cells) by 99%, at a concentration of 1 microgram/ml. The levels of individual mRNA specific for five of the virus genes were also reduced drastically, while the level of mRNA for a cellular housekeeping gene-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-was unaltered by fluorouracil treatment of infected cells. Both virus RNA and protein synthesis showed inhibition in a dose-dependent manner. The virions which budded out of 5-fluorouracil-treated cells also contained reduced amounts of virus proteins compared with virus particles from untreated cells.

Purified rinderpest virus was earlier shown to transcribe in vitro, all virus-specific mRNAs with the promoter-proximal N mRNA being the most abundant. Presently, this transcription system has been shown to synthesize full length monocistronic mRNAs comparable to those made in infected cells. Small quantities of bi- and tricistronic mRNAs are also synthesized. Rinderpest virus synthesizes in vitro, a leader RNA of not, vert, similar 55 nucleotides in length. Purified rinderpest virus also exhibits RNA editing activity during the synthesis of P mRNA as shown by primer extension analysis of the mRNA products.

Dendritic cells (DC) are the professional antigen-presenting cells and act as sentinels of the immune system. Therefore, the activation status of DC upon an encounter with vaccine antigens determines the intensity and duration of protective immune responses. DC are also critical for the transmission

Regular vaccinations with potent vaccine, in endemic countries and vaccination to live in non-endemic countries are the methods available to control foot-and-mouth disease. Selection of candidate vaccine strain is not only cumbersome but the candidate should grow well for high potency vaccine preparation. Alternative strategy is to generate an infectious cDNA of a cell culture-adapted virus and use the replicon for development of tailor-made vaccines. We produced a chimeric `O' virus in the backbone of Asia 1 and studied its characteristics. The chimeric virus showed high infectivity titre (>10(10)) in BHK 21 cell lines, revealed small plague morphology and there was no cross reactivity with antiserum against Asia I. The virus multiplies rapidly and reaches peak at 12 h post infection. The vaccine prepared with this virus elicited high antibody titres.

Infection of the skin or throat by Streptococcus dysgalactiae subspecies equisimilis (SDSE) may result in a number of human diseases. To understand mechanisms that give rise to new genetic variants in this species, we used multi-locus sequence typing (MLST) to characterise relationships in the SDSE population from India, a country where streptococcal disease is endemic. The study revealed Indian SDSE isolates have sequence types (STs) predominantly different to those reported from other regions of the world. Emm-ST combinations in India are also largely unique. Split decomposition analysis, the presence of emm-types in unrelated clonal complexes, and analysis of phylogenetic trees based on concatenated sequences all reveal an extensive history of recombination within the population. The ratio of recombination to mutation (r/m) events (11:1) and per site r/m ratio (41:1) in this population is twice as high as reported for SDSE from non-endemic regions. Recombination involving the emm-gene is also more frequent than recombination involving housekeeping genes, consistent with diversification of M proteins offering selective advantages to the pathogen. Our data demonstrate that genetic recombination in endemic regions is more frequent than non-endemic regions, and gives rise to novel local SDSE variants, some of which may have increased fitness or pathogenic potential., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Sequence analysis of part of the fusion protein gene from recent isolates of rinderpest virus revealed that distinct lineages of the virus exist which reflect the geographical location of their isolation in Africa and Asia. Current strains circulating in Kenya and Sudan were most similar, both in terms of nucleotide sequence and pathogenic nature, to viruses isolated in Egypt and in Nigeria in 1983/1984 and they were quite distinct from an East African isolate (RBT-1) from the 1960s. Two older isolates of the virus, the Japanese avianized/lapinized vaccine strain dating from the 1930s and the Old Kabete strain dating from 1911, each differed considerably from the other viruses. The sequence data were derived from the region where the precursor protein is cleaved to yield the biologically active F1/F2 heterodimer; all strains analysed had a highly basic connecting peptide which is required for efficient cleavage by endogenous host cell proteases. No correlation was found between amino acid changes at this site and the rinderpest virus pathogenicity unlike the association reported for Newcastle disease virus.

Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly. HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRV-HN specific cell-mediated immune responses were also detected in mucosally immunized sheep. (C) 2010 Elsevier B.V. All rights reserved.

Streptococcus pyogenes [group A streptococcus (GAS)], a human pathogen, and Streptococcus dysgalactiae subsp. equisimilis [human group G and C streptococcus (GGS/GCS)] are evolutionarily related, share the same tissue niche in humans, exchange genetic material, share up to half of their virulence-associated genes and cause a similar spectrum of diseases. Yet, GGS/GCS is often considered as a commensal bacterium and its role in streptococcal disease burden is under-recognized. While reports of the recovery of GGS/GCS from normally sterile sites are increasing, studies describing GGS/GCS throat colonization rates relative to GAS in the same population are very few. This study was carried out in India where the burden of streptococcal diseases, including rheumatic fever and rheumatic heart disease, is high. As part of a surveillance study, throat swabs were taken from 1504 children attending 7 municipal schools in Mumbai, India, during 2006-2008. GAS and GGS/GCS were identified on the basis of beta-haemolytic activity, carbohydrate group and PYR test, and were subsequently typed. The GGS/GCS carriage rate (1166/1504, 11%) was eightfold higher than the GAS carriage (22/1504, 1.5%) rate in this population. The 166 GGS/GCS isolates collected represented 21 different emm types (molecular types), and the 22 GAS isolates represented 15 different emm types. Although the rate of pharyngitis associated with GGS/GCS is marginally lower than with GAS, high rates of throat colonization by GGS/GCS underscore its importance in the pathogenesis of pharyngitis.

Background Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4+ Tregs. Significance Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Given the increasing aetiological importance of Streptococcus dysgalactiae subspecies equisimilis in diseases which are primarily attributed to S. pyogenes, molecular markers are essential to distinguish these species and delineate their epidemiology more precisely. Many clinical microbiology laboratories rely on agglutination reactivity and biochemical tests to distinguish them. These methods have limitations which are particularly exacerbated when isolates with mixed properties are encountered. In order to provide additional distinguishing parameters that could be used to unequivocally discriminate these two common pathogens, we assess here three molecular targets: the speB gene, intergenic region upstream of the scpG gene (IRSG) and virPCR. Of these, the former two respectively gave positive and negative results for S. pyogenes, and negative and positive results for S. dysgalactiae subsp. equisimilis. Thus, a concerted use of these nucleic acid-based methods is particularly helpful in epidemiological surveillance to accurately assess the relative contribution of these species to streptococcal infections and diseases.

Streptococcus pyogenes [group A streptococcus (GAS)], a human pathogen, and Streptococcus dysgalactiae subsp. equisimilis [human group G and C streptococcus (GGS/GCS)] are evolutionarily related, share the same tissue niche in humans, exchange genetic material, share up to half of their virulence-associated genes and cause a similar spectrum of diseases. Yet, GGS/GCS is often considered as a commensal bacterium and its role in streptococcal disease burden is under-recognized. While reports of the recovery of GGS/GCS from normally sterile sites are increasing, studies describing GGS/GCS throat colonization rates relative to GAS in the same population are very few. This study was carried out in India where the burden of streptococcal diseases, including rheumatic fever and rheumatic heart disease, is high. As part of a surveillance study, throat swabs were taken from 1504 children attending 7 municipal schools in Mumbai, India, during 2006–2008. GAS and GGS/GCS were identified on the basis of β-haemolytic activity, carbohydrate group and PYR test, and were subsequently typed. The GGS/GCS carriage rate (166/1504, 11 %) was eightfold higher than the GAS carriage (22/1504, 1.5 %) rate in this population. The 166 GGS/GCS isolates collected represented 21 different emm types (molecular types), and the 22 GAS isolates represented 15 different emm types. Although the rate of pharyngitis associated with GGS/GCS is marginally lower than with GAS, high rates of throat colonization by GGS/GCS underscore its importance in the pathogenesis of pharyngitis.

ErbB3 binding protein Ebp1 has been shown to downregulate ErbB3 receptor-mediated signaling to inhibit cell proliferation. Rinderpest virus belongs to the family Paramyxoviridae and is characterized by the presence of a non-segmented negative-sense RNA genome. In this work, we show that rinderpest virus infection of Vero cells leads to the down-regulation of the host factor Ebp1, at both the mRNA and protein levels. Ebp1 protein has been shown to co-localize with viral inclusion bodies in infected cells, and it is packaged into virions, presumably through its interaction with the N protein or the N-RNA itself. Overexpression of Ebp1 inhibits viral transcription and multiplication in infected cells, suggesting that a mutual antagonism operates between host factor Ebp1 and the virus.

Rinderpest virus belongs to the family of Paramyxoviridae, consisting of non-segmented negative sense RNA viruses. Viral transcription and replication are carried out by the RNA dependent RNA polymerase L protein which functions together with P protein as L-P complex. The exact events triggering the polymerase complex from transcription to replication function is poorly understood. In the present work, an in vitro transcription system has been described with partially purified L-P complex expressed in insect cells and viral genomic RNA. The relative abundance of each species of mRNA synthesized in vitro decreased from the 3' end of the genome to the 5' end similar to their abundance in virus infected cells. Recombinant L-P complex was unable to synthesize leader RNA suggesting the initiation of transcription from gene start site and not at the 3' end of the genome.

$CD8^+ T$ cells are thought to play an important role in protective immunity against tuberculosis. We report the identification of three peptides derived from Rv1818c, Rv3812 and Rv3018c proteins of Mycobacterium tuberculosis that bound to HLA-A*0201 molecules and their ability to induce in vitro T-cell response in peripheral blood lymphocytes from HLA-A*0201-positive healthy individuals (PPD+) and patients with TB. The peptide-specific cytotoxic T lymphocytes (CTL) generated were capable of recognizing peptide pulsed targets. Three 9-mer peptides bound with high affinity to HLA-A*0201 and displayed low dissociation rates of the bound peptide from HLA. Epitope-specific recognition was demonstrated by the release of perforin and γ-interferon. Overall, our results demonstrate the presence of HLA class I-restricted $CD8^+$ CTL against proteins from PE and PPE proteins of M. tuberculosis and identify epitopes that are strongly recognized by HLA-A*0201-restricted $CD8^+ T$ cells in humans. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.

Three proteins of PE and PPE families of Mycobacterium tuberculosis were evaluated for their ability to induce T cell responses in mice. To enhance immunity induced by protein immunization, we tested the efficacy of adjuvant Ribi (monophosphoryl lipid A+TDM), along with three proteins of the PE/PPE family. Balb/c mice were subcutaneously injected with recombinant proteins, encoded by Rv1818c, Rv3018c and Rv3812 genes of M. tuberculosis H37Rv, formulated with Ribi or IFA for comparative study. Sera from mice immunized with Ribi revealed an increase in the specific immunoglobulin G titers by twofold against Ribi than in mice immunized with IFA. Ribi also elicited stronger delayed-type hypersensitivity and cytotoxic T-lymphocyte activity against the recombinant proteins when compared with IFA. Antigen specific IgG subclass analysis showed that Ribi tends to facilitate IgG2a production, suggesting enhancement of predominant Th1 response which in turn may facilitate increased production of protective IFN-\gamma. Furthermore, Ribi preparation increased the number of T cells secreting IFN-\gamma. These results indicate that Ribi acts as an effective adjuvant for immune response to antigens of M. tuberculosis. For the first time, we demonstrate that Rv3018c, Rv1818c and Rv3812 proteins of PE/PPE family are T cell antigens with vaccine potential.

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation. (C)) 2007 Elsevier Inc. All rights reserved.