Identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain.

We recently generated a fully synthetic porcine reproductive and respiratory syndrome virus strain (designated as PRRSV-CON), which confers unprecedented levels of heterologous protection. We report herein that the synthetic PRRSV-CON possesses a unique phenotype in that it induces type-I interferons (IFNs) instead of suppressing these cytokines as most of the naturally occurring PRRSV isolates do. Through gain- and loss- of-function studies, the IFN-inducing phenotype of PRRSV-CON was mapped to the 3.3kb genomic fragment encoding three viral nonstructural proteins: nsp1α, nsp1ß and the N-terminal part of nsp2. Further studies indicated that a cooperation among these 3 proteins was required for effective induction of IFNs. Collectively, this study constitutes the first step toward understanding the mechanisms by which the synthetic PRRSV-CON confers heterologous protection.

Evaluation of core and NS4B synthetic peptide-based immunoassays for the detection of hepatitis C virus antibodies in clinical samples from Cameroon, Central Africa.

BACKGROUND: According to previous data, the antibodies produced during natural hepatitis C virus (HCV) infection frequently recognize amino acids 10-43 in the core protein and 1689-1740 or 1921-1940 in the non-structural 4B (NS4B) protein. The reactivity of these peptides with the corresponding antibodies has mainly been evaluated using serum samples from Western countries where HCV genotype 1 (HCV-1) is predominant, and no information is available concerning samples from sub-Saharan countries where high HCV variability has been reported. OBJECTIVE OF THIS STUDY: To evaluate the performance of HCV core and NS4B peptide-based immunoassays in the serodiagnosis of HCV infection in Cameroon subjects. STUDY DESIGN: Three core and four NS4B-based synthetic peptides derived from HCV genotypes 1b and 2a were designed and tested against a panel of 151 serum samples from Cameroon (40 positive for HCV-1, 32 for HCV-2, 39 HCV-4, and 40 HCV-negative). RESULTS: The three core peptides all demonstrated strong immunoreactivity, regardless of the HCV genotype from which they were derived, with greater than 90% and 92% sensitivity and specificity. In contrast, the NS4B-derived peptides exhibited lower sensitivities (24.3-65.8% depending on the HCV genotype) but higher specificities (100% for all four peptides tested). CONCLUSIONS: Our findings indicate that an HCV core peptide could be used for the diagnosis of chronic HCV infection. Among the NS4B peptides tested, a chimeric NS4B peptide encompassing both N- and C-terminal portions of the NS4B protein gave a much better performance than the two component N- and C-terminal peptides used individually.

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains.

The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354-389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

Design, expression, and processing of epitomized hepatitis C virus-encoded CTL epitopes.

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.

Influence of lipidation of GBV-C/HGV NS3 (513-522) and (505-514) peptide sequences on its interaction with mono and bilayers.

Two decapeptide fragments of the non-structural hepatitis G NS3 protein (GBV-C/HGV), 513-522 (RGRTGRGRSG) and 505-514 (SAELSMQRRG), as well as their palmitoylated derivatives were synthesized. The physico-chemical properties of the peptides were analyzed in both the absence and presence of the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), the negative 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) and the positive 1,2-dioeloyl-3-trimethylammonium-propane (DOTAP) lipid monolayers. Based on their high hydrophilic properties, neither parent peptide presented surface activity and their incorporation into lipid monolayers was low. In contrast, their palmitoylated derivatives showed concentration-dependent surface activity and could be inserted into lipid monolayers to varying degrees depending on their sequence. Compression isotherms showed that the presence of palmitoylated peptides in the subphase resulted in a molecular arrangement less condensed than that corresponding to the pure phospholipid. In concordance with the monolayer results, differential scanning calorimetry (DSC) demonstrated that the parent peptides did not have any effect on the thermograms, while the palmitoylated derivatives affected the thermotropic properties of DPPC bilayers.

Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine.

To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the non-structural protein 3D in swine, pentadecapeptides were tested in proliferation and Interferon-gamma ELISPOT assays using lymphocytes from two strains of inbred miniature pigs (c/c and d/d haplotype) experimentally infected with FMDV. Lymphocytes of c/c pigs recognized peptides from three different regions in 3D, d/d lymphocytes recognized peptides from two regions, one of them being adjacent to an epitope of c/c pigs and comprising amino acid residues 346-370. Analyses of the response of d/d lymphocytes against peptides representing the structural protein 1A revealed another novel T-cell epitope. Investigation of the phenotype of responding lymphocytes showed a response of CD4(+)CD8(+)MHC-class-II(+) cells, identifying them as activated T-helper cells. This is the first report on FMDV specific T-cell epitopes recognized by swine leukocyte antigen (SLA) inbred swine and provides information useful for the design of novel vaccines against FMDV.

Development of synthetic peptide ELISA based on nonstructural protein 2C of foot and mouth disease virus.

It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.

Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus.

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.

Development of synthetic peptide ELISA based on nonstructural protein 2C of foot and mouth disease virus

It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.

Immune response to an epitope of the NS4 protein of hepatitis C virus in HCV-related disorders.

NS4, a nonstructural protein of HCV, is a frequent target of antibodies in infected subjects. According to recent data, the antibodies frequently recognize the sequence 1921-40 of the NS4 protein. The aim of this work was to analyze antibody reactivity with the sequence 1921-40 in different HCV-related disorders. Although this sequence is located in a relatively invariant region of viral genome, two strain-specific sequences are described. Thus, three NS4 1921-1940 peptides were synthesized: the BK shared by most viral strains, the J6 (strain 2a), and the J8 (strain 2b). The peptides were used as antigens in the solid phase for measuring serum IgG antibodies in an ELISA assay. Antibodies reactive with the 1921-40 BK peptide were detected in 64% of sera from patients with autoimmune hepatitis (AIH), 51% from chronic hepatitis C (CHC), and 22% from mixed cryoglobulinemia (MC). The frequency of positive sera in MC was significatively lower than in AIH (P < 0.0001) or CHC (P < 0.0021). Similar results were obtained with the J6 and J8 peptides. All sera that did not react with the BK peptide were negative on J6 and J8 and conversely most sera reacting with the BK peptide also bound the J6 and the J8 peptides. No correlation was found between the genotype of the infecting virus and the presence of antibodies to any of the NS4 peptides. These results indicate that many HCV-infected subjects produce antibodies to the NS4 sequence 1921-40. The immune response to this sequence is not strain specific and varies with the different disorders associated with HCV infection.

Gene 5 of the IDIR agent (group B rotavirus) encodes a protein equivalent to NS34 of group A rotavirus.

Gene 5 of the IDIR strain of group B rotavirus was cloned, sequenced, and expressed in RBC lysates in order to identify the coding assignment. The complete nucleic acid sequence of IDIR gene 5 included 1306 bases and encoded a single long open reading frame of 390 amino acids with a predicted molecular weight of 44.8 kDa. Comparison of the predicted amino acid sequence indicated substantial identity with the NS34 protein of group A rotaviruses (GAR). Like GAR NS34, the product deduced from IDIR agent gene 5 was predicted to exhibit a high degree of alpha helix secondary structure and a relatively low pl (4.6). In vitro translation of gene 5 resulted in synthesis of a protein which was specifically immunoprecipitated by convalescent antibody obtained from infant rats following infection with the IDIR agent, confirming that this product was of viral origin. Under non-reducing conditions, products as large as 200 kDa were also noted in SDS-PAGE. Formation of oligomers by GAR NS34 has previously been observed, indicating that this feature is probably a general characteristic among rotavirus NS34 and may relate to the functional role of the protein in viral replication.

Isolate antibody to hepatitis C virus core antigen (C22) by RIBA-2: correlation with HCV-RNA and anti-NS5.

The presence of circulating hepatitis C virus genome (HCV-RNA), elevated ALT levels and antibodies to an NS5-derived synthetic peptide have been examined in 13 subjects with isolate positivity for antibodies to the HCV core antigen (C22) on RIBA-2 testing. All subjects were followed up for 8-18 months (mean 12.4 months). In seven subjects (54%), intermittent or persistent viremia was associated with abnormal ALT levels (6 subjects) and with positivity for antibodies to NS5-peptide (6 subjects). On the other hand, in 6 out of 13 subjects (46%) no viral replication, no liver cytonecrosis and no antibodies to NS5 were found. It is concluded that isolate reactivity to C22 by RIBA-2 is a heterogeneous condition that corresponds to two distinct categories of subjects: those with active HCV infection and those without evidence of virus replication. Although HCV-RNA determination is the most reliable means of identifying HCV carriers, antibodies to NS5 can be a useful marker of virus activity. In fact, antibodies to NS5 were detected in 6 out of 7 viremic patients, compared to 0 out of 6 non-viremic patients (P = 0.004). It remains to be elucidated whether the isolate reactivity to core antigen found in non-viremic subjects represents a specific, HCV-induced antibody response, or is an unrelated crossreactivity.