Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3D(pol)) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 313 (VTg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV ere has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 313 peptides has now been determined, and the role of the FMDV ere (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Prevalence of newcastle disease virus in broiler chickens (Gallus gallus) in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A survey for maintenance of virulent newcastle disease virus-free area in poultry production in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Protection levels of vaccinated pigeons (Columba livia) against a highly pathogenic newcastle disease virus strain

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Experimental infection of Newcastle disease virus in pigeons (Columba livia): Humoral antibody response, contact transmission and viral genome shedding

The aim of this study was to evaluate the humoral antibody response, the genome viral excretion and the contact transmission of pathogenic chicken origin Newcastle disease virus (NDV) from experimentally infected pigeons (Columba livia) to in-contact pigeon. The antibody response to infection was assessed by the hemagglutination inhibition (HI) test and the genome viral excretion was detected by RT-PCR. Viral strain induced high antibody levels, both in inoculated and in sentinel birds. The pathogenic viral strain for chickens was unable to produce clinical signs of the disease in experimentally infected pigeons, although it induced the Immoral antibody response and produced NDV genome shedding. NDV genome was detected intermittently throughout the experimental period, from 5 days post-infection (dpi) to 24 dpi. Therefore, viral genome shedding occurred for 20 days. The viral genome was detected in all birds, between I I and 13 dpi. Furthermore, the high infectivity of the virus was confirmed, as all non-inoculated sentinel pigeons showed antibody levels as high as those of inoculated birds. (C) 2007 Elsevier B.V. All rights reserved.

PREVALENCE of SALMONELLA SPP. ANTIBODIES TO TOXOPLASMA GONDII, and NEWCASTLE DISEASE VIRUS IN FERAL PIGEONS (COLUMBA LIVIA) IN THE CITY of JABOTICABAL, BRAZIL

The rock pigeon (Columba livia) may serve as a reservoir for several pathogenic agents that can be transmitted to poultry, wildlife, domesticated pets, and/or humans via excreta, secretions, or dust from feathers. In addition, ingestion of infected pigeons by wild and domestic animals can also transmit these pathogenic agents. The health status of 126 free-living pigeons in an urban area was evaluated by microbiologic culture for Salmonella and serologic testing for the presence of antibodies for Toxoplasma gondii and for Newcastle disease virus (NDV) from 120 and 109 pigeons, respectively. After drawing blood, the birds were euthanized, and fragments of the liver, spleen, lungs, and gonads, and feces were cultured for Salmonella spp. Salmonella spp. was isolated from 10 birds (7.94%), of which 8 were Salmonella typhimurium, one was Salmonella enterica subsp. enterica serotype 4,12 and one was Salmonella enterica subsp. enterica serotype 4,12,i. Six of 109 pigeons (5.50%) were positive for NDV antibodies when using the hemagglutination inhibition test. Toxoplasma gondii antibodies were detected by immunofluorescence in one of 120 sera tested (0.83%). The results indicate that feral rock pigeons were exposed to NDV and T gondii, although the exposure was low. In addition, these birds had Salmonella spp. and could disseminate this pathogen in the environment.

Replication of classical infectious bursal disease virus in the chicken embryo related cell line

Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination, A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48 h, Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597+/-0.02 and 0.010+/-0.01 after 12h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome, This molecular technique demonstrated that from 6 h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.

Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction

The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.

Liquid-phase blocking sandwich enzyme-linked immunosorbent assay for detection of antibodies against foot-and-mouth disease virus in water buffalo sera

Objective-To develop and apply the liquid-phase blocking sandwich ELISA (BLOCKING-ELISA) for the quantification of antibodies against foot-and-mouth disease virus (FMDV) strains O-1 Campos, A(24) Cruzeiro, and C-3 Indaial.Design-Antibody quantification.Sample Population-158 water buffalo from various premises of São Paulo Stale-Brazil. The sera were collected either from systemically vaccinated or nonvaccinated animals.Procedure-The basic reagents of BLOCKING-ELISA (capture and detector antibodies, virus antigens, and conjugate) were prepared and the reaction was optimized and standardized to quantify water buffalo antibodies against FMDV. An alternative procedure based on mathematical interpolation was adopted to estimate more precisely the antibody 50% competition liters in the BLOCKING-ELISA. These titers were compared with the virus-neutralization test (VNT) titers to determine the correlation between these techniques. The percentages of agreement, cutoff points, and reproducibility also were determined.Results-The antibody liters obtained in the BLOCKING-ELISA had high positive correlation coefficients with VNT, reaching values of 0.90 for O-1 Campos and C-3 Indaial, and 0.82 for the A(24) Cruzeiro (P < 0.0005). The cutoff points obtained by use of the copositivity and conegativity curves allowed determination of high levels of agreement between BLOCKLNG-ELISA and VNT antibody titers against the 3 FMDV strains analyzed.Conclusions-The results characterized by high cor relation coefficients, levels of agreement, and reproducibility indicate that the BLOCKING-ELISA may replace the conventional VNT for detection and quantification of antibodies from water buffalo sera to FMDV.

RAPID COAGGLUTINATION TEST FOR THE DETECTION AND TYPING OF FOOT-AND-MOUTH-DISEASE VIRUS

Protein A containing Staphylococcus aureus was used to develop a coagglutination (COA) test for the detection and typing of foot and mouth disease virus (FMDV) O, A and C serotypes in infected cells and tissues. Different batches and amounts of guinea pig anti-FMDV sera were assessed to optimize the preparation of COA conjugates. The sensitivity and specificity of the COA Test for the detection of FMDV O, A and C serotypes and heterologous viruses was also characterized. Comparison between the COA Test and complement fixation test for the detection and typing of FMDV obtained from extracts of tongue epithelial tissues from infected cattle revealed high agreement in the results and indicated a potential application of the COA Test for the direct diagnosis of viruses.

Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method, the cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement ( = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Application of a serum/protein-free medium for the growth of mammalian cell lines and high level production of classical, variant and very virulent strain of infectious bursal disease virus (IBDV)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Newcastle disease virus vaccine strains: Immunogenicity is not influenced by ICPI

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)