Amer Silim

Evidence for the multimeric nature and cell binding ability of avian reovirus sigma 3 protein.

It has been suggested that avian reovirus sigma 3 protein is analogous to sigma 1 trimer, the mammalian reovirus attachment protein. We have investigated the multimeric nature and cell binding ability of sigma 3 protein. The data presented here demonstrate that sigma 3 protein is a multimer in its undisrupted form as determined by SDS-PAGE in non-dissociating conditions. However, virion-associated sigma 3 protein and COS-7 cell-expressed protein behaved differently in SDS-PAGE, suggesting a need for virus-associated factor(s) to control the multimerization of the protein. The data also show that Escherichia coli expressed sigma 3 fusion protein (sigma 3-MBP) in its multimeric form is capable of attaching to Vero cells. The binding was found to be specific and receptor mediated by the fact that it was inhibited by a monoclonal antibody specific for sigma 3 protein and by competition with avian reovirus particles. As determined by a reverse experiment, sigma 3-MBP was also able to reduce the virus p.f.u. in monolayer cell cultures, indicating the important role of sigma 3 protein in the initiation of virus infection.

Molecular characterization of three new avian infectious bronchitis virus (IBV) strains isolated in Quebec.

Three unrecognized field isolates of Infectious Bronchitis Virus (IBV) were recovered from commercial broiler chickens vaccinated with live Mass viral strain (H120). These isolates were identified by immunofluorescence using monoclonal antibodies produced against reference serotypes: Mass, Conn, and Ark. RT-PCRs were performed on viral RNAs to amplify S1 gene using a specific set of primers S1OLIGO3′ and S1OLIGO5′. Restriction polymorphism (RFLP) of PCR products was determined by the use of HaeIII restriction enzyme. As expected, patterns of PCR products were different from common pattern of strains assigned to Mass serotype M41, Beaudette, H120, and Florida. Molecular analysis showed a nucleotide insertion in hypervariable region one (HVR-1) of S1 gene of only Quebec isolates (Qu16, Qu_mv and Q_37zm). However, New Brunswick IBV isolate (NB_cp) did not display these insertions. Major amino acid changes involved insertion of two stretches (aa118–119: Arg–Ser and aa141–145: Sys–Ser–Asn–Ala–Ser–Cys) located at N-terminal and C-terminal regions of HVR-2. It is speculated that cysteine residue located upstream and downstream of Cys–Ser–Asn–Ala–Ser–Cys segment might be involved in the formation of loop structure and disulfide bond that could trigger important epitope changes. Insertion of new NXT and NXS (X≠P) glycosylation motifs scattered along S1 region and insertion of cysteine residues in HVR are contributing to the antigenic shifting of Quebec isolates. Fragment insertions were thought to be induced by inter-serotype recombination between vaccine strain (H120) that belongs to Mass serotype and another strain belonging to Ark serotype. Phylogenetic tree based on amino acid sequences showed that Quebec isolates formed a new phylogenetic cluster.

Characterization of monoclonal antibodies against avian reovirus strain S1133

Monoclonal antibodies (MAbs) were produced against a vaccinal S1133 strain of avian reovirus. Characterization of six MAbs in Western blotting, radioimmunoprecipitation and gold immunoelectron microscopy revealed that the MAbs were specific to the outer capsid proteins, mu2/mu2c, sigma2 and sigma3. Two of three MAbs, directed against sigma2 protein, neutralized the virus infectivity in a broadly specific manner, whereas the third had no neutralizing activity. The only MAb which reacted with sigma3 protein showed a type-specific neutralizing activity. Two MAbs recognizing the mu2/mu2c proteins failed to neutralize the virus infectivity.

Comparison of neutralizing antigens of recent isolates of infectious bursal disease virus

SummaryMonoclonal antibodies (MAbs) to a local turkey isolate (QT-1) of infectious bursal disease virus (IBDV) were produced to identify the virus-specific neutralizing proteins. Radioimmunoprecipitation assays showed that all the MAbs were specific for major viral protein, VP2. Two of the MAbs neutralized the local turkey and chicken isolates along with a reference strain belonging to serotype 1 but not the reference strain of serotype 2. The reactivities of the neutralizing MAbs against two reference strains and some recent field strains of IBDV isolated in the province of Québec were studied by indirect enzyme-linked immunosorbent assay and virus neutralization tests. The variations in the reactivities of the MAbs observed suggest differences in the neutralizing epitopes of the different isolates. Competitive binding assay using the MAbs revealed the presence of a third epitope involved in the neutralization of IBDV belonging to serotype 1.

Biological roles of the major capsid proteins and relationships between the two existing serotypes of infectious bursal disease virus

SummaryNeutralizing monoclonal antibodies (n-MAbs) were produced against infectious bursal disease virus (IBDV) of serotypes 1 and 2. The n-MAbs recognizing the major antigenic proteins VP2 and VP3, were characterized using different strains of IBDV representing the existing two serotypes and a variant subtype of serotype 1. The biological properties of these viral antigens as defined by the MAbs in vitro, were studied utilizing post-adsorption virus neutralization tests and fluorescence-activated cell sorter analysis. The MAbs directed against the immunodominant epitopes on VP2 were capable of enhanced virus neutralization but did not inhibit the virus attachment to susceptible cells. These MAbs were able to neutralize the virus by interfering with an event subsequent to virus adsorption, possibly inhibiting virus penetration or uncoating. On the contrary, a MAb that immunoprecipitated the other capsid protein VP3 was able to prevent virus attachment although it possessed lower neutralization titers. Cross-immunoprecipitations of various virus strains by these MAbs and antisera revealed interrelationships between the two serotypes of IBDV.

Serological and pathogenic characterization of avian reoviruses isolated in Quebec

Reoviruses were isolated from intestinal contents of broiler chickens from nine flocks in Quebec with malabsorption syndrome. Serum neutralization test demonstrated the existence of antigenic differences between the isolates and the reference vaccine strain. The isolated reoviruses were inoculated orally and into the foot pad in one-day-old chicks, resulting in a transient, but significant depression in body weight gains. Chickens infected with isolate 615, showed in addition to growth problems, clinical signs and tissue lesions similar to those observed in field cases. When isolate 615 was inoculated into SPF chicks at one day of age, intestinal absorption of D-xylose in infected chicks at 7 days post-infection was significantly lower (P <0.05) than for corresponding controls. This study suggests the implication of some reovirus isolates, such as 615 which was serologically distinct from the vaccine strain S1133, as infectious agents associated with pathological conditions other than viral arthritis.

The 64 kDa lipoprotein of Mycoplasma gallisepticum has two distinct epitopes responsible for haemagglutination and growth inhibition

A major Mycoplasma gallisepticum polypeptide of 64 kDa (p64) was characterized using two distinct monoclonal antibodies (MAbs), MAb KI produced in our laboratory and MAb MyG 001 produced by Avakian & Ley (1993). The p64 antigen was shown to be a lipoprotein in a radioimmunoprecipitation assay using [(3)H] palmitic acid-labelled M. gallisepticum cultures. The two MAbs inhibited the growth of M. gallisepticum in liquid medium and reacted to two distinct epitopes on the same p64 antigen in competitive enzyme-linked immunosorbent and chemiluminescence Western immunoblot assays. MAb Kl inhibited haemagglutination of chicken and turkey erythrocytes whereas MAb MyG 001 did not. The results of our study indicate that p64 has two distinct epitopes involved in haemagglutination and growth inhibition of M. gallisepticum. MAb Kl also inhibited the attachment of the mycoplasma to TLT lymphoblastoid chicken B cell line, suggesting that p64 is a cytadhesin.

Radioimmunoprecipitation of avian reovirus polypeptides using virus-specific IgM and IgG murine monoclonal and chicken polyclonal antibodies

A simple and improved procedure for radioimmunoprecipitation (RIPA) for the identification of major immunogenic proteins of avian reovirus using murine monoclonal and chicken polyclonal antibodies is described. Bacterial proteins (Staphylococcus aureus-Protein A or Streptococcus species-Protein G) commonly used in RIPA procedures lack reactivities with low avidity monoclonals belonging to immunoglobulin (Ig)G or IgM subtypes, as well as avian Ig. Hence we used an indirect approach utilizing species-specific anti-IgM, IgG or chicken Ig antibodies followed by the precipitation of the immune complexes using Protein A. This improved the sensitivity of the RIPA enabling the antigenic analysis of the major antigenic proteins of avian reovirus. The results indicated that the virus is highly immunogenic in the natural host, chicken than in mice. Furthermore, there exists a direct correlation between a strong neutralizing antibody response and an increased precipitation of the sigma (sigma) proteins of the virus. The results also demonstrate a strong association between the conformational viral epitopes of the three classes of proteins, large (lambda), medium (mu) and small (sigma).

Prediction of Optimal Vaccination Timing for Infectious Bursal Disease Based on Chick Weight

Abstract Growth rate in broiler birds has increased substantially in the last decade due to improvement in genetics, feed formulation, cleaner environment, and vaccine formulations. As a result, it has become necessary to review and revise prediction method for vaccination in chicks. This study was undertaken to determine the possible use of the rate of weight gain rather than age in predicting vaccination time. Two groups of 1-day-old broilers originating from old and young breeders, respectively, and with different levels of maternal antibodies against infectious bursal disease virus (IBDV) were used in this study. The chicks were divided into four groups and subjected to two feed regiments: groups A1 and B1 were fed broiler feed for normal growth rate, and groups A2 and B2 were fed breeder feed for slower growth rate. At 1, 4, 8, 12, 16, 22, 29, and 36 days of age, 22 chicks in each group were weighed, and blood samples were collected. Serum samples were tested for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA) and virus neutralization test. Maternal antibody decline curves for each group were plotted according to chick age and chick weight. Fast-growing birds in groups A1 and B1 showed a faster rate of antibody decline, whereas slow-growing birds in groups A2 and B2 had a slower rate of antibody decline. Based on the effect of weight gain on maternal antibody decline, a new way of predicting vaccination time for IBDV based on measuring maternal antibody titers at 4 days of age was proposed and tested. The predicted antibody decline was shown to correspond to the real ELISA titers measured in our experiments (R  =  0.9889), whereas a lower correlation (R  =  0.8355) was detected between real ELISA titers and the titers predicted by the current method using age-based Deventer formula.

Lymphoproliferative responses of specific‐pathogen‐free chickens to Mycoplasma gallisepticum strain PG31

Mycoplasma gallisepticum (MG) is one of the aetiologic agents of chronic respiratory disease in chickens and infectious sinusitis in turkeys. We investigated humoral and cellular immune mechanisms following experimental infection with four different strains of MG. Peripheral blood leukocytes (PBL) obtained from chickens were examined for proliferation using antigen preparations of whole cell MG as stimuli in vitro. A consistent lymphoproliferative response was observed against the homologous whole cell antigens in the group of chickens infected with strain PG31. Significant lymphoproliferation was detected as early as 1 week post-infection. We further characterized antigen-specific proliferation by measuring the production of interferon and nitric oxide by the PBL of infected chickens. Consistent with lymphoproliferation, we also detected the presence of interferon and nitric oxide in vitro in antigen-stimulated cultures. These results indicate a possible role of cell-mediated immune responses in the development of immunity following MG infection in chickens.