The peptide motif of the single dominantly expressed class I molecule of the chicken MHC can explain the response to a molecular defined vaccine of infectious bursal disease virus (IBDV)

In contrast to typical mammals, the chicken MHC (the BF-BL region of the B locus) has strong genetic associations with resistance and susceptibility to infectious pathogens as well as responses to vaccines. We have shown that the chicken MHC encodes a single dominantly expressed class I molecule whose peptide-binding motifs can determine resistance to viral pathogens, such as Rous sarcoma virus and Marek’s disease virus. In this report, we examine the response to a molecular defined vaccine, fp-IBD1, which consists of a fowlpox virus vector carrying the VP2 gene of infectious bursal disease virus (IBDV) fused with ?-galactosidase. We vaccinated parental lines and two backcross families with fp-IBD1, challenged with the virulent IBDV strain F52/70, and measured damage to the bursa. We found that the MHC haplotype B15 from line 15I confers no protection, whereas B2 from line 61 and B12 from line C determine protection, although another locus from line 61 was also important. Using our peptide motifs, we found that many more peptides from VP2 were predicted to bind to the dominantly expressed class I molecule BF2*1201 than BF2*1501. Moreover, most of the peptides predicted to bind BF2*1201 did in fact bind, while none bound BF2*1501. Using peptide vaccination, we identified one B12 peptide that conferred protection to challenge, as assessed by bursal damage and viremia. Thus, we show the strong genetic association of the chicken MHC to a T cell vaccine can be explained by peptide presentation by the single dominantly expressed class I molecule

The effects of classic and variant infectious bursal disease viruses on lymphocyte populations in specific-pathogen-free White Leghorn chickens

Infectious bursal disease virus (IBDV) is a pathogen that primarily infects B lymphocytes in domestic avian species. This viral infection has been associated with immunosuppression, clinical disease/mortality, and enteric malabsorption effects. The purpose of this experiment was to compare the effects of a classic (USDA-STC) and a new variant IBDV (RB-4, known to induce primarily the enteric disease) on immune cell populations in lymphoid organs. Seventeen-dayold specific-pathogen-free (SPF) White Leghorn chickens were either not infected (control) or inoculated with either USDA-STC or RB-4 IBD viral isolate. On days 3 and 5 post-inoculation (PI), lymphoid tissues were collected to prepare cell suspensions for immunofluorescent staining and cell population analysis by flow cytometry. Portions of the tissues were snap frozen for immunohistochemistry to localize various immune cells and IBD virus in the tissues. Tissue homogenates were prepared to test for IBDV by quantitative MTT assay. Both the USDA-STC and RB-4 viruses greatly altered lymphocyte populations in the spleen and bursa. At 5 d PI, bursal B cells were approximately 25% and 60% of lymphocytes in chicks infected with USDA-STC and RB-4, respectively, whereas in control birds, B cells constituted 99% of bursal lymphocytes. This reduction in the proportions of bursal B cells was associated with an infiltration of T cells. In the spleen, IBDV infection also reduced the percentage of B cells and increased the percentage of T cells. The differential effects of classic and variant IBDV infection on immune cell populations in lymphoid organs may explain the differences in clinical effects induced by these viruse

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

BACKGROUND: Eimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens. RESULTS: In this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5’Et-Actin or 5’Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens. CONCLUSIONS: E. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1756-2) contains supplementary material, which is available to authorized users

A comparative study of acute phase protein concentrations in historical and modern broiler breeding lines

Acute-phase proteins (APP) are secreted from the liver as a result of inflammation or infection and are measurable in serum and plasma. To determine whether the constitutive APP serum amyloid A (SAA), alpha-1-acid glycoprotein (AGP), ceruloplasmin (Cp), and ovotransferrin (Ovt) have changed as a result of selection for improved production and growth characteristics over the last 40 yr two historical broilers lines were compared to a modern line of the same lineage. Serum was harvested from blood samples taken from the 3 broiler lines on days 10, 17, and 20, and the APP concentrations were determined using immunoassay methods. Most of the significant changes observed were age related, with SAA and Cp having significantly lower concentrations at day 20 than days 10 and 17 in all lines. The only significant difference between lines was observed at day 20 on which both Cp (P = 0.01) and AGP (P = 0.03) were significantly higher in the modern line than the 90s line, though no significant differences were noted between the modern and 70s line. When evaluating the difference in APP concentrations between males (Cx) and females (Px) across all 3 lines, females had a higher SAA at day 17 and lower SAA at day 20, P = 0.0078 and 0.0327 respectively, and males had a significantly higher Ovt on days 17 and 20 (P = 0.0002 and P = 0.003 respectively). These results reveal that APP concentrations fluctuate over this early period of growth and that the changes in APP serum concentration appear uniform between 3 lines with very contrasting selection history, suggesting the improvements made in meat production efficiency since the 1970s have not affected the circulating concentrations of these constitutively expressed APP

A protein with simultaneous capsid scaffolding and dsRNA-binding activities enhances the birnavirus capsid mechanical stability

Viral capsids are metastable structures that perform many essential processes; they also act as robust cages during the extracellular phase. Viruses can use multifunctional proteins to optimize resources (e.g., VP3 in avian infectious bursal disease virus, IBDV). The IBDV genome is organized as ribonucleoproteins (RNP) of dsRNA with VP3, which also acts as a scaffold during capsid assembly. We characterized mechanical properties of IBDV populations with different RNP content (ranging from none to four RNP). The IBDV population with the greatest RNP number (and best fitness) showed greatest capsid rigidity. When bound to dsRNA, VP3 reinforces virus stiffness. These contacts involve interactions with capsid structural subunits that differ from the initial interactions during capsid assembly. Our results suggest that RNP dimers are the basic stabilization units of the virion, provide better understanding of multifunctional proteins, and highlight the duality of RNP as capsidstabilizing and genetic information platformsThis work was supported by grants from the Spanish Ministry of Economy and Competitivity (FIS2011-29493 to PJP, BFU2011-29038 to JLC and BFU2014-55475R to JRC) and Comunidad Autónoma de Madrid (S2013/MIT-2850 to JLC and S2013/MIT-2807 to JRC

Development of Real-Time Polymerase Chain Reaction Assays for the Detection and Differentiation of Infectious Bursal Disease Virus Subtypes

Two different real-time polymerase chain reaction (PCR) detection approaches based on SYBR Green I dye and Taqman probe based assays were developed for the detection and differentiation of infectious bursal disease virus (IBDV) subtypes. Both approaches were able to detect and differentiate IBDV subtypes based on the use of subtype-specific primers or subtype-specific probes where the primers were designed based on single nucleotide polymorphism (SNP) concept. After optimization of the primer combinations and PCR parameters, very virulent-specific primer, IF & IVIR, and classical-specific primer, IF & RCLA were used in the SYBR Green I real-time RT-PCR assay. Plasmid DNA carrying the VP4 gene of the references IBDV strains: very virulent strain UPM94/273 and classical strain D78 were established and used as positive controls in the real time RT-PCR. The developed assay had a dynamic detection limit which spans over 5 log10 concentration range for very virulent and spans over 7 log10 concentration range for classical strain, respectively. The correlation coefficient for amplification of very virulent and classical strain was R2 = 0.9918 and R2 = 0.9977, respectively. No amplification was found when the subtype-specific primers were used to amplify other avian RNA viruses. The performance of the SYBR Green I based assay was tested on various IBDV isolates including 10 previously characterized IBDV and 11 commercial vaccine strains. The very virulent-specific primer only detected and amplified the very virulent IBDV with threshold cycle (CT) ranged from 14.93 to 21.52 and melting temperature (Tm) between 85.6°C to 88.0°C. The classical-specific primer was only able to amplify the classical IBDV with CT value ranged from 11.99 to 20.89 and Tm between 85.6°C to 86.8°C. The diagnostic efficacy of the developed assay was also evaluated using bursal samples obtained from experimentally infected chickens. Bursal samples collected from D78 vaccine infected chickens at day 3 and 5 p.i were positive for IBDV with average CT of 23.05+1.31, Tm of 85.8+0.17°C and average CT 21.82+1.42, Tm of 86.0+0.28°C , respectively. Bursal samples collected at day 10 p.i from this group were also found positive for IBDV with average CT of 24.42+1.20 and Tm of 85.9+0.18°C. On the other hand, only bursal samples collected at day 3 and 5 p.i were found positive for yery virulent IBDV with average CT 19.39+0.72, Tm of 86.6+0.14°C and average CT 23.55+1.39, Tm of 86.5+0.19°C, respectively. In the case of samples from dual infection with different IBDV subtypes, viral RNA was detectable only on day 3 and 5 p.i. In general, majority of the bursal samples have higher very virulent virus with an average CT value ranged from 21.24+0.68 to 22.19+0.97 compared to vaccine virus with Ct value ranged from 23.88+0.74 to 25.36+1.19. The performance of the developed SYBR Green I based assay was analyzed with other standard diagnostic methods. In the uninfected control group, no obvious microscopic lesions were found in the bursa and the lesions score was less than 1.0. However, mild bursal lesions without signs of inflammation with lesions score less than 3.0 was detected from bursal tissue obtained from chickens inoculated with vaccine strain D78. Based on the lesion score, it was clear that bursal pathology developed rapidly, with complete loss of tissue architecture by day 3 p.i. when the chickens were infected with virulent IBDV. The correlation between ELISA antibody titers and real-time CT values were inversely related, where the lower titers of antibodies associated with higher level of viral RNA as found in chickens infected with very virulent strain UPM94/273. On the other hand, vaccine strain D78 induced higher detectable antibody titers than UPM94/273, which indirectly support less virus replication with late positive amplification in real-time RT-PCR. Thus, the level of viral RNA in bursal samples obtained from D78 infected chickens was lower than UPM94/273 infected chickens. A total of 37 bursal samples from IBD suspected field cases were collected and then tested on the developed assay. The developed SYBR Green I based PCR assay was able to detect 9 samples positive for very virulent, 4 positive for classical IBDV and 12 samples positive for both very virulent and vaccine strains of IBDV. Sequence analysis of the hypervariable region of the VP2 gene of the IBDV samples revealed that the residues involved in determining the virulence of VV IBDV and CL IBDV were highly conserved. For the Taqman based duplex real-time PCR assay development, a new set of primers FWDC and RVSC were designed from the conserved region of VP4 of both very virulent and classical strains. A dual-labeled fluorescent probe each specific for very virulent IBDV (ProVV) and vaccine IBDV (ProCL) were designed The performance of the developed Taqman assay was compared with other PCR methods namely conventional RT-PCR and previously developed SYBR Green I assay. The Taqman assay was found far more superior in terms of turn around time and sensitivity. With the aid of β-actin gene, the Taqman assay was also used to determine the viral load fold changes in bursal samples that were positive for both vaccine and very virulent IBDV. Majority of these samples have higher viral load fold change in very virulent than the classical strain except for three samples MB078/04, MB001/05 and MB033/05 which showed higher fold change in classical strain than very virulent strain. In conclusion, this study has successfully developed SYBR Green I based and Taqman based one-step real-time PCR assays for rapid detection and differentiation of IBDV subtypes in particular very virulent and classical IBDV strains

Molecular and Biological Characterization of Two Very Virulent Infectious Bursal Disease Virus Isolates, Upm94/273 and Upm97/61

An atypical very virulent (w) strain (UPM94/273) and typical vv strain (UPM97/61) of infectious bursal disease virus (IBOV) isolated in Malaysia, were characterized both in vivo and at the molecular level. Comparison of the deduced amino acid sequences with other serotype 1 and 2 sequences revealed 16 amino acid residues, which were conserved only in the vvIBDV. Among the 16 unique amino acid differences, 8 were in VP1 (146 Asp, 147 Asn, 242 Glu, 390 Met, 393 Asp, 562 Pro, 687 Pro and 695 Arg), 3 were in VP2 (222 Ala, 256 lie and 294 lie), 2 were in VP3 (990 Val and 1005 Ala) and 3 were in VP4 (685 Asn, 715 Ser and 751 Asp). The importance of these unique amino acid residues is not known but they could affect the virulence of vvIBOV. The UPM94/273 also demonstrated 6 unique amino acid residues at segment A at positions Ser254, Glu270, Lys588, Ser745, Phe838 and Lys863 and 8 unique amino acid residues at segment B at positions Ala92, Ser100, Va1208, Asp253, Asp560, Asn565, Gly750 and Gly876. In addition, these amino acid substitutions have not been reported before in vvlBDV and were found only on variant, classical and/or serotype 2 strains. However, the VP5 region of both vvlBDV strains was conserved. The UPM97/6 1 demonstrated 7 unique amino acid substitutions at segment A and 4 unique amino acid substitutions at segment B. However, none of the amino acids changes have been reported elsewhere in other IBDV strains. Although the actual functions of the amino acid substitutions are not know, the unusual amino acid substitutions at segment A and/or B of both isolates may be important in virus virulence. Alignments of the nucleic acid and amino acid sequences of segment A and B followed by distance analysis allowed the generation of phylogenetic trees. Phylogenetic analysis based on segment A and B revealed that all the vvlBDV strains including U PM94/273 isolate can be clustered in a group that is distinct from classical, variant, attenuated and serotype 2 strains. However, the tree branching patterns were quite different between segment A and segment B. In addition, the vvlBDV strains showed several conserved amino acid substitutions at segment B as found in the Australian 002-73 and serotype 2 strains. These findings indicate that probably a genetic reassortment may have play an important role in the emergence of vvIBDV. Flow cytometry and real time peR assays, indicated that chickens infected with UPM97/61 induced higher percentages of apoptotic cells but lower level of viral load whereas UPM94/273 induced lower percentages of apoptotic cells but higher level of viral load, suggesting a negative correlation between viral load and apoptosis. These results indicated that U PM97/61 was more virulent than UPM94/273

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast.

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world?s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development

Analysis of the early immune response to infection by infectious bursal disease virus in chickens differing in their resistance to the disease

Chicken whole-genome gene expression arrays were used to analyze the host response to infection by infectious bursal disease virus (IBDV). Spleen and bursal tissue were examined from control and infected birds at 2, 3, and 4 days postinfection from two lines that differ in their resistance to IBDV infection. The host response was evaluated over this period, and differences between susceptible and resistant chicken lines were examined. Antiviral genes, including IFNA, IFNG, MX1, IFITM1, IFITM3, and IFITM5, were upregulated in response to infection. Evaluation of this gene expression data allowed us to predict several genes as candidates for involvement in resistance to IBDV. © 2015, American Society for Microbiology

Genome sequence analysis of a distinctive Italian infectious bursal disease virus.

ABSTRACT In a recent study, an emerging infectious bursal disease virus (IBDV) genotype (ITA) was detected in IBDV-live vaccinated broilers without clinical signs of infectious bursal disease (IBD). VP2 sequence analysis showed that strains of the ITA genotype clustered separately from vaccine strains and from other IBDV reference strains, either classic or very virulent. In order to obtain a more exhaustive molecular characterization of the IBDV ITA genotype and speculate on its origin, genome sequencing of the field isolate IBDV/Italy/1829/2011, previously assigned to the ITA genotype, was performed, and the sequences obtained were compared to the currently available corresponding sequences. In addition, phylogenetic and recombination analyses were performed. Interestingly, multiple amino acid (AA) sequence alignments revealed that the IBDV/Italy/1829/2011 strain shared several AA residues with very virulent IBDV strains as well as some virulence markers, especially in the VP1 protein. Nevertheless, sequence analysis demonstrated the presence of several residues typical of IBDV strains at a low degree of virulence in the IBDV/Italy/1829/2011 strain. Although homologous recombination and reassortant phenomena may occur naturally among different IBDV strains, no evidence of those events was found in the genome of the IBDV/Italy/1829/2011 strain, which was confirmed to be a genetically distinctive IBDV genotype