IBDV Strains Research Articles

English

In recent years very virulent (VV) IBDV strains and classical (CV) IBDV strains re-emerged and caused devastating outbreaks in different parts of the world. In this study, genetic evolution of fifteen IBDVs collected in Senegal in 1979, 1999, 2007, 2012, 2013 and 2014 was characterized to gain information for a better control of IBD. Following RT-PCR, nucleotide sequence of the VP2 hypervariable region was determined and compared with sequences available in GenBank. Phylogenetic analysis showed that the viruses diverged into two genotypes: Very virulent (VV) IBDV and classical virulent (CV) IBDV. The Senegalese field strains of the first genotype (VV) IBDV had 98.9 to 100% identity among themselves, whereas their identity with reported Nigerian (VV) IBDVs ranged between 96.7 and 99%. The close phylogenetic relationship of the Senegalese and Nigerian strains suggests that they likely derived from a common ancestor. In the phylogenetic tree, all the Senegalese (VV) IBDV strains belonged to the African very virulent types (VV2). The genotyping of senegalese field IBDV strains indicated that the majority of viruses circulating in Senegal are (VV) IBDVs and highlights a genetic stability. Key words: Infectious bursal disease, phylogenetic analysis, Senegal.

Pathogenicity, tissue distribution, shedding and environmental detection of two strains of IBDV following infection of chickens at 0 and 14 days of age

ABSTRACTInfectious bursal disease virus (IBDV) is endemic to most poultry-producing countries worldwide. Immunosuppressive classical and variant IBDV strains endemic to Australia are genetically distinct from other international strains. We report the results of infection experiments with Australian classical strain 06/95 and variant strain 02/95 in SPF chickens. We tested the effects of strain and age of infection on bursal atrophy, viral RNA (vRNA) load in bursa of Fabricius (bursa), spleen, thymus, caecal tonsils, faeces, litter and exhaust dust as determined by real-time reverse transcriptase polymerase chain reaction. The two IBDV strains did not differ in the degree of bursal atrophy induced, lymphoid organ distribution and faecal shedding but variant strain 02/95 induced a greater antibody response to the infection than classical strain 06/95 which was associated with a more rapid decline in IBDV vRNA genome copy number (VCN) in lymphoid organs and faeces. Infection at 14 days of age induced greater bursal atrophy and higher vRNA copy number in lymphoid tissues than infection on the day of hatching, indicating true age susceptibility independent of maternal antibody (Mab) status. The direction of the association between rankings for IBDV vRNA load in bursa and relative bursal weight changed from positive at 3 and 6 days post-infection to negative at 28 days post-infection. Intra-tracheal administration of dust collected from chickens infected with IBDV resulted in successful transmission of IBDV. IBDV vRNA was detected successfully at high levels in the environmental litter and dust samples.

Identification of a European interserotypic reassortant strain of infectious bursal disease virus

ABSTRACTInfectious bursal disease virus (IBDV, family Birnaviridae) is a bi-segmented double-stranded RNA virus for which two serotypes are described. Serotype 1 replicates in the bursa of Fabricius and causes an immunosuppressive and potentially fatal disease in young chickens. Serotype 2 is apathogenic in poultry species. Up to now, only one natural event of interserotypic reassortment has been described after the introduction of very virulent IBDV (vvIBDV) in the USA in 2009, resulting in an IBDV strain with its segment A related to vvIBDV and its segment B related to US serotype 2 strain OH. Here, we present the first European isolate illustrative of interserotypic reassortment. The reassorting isolate, named 100056, exhibits a genomic segment A typical of current European vvIBDV but a segment B close to European serotype 2 viruses, supporting an origin distinct from US strains. When inoculated into SPF chickens, isolate 100056 induced mild clinical signs in the absence of mortality but caused a severe bursal atrophy, which strongly suggests an immunosuppressive potential. These results illustrate that interserotypic reassortment is another mechanism that can create IBDV strains with a modified acute pathogenicity. As a consequence, and for a more precise inference of the possible phenotype, care should be taken that the molecular identification of IBDV strains is targeted to both genome segments.

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy

ABSTRACTA distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.

Relationship between Virus Replication and Apoptosis Events in IgM + Cells from Chicken Spleen and Bursa of Fabricius Infected with Malaysia Strain of Very Virulent Infectious Bursal Disease Virus

Background: Infection of IBDV was reported to be endemic in worldwide including Malaysia and can be spread orally thru polluted fodder and water source, thus causing economic losses especially in commercial poultry industry. The infection resulted in depletion of B lymphocytes and subsequently destruction of the bursa which leaded to immunosuppression of the bird and it was postulated that the depletion of cells in the bursa was due to induction of apoptosis. In the current study, the infection of Malaysia isolated very virulent IBDV UPM0081 on IgM bearing B lymphocytes (IgM+ cells) from chicken spleen and bursa was compared.Materials, Methods & Results: A total of sixty eggs were obtained and raised until the age of 3 weeks old. The birds were divided into two groups (n = 30), which one of them served as control while IBDV strain UPM0081 was used to infect another group of birds at the concentration of 103 ELD50. The birds were observed and sacrificed at day 2, 4 and 5 post infections. Spleen and bursa of Fabricius were harvested and subjected to IgM+ cell enrichment using microbeads. The cell viability of enriched cells was assayed using MTT and cell cycle was analyzed using propidium iodide. Annexin V FITC and acridine orange/propidium iodide double stain assays were used to determine the event of apoptosis in the enriched IgM+ cells. Also, the IBDV viral load was also quantified by using real time PCR to evaluate the relationship between virus replication and apoptosis events in the infected chickens. Current results showed that the apoptotic events were observed to be significantly higher in IgM+ cells isolated from chicken bursa as compared to the cells isolated from spleen. The bursal B lymphocytes cell viability was observed to be decreasing following the infection of very virulent IBDV. The cells were then investigated of their apoptotic rate and data showed that increasing apoptotic cells (early and late apoptosis) were observed in AO/PI double stain as well as increment of SubG0/G1 population in the cell cycle analysis and also increment of Annexin V FITC bound cells in the apoptosis study. As for B lymphocytes from chicken spleen, the magnitude of damage caused by very virulent IBDV was not as severe as what being observed in the chicken bursa, with the cell viability drastically decreased on day 4 following IBDV infection.Discussion: IBDV caused severe destruction in bursa of Fabricius compared to spleen, in which cell death events in the former was reported to be directly caused by the virus. Apoptotic event in chicken spleen following IBDV infection was observed to be caused by oxidative stress. Thus, viral replication played a role in inducing bursal IgM+ cells death while such phenomenon was not observed in spleen isolated IgM+ cells. In summary, the cell death events of IgM+ cells in chicken spleen and bursa of Fabricius may be accounted by different factors upon infection with Malaysia strain of IBDV UPM0081. It is obvious that IgM+ cells from chicken bursa suffered from apoptotic cell death in an increasing manner considerably with time of infection and RNA load detected in the cells, which supported by previous literature that IBDV induces host cells apoptosis, with both VP2 and VP5 playing a role in binding and apoptosis. Meanwhile, the cell death events of B lymphocytes in chicken spleen was observed to be more relevant to other factors such as the oxidative stress or proinflammatory cytokines that caused by the virus infection rather than the viral RNA load.

Further evidence for very virulent infectious bursal disease virus in vaccinated chickens in Nigeria.

Reverse transcription polymerase chain reaction (RT-PCR) and partial sequencing of the VP2 hypervariable region was performed on clinical samples from two infectious bursal disease (IBD) outbreaks in Plateau state, Nigeria. IBD virus RNA was detected in all four bursa of Fabricius samples. Nucleotide sequencing and analysis of the four samples revealed high similarity to previous IBDV sequences from northern and southern Nigeria. The deduced amino acid sequences were compared to reference IBDV strains retrieved from the GenBank; virulence markers A222, I256, and I294 were conserved in both outbreak and reference sequences. Amino acid residue S254 was conserved in the outbreak viruses and previous viruses from northern Nigeria. Phylogenetic analysis revealed that all four viruses were very virulent IBDVs. These viruses clustered with vv2-1 variant viruses from Oyo and Ogun states and less closely with vv2-2 isolates from Tanzania. The nucleotide identity of the sequences in this study ranged from 99.6 to 100 % with each other. These findings are further evidence of IBD outbreaks in vaccinated chicken flocks in Nigeria.

Evaluation of a Phylogenetic Marker Based on Genomic Segment B of Infectious Bursal Disease Virus: Facilitating a Feasible Incorporation of this Segment to the Molecular Epidemiology Studies for this Viral Agent.

BackgroundInfectious bursal disease (IBD) is a highly contagious and acute viral disease, which has caused high mortality rates in birds and considerable economic losses in different parts of the world for more than two decades and it still represents a considerable threat to poultry. The current study was designed to rigorously measure the reliability of a phylogenetic marker included into segment B. This marker can facilitate molecular epidemiology studies, incorporating this segment of the viral genome, to better explain the links between emergence, spreading and maintenance of the very virulent IBD virus (vvIBDV) strains worldwide.Methodology/Principal FindingsSequences of the segment B gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank Database; Cuban sequences were obtained in the current work. A phylogenetic marker named B-marker was assessed by different phylogenetic principles such as saturation of substitution, phylogenetic noise and high consistency. This last parameter is based on the ability of B-marker to reconstruct the same topology as the complete segment B of the viral genome. From the results obtained from B-marker, demographic history for both main lineages of IBDV regarding segment B was performed by Bayesian skyline plot analysis. Phylogenetic analysis for both segments of IBDV genome was also performed, revealing the presence of a natural reassortant strain with segment A from vvIBDV strains and segment B from non-vvIBDV strains within Cuban IBDV population.Conclusions/SignificanceThis study contributes to a better understanding of the emergence of vvIBDV strains, describing molecular epidemiology of IBDV using the state-of-the-art methodology concerning phylogenetic reconstruction. This study also revealed the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvIBDV strains. Therefore, it highlights the need to obtain information about both genome segments of IBDV for molecular epidemiology studies.

Isolation and Molecular Characterization of Vp2 Gene of Infectious Bursal Disease: Demonstrated No Predominant New Strains Circulating in Bangladesh

The present study was conducted for molecular characterization of Bangladeshi isolates of Infectious Bursal Disease virus to establish genetic diversity with other European and Asian strains. A total of 129 bursa of Fabricius were collected after post mortem from IBD infected chicken. Only 30 samples were confirmed for IBDV by amplification of a 729-bp fragment of the VP2 gene hyper variable region using RT-PCR. The hyper variable region (aa 184-426) of amplified VP2 fragments of the 30 IBDV isolates were sequenced and compared with 10 strains published in Gene Bank and their phylogenic relationship were analyzed. Amino acid sequences analysis of the hyper variable region of the VP2 gene revealed that 27 isolates were 99% identical with European and Asian vvIBDV strains and 3 isolates showed close relationship with classical strains of IBDV. Twenty seven Bangladeshi vvIBDV isolates encode amino acids 222A, 2421, 2561, 294I, and 299S, which are conserved among other vvIBDV strains. The 3 Classical isolates showed amino acid substitutions at 222A→P, 256I→V, and 299 S→N positions. All isolates had the same heptapeptide 279D, S-W-S-A-S-G-S (326→332), and 284A. Phylogenetic analysis based on the nucleotide sequence revealed that the Bangladeshi isolates were closely related to European and Asian vvIBDV. Taken together, there was no predominant new strain circulating in Bangladesh during June 2009-May 2011. Moreover, in order to protect birds from the sudden emergence of new predominant strains, continuing surveillance of IBDV strains circulating in the field is of utmost importance.

Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens.

The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected.

Simultaneous detection of vaccinal and field infectious bursal disease viruses in layer chickens challenged with a very virulent strain after vaccination

Infectious bursal disease virus is an important poultry pathogen. It is distributed worldwide and causes significant economic losses. In this study, a system was adopted for the simultaneous monitoring of vaccine and virulent strains using reverse transcription polymerase chain reaction (RT-PCR). After the decay of maternal antibodies, chickens were vaccinated at the age of 37 days with a virus of intermediate virulence and challenged at 5, 10 and 14 days post vaccination (dpv). The challenge was done with IBDV strain CH/99. Sequencing of the hypervariable region of VP2 has shown that CH/99 belongs to the very virulent group of viruses. The vaccine virus could be found in the bursa of Fabricius, spleen, thymus and bone marrow until 24 dpv. The CH/99 challenge virus was found in the bursa and lymphoid organs when chickens were challenged at 5 and 10 dpv. When challenge was performed at 14 dpv, the pathogenic virus could not be found in the bursa and other lymphoid organs.

Pathogenicity and immunosuppresive properties of GM-97 strain of infectious bursal disease virus in commercial broiler chicken

The current study was conducted to evaluate the pathogenicity and immunosuppressive effects of GM-97 strain of infectious bursal disease virus in commercial broiler chickens. A total of 500 broiler chickens were vaccinated with the virus through oral route at 10 and 17 days of age (10 2 -10 3 EID50/dose). Chickens were also vaccinated with Newcastle disease virus (Hitchner B1) orally at 14 and 21 days old. Chickens were euthanized (at 12, 14, 16, 20, 23, 26 days of age) after measuring body weight. Bursa of Fabricius was examined for any gross lesion, weighed and processed for histological investigations. Bursa to body weight ratio and bursal lesion scoring were made to evaluate pathogenicity of the virus. Blood samples were analyzed for antibody response to ND vaccine virus using HI test. Results showed that the GM-97 strain of IBDV induced mild to moderate depletion of lymphoid cells in the center of bursal follicles and non-significant difference in bursa to body weight ratio amongst vaccinated and unvaccinated chickens. Chickens responded well to ND vaccine by mounting high level of serum NDV specific HI antibody titers. It can be concluded from the present study that GM-97 strain of IBDV has mild pathogenicity but is not immunosuppressive.

Both Genome Segments Contribute to the Pathogenicity of Very Virulent Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.

Infectious bursal disease virus rescued efficiently with 3′ authentic RNA sequence induces humoral immunity without bursal atrophy

A reverse genetics infectious bursal disease virus (RG-IBDV) that contained authentic 3′ RNA sequence was characterized both in vitro and in vivo. LP1-IBDV, a cell line-adapted IBDV strain variant E (VE) was used as the parent virus for constructing viral cDNA clones. Authentic 3′ RNA sequence was generated by using cis-acting hepatitis delta virus ribozyme (HDR). The absence of HDR in the clones did not affect viral protein expression, but the obtained viral titers were reduced by 200-folds when compared to the clones with HDR sequence. RG-IBDV generated from clones with HDR sequence was similar to LP1-IBDV by plaque size, growth kinetics and electron microscopic morphology. RG-IBDV did not cause bursal atrophy in 3-week-old chickens at 3, 7 and 17 days post infection (DPI) and induced high ELISA and neutralizing antibody titers to IBDV at 7 and 17 DPI. The results indicated that RG-IBDV can be generated efficiently with an authentic 3′ RNA terminus and the obtained RG-IBDV is non-pathogenic and immunogenic with the potential as a vaccine strain that can be further genetically modified to broaden its application.

Pathological Study of SPF Chickens Experimentally Infected with a Chinese IBDV Strain BC6/85

Pathological Study of SPF Chickens Experimentally Infected with a Chinese IBDV Strain BC6/85

Sequence variability and evolution of the terminal overlapping VP5 gene of the infectious bursal disease virus

The infectious bursal disease virus (IBDV; Birnaviridae family) constitutes one of the main threats to the poultry industry worldwide. Most of the progress in the molecular epidemiology of this virus has been achieved through the study of the coding region of the capsid protein VP2. Little research has been done regarding the molecular evolution and the epidemiological implications of genetic variability of other IBDV genome regions. In this article, the gene that codes the non-structural protein VP5 was analyzed. Although this protein is not essential for the virus replication, recent evidence indicates that it could be related to the virulent phenotype and the adaptive capacity of the virus. The VP5 gene is also of evolutionary interest because it has an open reading frame that terminally overlaps with the pVP2-VP4-VP3 polyprotein coding region. In the first part of this study, the full VP5 gene of a South American strain was characterized. The results revealed that the VP5 gene of Uruguayan hypervirulent IBDV strains (vvIBDV) lacks the alternative AUG start codon characteristic of the vvIBDV strains that have been described to date. Instead, as occurs in classic and variant strains, this VP5 gene has an AUG start site located four codons downstream and, consequently, it codes for a 145 amino acid long protein rather than the putative 149 amino acid long protein of other vvIBDV. In spite of this, these viruses conserved the VP5 and VP2 amino acid signature of the hypervirulent strains and clustered with reference vvIBDV sequences. This finding may represent evidence that the VP5 gene could be evolving by changing the translation initiation site. In the second part of this study, an evolutionary analysis including the sequences reported in this study together with most of VP5 sequences available in the GenBank, showed the existence of a complex system of selective pressures controlling the evolution of the VP5 gene. Using the dN/dS index, we found a strong purifying selection exerted on the 5' terminal overlapping region of VP2 that would be constraining the evolution of VP5. These results reinforce the hypothesis that the VP5 gene was originated late in the IBDV evolution by a mechanism of genetic overprinting. The results described in this study provided new information about the dynamics of the IBDV genome and revealed some of the mechanisms at play in the evolution of this virus. Since VP5 seems to be related to viral pathogenicity, this evolutionary information might be useful to highlight the impact of the genetic variation of this protein on the epidemiology of IBDV.

Immunostimulatory Effects of Arginine in Broiler Chickens Challenged with Vaccine Strain of Infectious Bursal Disease Virus

Infectious Bursal Disease (IBD) continues to pose potential threat to poultry industry all over the world. The disease can spell disaster not only through its infection but also by break of immunity in chickens vaccinated for other diseases. On the other hand, arginine (Arg), a ubiquitous, semi-essential amino acid has emerged as an imunostimulant from variety of human and animal studies. In the present study, we demonstrate the stimulatory effects of Arg on systemic immune response in chickens challenged by orally administration of intermediate plus strain of IBD virus at 28 days of age. A corn-soybean meal based diet containing different levels of Arg (0, 0.67, 1.37, 2.07 and 2.77) for the starter, (0, 0.53, 1.1, 1.68 and 2.25) for the grower and (0, 0.52, 1.04, 1.56 and 2.08) for the finisher was used. In a completely randomized design with five treatments of five replicates each and 10 chickens per replicate, 250 Cobb500 male broiler chickens from 0-49 days of age were used. To measure the innate, cellular and humoral immunity indicators (interferon-α, interferon-γ, immunoglobulin G) at 27, 35, 42 and 49 days of age, serum samples from each replicate of treatments were collected and subjected to ELISA. The result showed that Arg supplementation in the chickens basal diets significantly increased the serum levels of interferon-α, interferon-γ, immunoglobulin G at 35, 42 and 49 days of age (p<0.05). The different levels of Arg at 27 days of age did not significantly affect interferon-α, interferon-γ, whereas Arg at 27 days of age significantly increased immunoglobulin G (p<0.05). These results revealed that arginine stimulates systematic immune response against intermediate plus strain of IBDV.

Characteristics of a Very Virulent Infectious Bursal Disease Virus from California

An outbreak of infectious bursal disease (IBD) in two California layer flocks resulted in the isolation of two infectious bursal disease viruses designated rA and rB. Increased mortality plus gross and histopathology in the layer flocks suggested rA and rB could be very virulent infectious bursal disease virus (vvIBDV). Preliminary studies indicated that high mortality resulted when bursa homogenates from the layer farms were used to inoculate specific-pathogen-free (SPF) chicks. In addition, rA and rB contained VP2 amino acid sequences typically seen in vvIBDV. Molecular and in vivo studies were conducted to more thoroughly identify and characterize the rA and rB viruses. Nucleotide sequence analysis demonstrated that rA and rB had identical sequences across the hypervariable VP2 (hvVP2) and segment B regions examined, and their amino acid sequences in the hvVP2 region were identical to the vvwIBDV type strains UK 661, OKYM, and Harbin. Furthermore, the genome segment B nucleotide sequences examined for rA and rB were a 98.1% match with vvIBDV and only an 88.0% match with classic IBDV strains. Phylogenetic analysis placed the rA and rB viruses with other vvIBDV and confirmed these viruses were close genetic descendants of vvIBDV seen around the world. Pathogenicity studies in 4-wk-old SPF chicks demonstrated that at a high dose (105.5 50% egg infective dose [EID50]) and a low dose (102.0 EID50) of rA and rB, mortality ranged from 91% to 100%. A pathogenic classic virus, standard challenge (STC), at similar doses did not cause mortality in the SPF chicks. In addition, mortality occurred in three out of four SPF birds exposed by direct contact to rA and rB inoculated chicks. Serum from convalescent birds inoculated with rA had high titers to IBDV but were negative for antibodies to infectious bronchitis virus, avian influenza virus, chicken anemia virus, Newcastle disease virus, Mycoplasma gallisepticum, and Mycoplasma synoviae. Virus isolation attempts on the rA and rB bursa homogenate inocula also indicated that no contaminating microorganisms contributed to the high mortality and pathology observed in the SPF chicks. In one experiment, broilers with maternal immunity to IBDV were protected from infection and disease when they were challenged with 10(2) EID50 and 10(5) EID50 of the STC virus. When challenged with 10(2) EID50 of the rA virus, the maternally immune broilers were protected from disease but not infection as evidenced by a positive reverse transcription-polymerase chain reaction (RT-PCR) assay for the virus. When the broilers were challenged with 10(5) EID50 of the rA virus, they had typical gross and histopathologic signs of IBD but no mortality by 7 days postinoculation. It was concluded that the rA and rB viruses meet the genotypic and phenotypic characteristics of a vvIBDV.

Response of embryonic chicken lymphoid cells to infectious bursal disease virus

We exposed chicken embryos at embryonation day 18 (ED18) to a classical virulent infectious bursal disease virus (IBDV; cIBDV) and an attenuated strain of IBDV (aIBDV) and examined the response of embryonic lymphoid cells to these viruses. Embryos responded much more vigorously to cIBDV than to aIBDV. Following cIBDV exposure, embryonic thymus and bursa showed cellular destruction, enhanced rate of apoptosis and presence of viral proteins detectable by immunohistochemistry. At ED21, thymocytes from cIBDV-exposed embryos were severely deficient ( P < 0.05) in responding to stimulation in vitro with mitogens containing mouse anti-chicken CD28 mAb, PMA and ionomycin. Because purified CD3 + T cells were also refractory to the mitogens, the mitogenic inhibition of embryonic thymocytes was not attributed to the presence of non-T cell suppressors. Cell suspensions prepared from embryonic thymus and spleen had upregulated gene expression of IFN-γ and IL-6 cytokines and of chemokine IL-8. In sharp contrast to cIBDV, embryos exposed to aIBDV had minimal detectable changes in the thymus and bursa, although the rate of apoptosis was enhanced in the thymus. Viral antigen was not detectable in the bursa until after hatch. Thymocytes from these embryos responded vigorously to the mitogens, similar to the response of thymocytes from unexposed control embryos. In addition, aIBDV induced a modest gene upregulation of IFN-γ, IL-6 and IL-8 in thymus and spleen. Relatively modest response of the embryo to aIBDV is significant because in ovo vaccination with aIBDV-type viruses and several other non-pathogenic viruses result in protective immunity that is well pronounced at hatch.

Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity

Three classic IBDV strains were previously isolated from commercial layer chicken flocks and shown to be phylogenetically related to vaccine strains but pathogenic in susceptible chickens. In this study, their viral genomes were sequenced and compared to sequences of vaccines being used in those flocks. The vaccine strains examined were sequenced directly from the manufacturer and had identical genome segment B sequences. Compared to these vaccines, the GA-1, H-30 and CS-2-35 isolates each had one silent mutation in the gene that encodes VP1. Compared to the two vaccines used at the time CS-2-35 was isolated, the segment A sequence of CS-2-35 contained numerous nucleotide and amino acid mutations suggesting the CS-2-35 virus was not closely related to these vaccines. This virus however did have amino acid mutations in VP2 that are reported to be necessary for replication in cell culture and lacked two of the three amino acid mutations previously shown to be necessary for virulence. These data suggest that CS-2-35 was a descendant from an attenuated strain of IBDV. When the segment A genomic sequences of the GA-1 and H-30 viruses were compared to the vaccines being used in those flocks they were most closely related to the attenuated D78 vaccine strain. In genome segment A, three nucleotide mutations in GA-1 and four in H-30 were observed compared to the D78 classic vaccine. These nucleotide mutations caused one amino acid (H253N) change in the GA-1 virus and two amino acids (H253Q and G259D) were different in the H-30 virus. In addition, both the GA-1 and H-30 viruses had the amino acid G76 in VP2 that appears to be unique to the vaccine D78. The data suggest that GA-1 and H-30 are genetically related and have a common ancestor even though they were isolated from geographically distant flocks. The evidence also suggests that GA-1, H-30 and CS-2-35 could be reversions from attenuated vaccine viruses or by coincidence genetically resemble classic IBDV vaccines. It should be noted that some of the classic virus vaccines were not being used according to the manufacturer's recommendations at the time the GA-1, H-30 and CS-2-35 strains were isolated. Together, the molecular and pathogenicity data indicate that a single amino acid mutation from Histidine (H) to Glutamine (Q) or Asparagine (N) at position 253 in VP2 will markedly increase the virulence of an attenuated IBDV.

Molecular and Structural Bases for the Antigenicity of VP2 of Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is responsible for a highly contagious and economically important disease causing immunosuppression in chickens. IBDV variants isolated in the United States exhibit antigenic drift affecting neutralizing epitopes in the capsid protein VP2. To understand antigenic determinants of the virus, we have used a reverse-genetics approach to introduce selected amino acid changes-individually or in combination-into the VP2 gene of the classical IBDV strain D78. We thus generated a total of 42 mutants with changes in 8 amino acids selected by sequence comparison and their locations on loops P(BC) and P(HI) at the tip of the VP2 spikes, as shown by the crystal structure of the virion. The antibody reactivities of the mutants generated were assessed using a panel of five monoclonal antibodies (MAbs). Our results show that a few amino acids of the projecting domain of VP2 control the reactivity pattern. Indeed, the binding of four out of the five MAbs analyzed here is affected by mutations in these loops. Furthermore, their importance is highlighted by the fact that some of the engineered mutants display identical reactivity patterns but have different growth phenotypes. Finally, this analysis shows that a new field strain isolated from a chicken flock in Belgium (Bel-IBDV) represents an IBDV variant with a hitherto unobserved antigenic profile, involving one change (P222S) in the P(BC) loop. Overall, our data provide important new insights for devising efficient vaccines that protect against circulating IBDV strains.