Characterization of infectious bursal disease viruses isolated in 2007 from Delmarva commercial broiler chickens.

A study was performed in 2007 to isolate and characterize infectious bursal disease viruses (IBDVs) in commercial broilers grown in the Delmarva (DMV) Peninsula region of the United States. Bursae of Fabricius were collected weekly from 1 to 4 wk of age from broilers on 10 farms with a history of poor performance. Microscopic pathology was used to determine the infectious bursal disease (IBD) status of the broilers. Bursae from 1- and 2-wk-old broilers did not show IBD microscopic lesions. Moreover, broilers on 1 of the 10 farms were IBD lesion free at 3 and 4 wk of age. However, 3 of 9 and 9 of 9 farms yielded broilers with IBD-affected bursae from 3- and 4-wk-old commercial broilers, respectively. Ten IBDV isolates were recovered from 3 of 3 lesion-positive bursal pools at 3 wk of age and 7 of 9 lesion-positive bursal pools at 4 wk of age. Analysis of the viral protein (VP) 2 genes identified all isolates as serotype 1 Delaware (Del) variant viruses. Five field isolates, each representing different molecular clades of the Delaware variant viruses, were selected for further study. Experimental infection of specific-pathogen-free white leghorn chickens with isolates DMV/4813/07, DMV/4947/07, DMV/4955/07, DMV/5038/07, and DMV/5041/07 produced gross and microscopic pathology of the bursa consistent with Delaware variant infection. Monoclonal antibody testing showed DMV/4813/07, DMV/4947/07, DMV/ 4955/07, and DMV/5041/07 to be similar to previous recognized variant viruses. However, DMV/5038/07 was found to be unreactive with the monoclonal antibodies that typically recognize reference strains STC, Del E, GLS, RS593, and AL2. In a challenge of immunity study, 10-day-old progeny from breeders immunized with a commercially available inactivated IBDV vaccine containing the Del E and classic strains were protected to a lesser degree against isolate DMV/5038/07 compared to Del E challenge based on microscopic lesion scores (P < 0.01) of the bursa. This result suggests the virus is antigenically different from the Del E strain contained in the vaccine. Collectively, the monoclonal antibody and progeny challenge of immunity findings suggest DMV/5038/07 is antigenically different from the Del E strain contained in the vaccine.

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.

Real-time reverse transcriptase-polymerase chain reaction detection and sequence analysis of the VP2 hypervariable region of Indian very virulent infectious bursal disease isolates.

Bursal samples collected from different field outbreaks in commercially reared chicken flocks from India that were suspected of very virulent (vv) infectious bursal disease (vvIBD) were tested. Two vaccine strains that are commonly being used in India also were included to ascertain their relatedness with the field isolates. When tested with real-time reverse transcriptase-polymerase chain reaction (RT-PCR), 14 of the 15 samples were found to be positive for vvIBD virus (vvIBDV) genetic sequences as determined by the vv232 and vv256 vvIBDV-specific probes. A melting temperature of 50 C and above was characteristic of vvIBDV strains. The vaccine strain infectious bursal disease intermediate (IBDI)-plus (IBDI+) had a higher melting temperature compared with IBDI, suggesting more relatedness to the vvIBDV strains. The real-time RT-PCR technique can be a useful tool in differentiating classic and vvIBDV strains and thereby assist in adopting more effective control strategies. Sequencing of the VP2 hypervariable region of these isolates further confirmed the results of real-time RT-PCR. All the suspected vvIBDV samples were found to share unique amino acid substitutions at positions 222 A, 256 I, 294 I, and 299 S characteristic of the very virulent strains. More sequence differences occurred at the nucleotide level among the vvIBDVs. They shared exactly the same amino acid sequence among themselves and also with the Bangladesh isolate BD-3-99 and some of the Nigerian isolates. They differed by one amino acid from earlier published Indian, Asian, and European vvIBDV VP2 sequences. The nucleotide sequence of IBDI+ vaccine showed more similarities with vvIBDV sequences; hence, it may be of more value in the control of these very virulent strains.

Molecular and phenotypic characterization of infectious bursal disease virus isolates.

Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens.

The use of gamma irradiation for the inactivation of infectious bursal disease viruses.

The maximum dosage of gamma irradiation approved by the U.S. Food and Drug Administration (FDA) for poultry is 3.0 kiloGrays (kGy). This treatment is designed to reduce bacterial contamination on uncooked poultry carcasses and meat products. The possible presence of infectious bursal disease virus (IBDV) on poultry postharvest has prompted some countries to study the risk associated with introducing nonnative strains of the virus from imported commodities. The goal of this study was to determine if this risk could be reduced using gamma irradiation to inactivate IBDV. At the dosage approved by the FDA, the titers of IBDV vaccine strains were reduced between 0 and 1 log10. Titers of the pathogenic IBDV strains tested were not reduced after the 3.0 kGy exposure. Furthermore, titers of pathogenic viral strains were not reduced following exposure up to 5.0 kGy. As the exposure to gamma irradiation increased, the titers of the vaccine strains decreased. At the maximum dosage tested (10 kGy), the 89/03 variant virus vaccine was completely inactivated. Titers of the three classic IBDV vaccine strains were reduced between 1.6-2.0 logs after the 10 kGy exposure; however, these viruses remained viable after this treatment. Gamma irradiation is not an effective intervention to reduce the risk of IBDV introduction via processed poultry.

Characterization of infectious bursal disease viruses from four layer flocks in the United States.

Twenty bursal samples were obtained from four infectious bursal disease virus (IBDV)-vaccinated layer flocks experiencing problems with immune suppression that was thought to be infectious bursal disease. All the samples were found to be positive for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Restriction fragment length polymorphism analysis of the samples identified them as classic molecular group 3 and group 4 viruses. Two samples from each of the four flocks were sequenced, and within a flock, these sequences were identical; however, between flocks, some differences were observed. One virus from each of the four flocks was selected for further analysis. The VP2 hypervariable sequence region of samples GA-1, H-30, and CS-2-35 had nucleotide and amino acid similarities with the D78 and Vi Bursa G classic vaccines that were used in those flocks. The sequence of HPR-2 was similar to the Bursa Vac 4 vaccine used in that flock and the STC virulent classic IBDV strain. The deduced amino acid sequence of these isolates revealed that all the isolates had proline at position 222, which is characteristic of U.S. classic viruses. The phylogenetic analysis of these isolates on the basis of the VP2 hypervariable amino acid sequence clustered GA-1, H-30, and CS-2-35 with the D78 vaccine and HPR-2 with STC. The pathogenicity of these isolates was tested in specific-pathogen-free chickens. Bursa-body weight (B-BW) ratios and histopathologic lesion scores in the bursa were determined. Gross lesions were observed in the bursa, and the B-BW ratios of the birds infected with all four wild-type viruses were significantly different compared with the D78 vaccine and uninoculated control groups. Histopathology of the bursa from groups infected with GA-1, H-30, CS-2-35, and HPR-2 showed different degrees of follicular depletion and necrosis. A very slight lymphoid depletion was observed in the D78-infected group at 5 days postinoculation, and no microscopic lesions were observed in this group at 8 days postinoculation or at any time in the uninoculated control group. The bursa collected from the field virus and D78-infected birds at necropsy revealed the presence of IBDV via RT-PCR, and the VP-2 hypervariable nucleotide sequences of the GA-1, H-30, CS-2-35, HPR-2, and D78 samples were identical to the original viral isolates and vaccine, respectively.

Chicken recombinant antibodies specific for very virulent infectious bursal disease virus.

A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 x 10(7) clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.

Molecular characteristics of infectious bursal disease viruses from asymptomatic broiler flocks in Europe.

Infectious bursal disease virus (IBDV) exists in several different antigenic and pathogenic forms. The immune suppression caused by this virus in young chickens is not always associated with clinical signs of disease. The antigenic variant viruses originally described in the United States typically do not cause clinical signs of disease but can cause a marked immune suppression via the destruction of B lymphocytes. Using a reverse transcription-polymerase chain reaction (RT-PCR) assay we conducted a survey of asymptomatic broiler chicken flocks in Europe for IBDV. Restriction fragment length polymorphisms in the viral protein 2 (VP2) gene of four isolates from Spain and four isolates from France indicated they may be different from the classic and very virulent (vv) IBDV strains found throughout Europe. Nucleotide sequence and phylogenetic analysis of the hypervariable region of the VP2 gene indicated that all eight viruses were more similar to U.S. variant viruses than classic viruses. In two viruses, one from France and one from Spain, threonine was observed at amino acid position 222 and serine was found at position 254. These two substitution mutations are characteristic of Delaware variant viruses. In addition, all eight viruses had mutated amino acid position 318 from glycine to aspartic acid, another substitution mutation commonly found in U.S. variant viruses. Although importation restrictions prevented us from directly testing the antigenicity of these viruses, their nucleotide and predicted amino acid sequences suggest they could be antigenically distinctive compared to classic and vvIBDV commonly found in Europe. Confirmation of the presence of antigenic variant IBDV strains in Europe requires additional immunologic studies to elucidate the exact nature of the viral epitopes. Our data support the need for these immunologic studies.

Molecular studies on suspect very virulent infectious bursal disease virus genomic RNA samples.

Infectious bursal disease (IBD) associated with high mortality was first observed in Europe in the mid-1980s. The viruses identified in those outbreaks were described as being very virulent infectious bursal disease virus (vvIBDV) strains. These viruses have spread to nearly every continent but have not yet been identified in North America, Australia, and New Zealand. There is a real and immediate concern that the very virulent form of IBDV will continue to spread until it is present on every continent. Genomic RNA samples from IBDV strains suspected of being very virulent were submitted to our laboratory for molecular analysis. Nucleotide sequences of the VP2 gene hypervariable sequence region were determined for 18 of these viruses. A comparison with published vvIBDV sequences indicated that all but one sample (Thai 4) had nucleotide and predicted amino acid sequences consistent with vvIBDV strains. Published sequences and the nucleotide sequences of our 17 putative vvIBDV strains were used to identify unique nucleotides in the VP2 gene. Probe pairs for a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay were designed based on these unique sequences and then used to test the 17 genomic samples that were identified by nucleotide sequencing to be consistent with vvIBDV, plus the one Thai 4 sample that was not consistent with vvIBDV. Using melting temperature (Tm) analysis following real-time RT-PCR, two probe pairs (vv232 and vv256) successfully identified the 17 putative vvIBDV strains and distinguished them from the Thai 4 sample. An additional 26 genomic RNA samples submitted as suspect vvIBDV strains were then tested using the vv232 and vv256 probes. Based on the melting point analysis of these two probes, all 26 samples contained nucleotide sequences consistent with vvIBDV strains. The specificity of the vv232 and vv256 probe pairs was evaluated using 19 non-vvIBDV strains. In every case, the probes distinguished the 19 classic and variant (non-vvIBDV) strains from the putative vvIBDV strains. Diagnostic assays that can reliably identify vvIBDV strains are needed for surveillance programs designed to monitor the spread of these viruses.

Molecular epidemiology of infectious bursal disease viruses: distribution and genetic analysis of newly emerging viruses in the United States.

Genetic mutations in the genome of infectious bursal disease virus (IBDV) have resulted in antigenic and pathogenic variants that continue to cause disease in commercially reared chickens. The extent of the genetic diversity among IBDV strains circulating in the United States is unknown. This study was designed to identify newly emerging viruses infecting chickens on poultry farms experiencing immune suppression-related problems. Fifty IBDV-positive samples were identified from 273 bursa samples using a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Mutation probes were designed to the hydrophilic B coding region of the VP2 gene. Six mutation probes used in this study were based on the nucleotide sequences of the Del-E, Bursine 2, D-78, STC, G6, and T1 IBDV strains. Following real-time RT-PCR, these mutation probes identified 11 of the 50 viruses in the melting temperature (Tm) analysis. The results indicated that the remaining 39 viruses had one or more nucleotide mutations compared with the six mutation probes in this region of the VP2 gene. Thirty-eight viruses were chosen for nucleotide sequence analysis across the hypervariable region of the VP2 gene. Within this group of 38 viruses, four were identified by the mutation probes and their nucleotide sequences confirmed that real-time RT-PCR data. In the remaining 34 viruses, nucleotide mutations were observed in as many as 8 of 23 nucleotides across the hydrophilic B epitope coding region. Furthermore, every amino acid position except one between 316 and 324 had at least one substitution mutation. Phylogenic analysis placed two of the 38 viruses sequenced on branches with classic viruses and the remaining 36 viruses were placed on four distinct branches. Branches 1 and 2 contained a majority of the viruses, which were distributed across most of the major poultry-producing states in the United States. These branches contained previously characterized variant IBDV strains. Viruses in branches 3 and 4 were confined to three states and did not contain any previously characterized IBDV strains.

Real-time RT-PCR analysis of two epitope regions encoded by the VP2 gene of infectious bursal disease viruses.

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in chickens and leads to severe economic losses in the poultry industry. Vaccination may not be effective if there is exposure of the vaccinated flock to a different antigenic subtype, which reinforces the importance of identification of new IBDV variants. The virus outer capsid is constituted of VP2, in which the major neutralizing epitopes are located. Forty-eight bursa samples collected from IBDV infected commercial broiler flocks in the US were analyzed by real-time RT-PCR using probes designed for two epitope regions of VP2 denominated minor peak 1 and peak B. It was observed that 23, 48 and 44 samples tested with the minor peak probes Del-E, STC and F15, respectively, had a lower melting temperature (Tm) than expected. Furthermore, 44, 41 and 48 samples tested with the Del-E, STC and F15 peak B probes, respectively, had a lower Tm compared to the control, which indicates the presence of one or more nucleotide mutations in the samples. This fact was confirmed by nucleotide sequencing which also demonstrated that most mutations resulted in amino acid substitutions. Real-time RT-PCR can be a useful tool to assist in the development of more effective vaccination strategies.

Real-time reverse transcriptase-polymerase chain reaction detection and analysis of nucleotide sequences coding for a neutralizing epitope on infectious bursal disease viruses.

We used real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) to detect infectious bursal disease virus (IBDV) strains. The LightCycler (Roche) and hybridization probe system (Roche, Molecular Biochemicals) were used. A mutation probe labeled with fluorescein and an anchor probe labeled with Red-640 dye were prepared for each of the STC, Del E, D78, and Bursine 2 viral sequences. The mutation probes were designed to hybridize to nucleotides that encode the hydrophilic B region of VP2 for each virus. The anchor probes were designed to a relatively conserved region immediately downstream from the mutation probes. When hybridized to the RT-PCR product, a mutation and anchor probe pair produced fluorescence resonance energy transfer that was detected by the LightCycler instrument. Because they were designed to have a lower melting temperature (Tm), the mutation probes dissociated from the template before the anchor probes. The Tm values of the four mutation probes for each of their homologous viruses (exact sequence match) were STC, 69.3 +/- 1.2 C; D78, 67.8 +/- 0.9 C; Del E, 65.5 +/- 0.6 C; and Bursine 2, 71.7 +/- 0.4 C. These values were compared with the Tm values observed for a particular probe and heterologous virus. If the Tm values observed for heterologous viruses were within two standard deviations of the Tm for the probe and its homologous virus, the nucleotide sequences of the viruses were considered to be similar. If they were below two standard deviations, they were considered to have one or more nucleotide mutations. The results indicated that the STC and Variant Vax BD viruses have similar genetic sequences at the hydrophilic B region. Likewise, Bursine 2, Bursine, Bursine+, BioBurs, BioBurs W, BioBurs AB, and IBDV Blen have similar nucleotide sequences in this region. The Tm values obtained for the D78 and Del E mutation probes with heterologous viruses indicated that none of the viruses tested had nucleotide sequences that matched these probes. Because the mutation probes were designed to bind to a region that encodes a neutralizing epitope, viruses with similar sequences were expected to have antigenically similar epitopes.

Virulent vaccine strains of infectious bursal disease virus not distinguishable from wild-type viruses with marker the use of a molecular.

Molecular techniques have not only made timely and accurate detection of infectious bursal disease viruses (IBDVs) possible but also have allowed the identification of viral strains. Previously, we identified a genetic marker that distinguished wild-type IBDV strains from vaccine strains of the virus. The marker was an NgoM IV restriction enzyme site in the VP2 gene that was present in 10 wild-type viruses but not 16 vaccine strains of IBDV. On the basis of that study, we concluded that the NgoM IV marker could be useful in the identification of wild-type potentially pathogenic strains of this virus. Because virulent (hot) vaccine strains of IBDV are used to vaccinate commercial poultry, it was important to determine if the NgoM IV marker was present in these virulent vaccines. The infectious bursal disease Blen and Bursa Vac virulent vaccines were examined and determined to contain the marker. We concluded that the presence of this marker was not unique to wild-type strains of the virus. The absence of the NgoM IV marker, however, was consistent with some level of attenuation, and its presence appears to be consistent with virulent IBDV strains.

Use of a genetic marker for wild-type potentially pathogenic infectious bursal disease viruses.

An amino acid mutation at residue 284 (Ala to Thr) in the VP2 protein of infectious bursal disease viruses (IBDVs) has been correlated with the ability to replicate in cell culture. In this study, we designed a molecular test for this mutation. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to amplify a 743-bp region of the VP2 gene that contained the codon for amino acid 284. The restriction endonuclease NgoMIV was selected for this study because the first three nucleotides of its six-base recognition sequence are the codon responsible for the amino acid alanine at residue 284. The RT/PCR products from 10 known pathogenic and 16 vaccine strains of IBDV were examined for the presence or absence of the NgoMIV site. We also examined 189 field strains of IBDV for the NgoMIV site. All 10 known pathogenic IBDV strains contained the NgoMIV site, indicating they contained alanine at residue 284. None of the vaccine strains had the NgoMIV site, suggesting they had threonine or another amino acid at residue 284. The results suggest that the presence of this NgoMIV site can be used as a marker for the identification of wild-type (nonvaccine) IBDV strains. The RT/PCR products from 152 (80.4%) of the field strains had the NgoMIV site and thus have the potential to be wild-type pathogenic viruses. The RT/PCR products from 37 (19.6%) of the field strains were not cleaved by NgoMIV and thus are potentially attenuated vaccine strains. Molecular diagnostic assays have been used to place IBDV strains into genetically related groups. The identification of this genetic marker now makes it possible to identify viruses that are wild-type strains that have the potential to be pathogenic viruses.

Amino acid comparison of infectious bursal disease viruses placed in the same or different molecular groups by RT/PCR-RFLP.

Infectious bursal disease virus (IBDV) strains have been identified and placed into molecular groups by a reverse transcriptase (RT)/polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay. The predicted amino acid sequences corresponding to the region of the genome examined by RFLP were determined and compared for 14 IBDV strains from different molecular groups and 11 IBDV strains that were identified in molecular group 6. Among the viruses within molecular group 6, 13 amino acid positions had mutations, and among the viruses in different molecular groups, 27 amino acid positions had mutations. In addition to having more mutations, viruses compared from different molecular groups also had mutations at key positions that were previously reported to be important for the formation of neutralizing epitopes. Three of these IBDV strains with unique RFLP patterns were used to challenge 1-wk-old broiler chickens with maternal immunity to IBDV. One of these viruses, T1, broke through this maternal immunity as evidenced by detection of the virus by RT/PCR-RFLP and production of an active virus neutralizing antibody response to classic and variant IBDV strains. Unique amino acid mutations in the T1 virus that may have contributed to its ability to break through this maternal immunity were observed at amino acids 318 and 322. The results indicate that RFLP profiles and nucleotide sequences can be used to predict the relative similarities and differences among IBDV strains, but determining the actual antigenic differences among viruses requires testing in vivo.

Genetic stability of the VP2 hypervariable region of four infectious bursal disease virus isolates after serial passage in specific-pathogen-free chicken embryos.

Infectious bursal disease virus strains U2, 586, L1, and Q2 were isolated from pooled bursal samples collected from commercially reared broilers. These viruses were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for 24 or 25 passages. Nucleotide sequences of a 743-bp reverse transcription (RT)/polymerase chain reaction (PCR) product containing the VP2 hypervariable region were compared before and after passage of the viruses in embryonated chicken eggs. To determine the genetic stability of the viruses, each isolate was compared with its egg-passed ancestor; virus isolates were not compared with each other. When the restriction enzymes BstNI and MboI were used, no differences were observed in the restriction fragment length polymorphism profiles of the RT/ PCR products after embryo passage. After embryo passage, six nucleotide changes were identified in the viruses. Among the four viruses examined, these nucleotide changes resulted in a total of five amino acid changes. The amino acid changes were S-222-L in virus 586, K-249-N in viruses U2, L1, and Q2, and G-281-V in virus Q2. Three of the five amino acid changes occurred at residue 249. The convergent nature of this residue shift in three of four of the chick embryo-passed viruses suggests the occurrence of a functional, as opposed to random, mutation. The original isolates caused typical signs of infectious bursal disease in 3-wk-old SPF chicks. Their embryo-passed ancestors also produced typical signs of infectious bursal disease in 3-wk-old SPF chicks, suggesting the amino acid mutations observed did not affect virulence of the viruses.

Molecular typing of infectious bursal disease virus of Israeli field and vaccine strains by the reverse transcription/polymerase chain reaction/restriction fragment length polymorphism assay.

Infectious bursal disease viruses (IBDVs) were examined by testing bursa samples from 37 commercially reared chicken flocks and three vaccine strains by the reverse transcription (RT)/polymerase chain reaction (PCR)/restriction fragment length polymorphism assay (RFLP). The assay was conducted with a 717-bp fragment of the VP2 gene with the restriction enzymes BstNI and MboI. The presence of a restriction site for SspI was used to predict a very virulent phenotype. Results indicated the existence of two molecular groups within the field isolates; four samples showed one pattern of RFLPs, and the majority, 30 out of the 37 tested, showed a second RFLP pattern. Three samples tested negative for IBDV. Eight bursa samples, representing the two molecular groups, were also tested by the RT/PCR/RFLP assay as developed by Jackwood. A comparison of the RFLP profiles by the two methods indicated that four isolates belonged to molecular group 6 and 30 isolates belonged to a new molecular group. All field isolates had a very virulent phenotype. One vaccine strain, produced from a local isolate, was classified as molecular group 6. The other two vaccine strains had RFLPs that differed from those of the field isolates.

Sequence of precursor polyprotein gene (segment A) of infectious bursal disease viruses isolated in Korea.

The coding regions of segment A of two recent Korean very virulent (vv) infectious bursal disease virus (IBDV) isolates (KK1 and KSH) and one atypical IBDV isolate (K310) were amplified by reverse transcriptase-polymerase chain reaction, sequenced, and compared with published sequences for IBDV. The overall amino acid sequence similarity of the KK1 and KSH strains compared with foreign vvIBDV strains was between 97.43% and 98.02%. The KK1 and KSH strains, like vvIBDV strains, share unique amino acid residues at positions 222(A), 256(I), 294(I), and 299(S). The sequence of K310 strain was markedly different from other IBDV strains. The K310 strain had 12, 2, and 1 unique amino acid substitutions in the VP2 hypervariable region, VP4, and VP3 gene, respectively, and 3 of 12 substitutions in a VP2 hypervariable region were found in two hydrophilic regions known to be involved in antigenic determination. Also, the K310 strain had 222(S) and 254(S), which were found in variant IBDV strains. The SWSASGS heptapeptide is conserved in all Korean IBDV isolates. By phylogenetic analysis, KK1 and KSH were categorized in one group with foreign vvIBDV isolates, but K310 isolate was categorized in a separate group that was differentiated from the other IBDV strains compared. The K310 strain seemed to be evolved from a separate lineage of IBDV strain.

Heat lability of five strains of infectious bursal disease virus.

An extensive world trade environment has created a need for many nations to protect their food animal industries against the importation of pathogenic microorganisms. We initiated studies to determine how cooking conditions would affect the viability of different infectious bursal disease virus (IBDV) strains. Five different viral strains were tested. The strains included two serotype 1 variant viruses, Del-A and MD; two serotype 1 classic viruses, STC and D78; and a serotype 2 virus, OH. A 100-microL aliquot of each viral stain in Dulbecco's Modified Eagle Medium with 2% fetal calf serum was heated to 37,65, 71, 74,77, 82, or 100 C for 1 min. Following heat treatment, the virus titers were determined in a BGM-70 cell culture. Virus titers declined following each incubation, and there were no appreciable differences among the five viral strains tested. The greatest decline in titer occurred at temperatures above 65 C. After 1 min at 65, 71, and 100 C, reduction of viable viruses were > or =90, > or =99, and >99.9%, respectively, compared to nonheated controls. A viral reduction curve similar to the first heating trial was observed when viruses were heated at 71 or 74 C for 6 min. Drumsticks and boneless chicken patties were seeded with the virus at a mean value of 10(5.5) TCID50/25 microL of IBDV and were cooked to internal temperatures of 71 and 74 C, respectively. All samples were quickly cooled after achieving target temperatures. Viable virus was recovered from both products following cooking.