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.

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.

Antibody titers to infectious bursal disease virus in broiler chicks after vaccination at one day of age with infectious bursal disease virus and Marek's disease virus.

The effect of day-of-age vaccination with infectious bursal disease virus (IBDV) alone or in combination with Marek's disease virus (MDV) in broiler chicks was investigated. One-day-old commercial broiler progeny obtained from IBDV-immunized breeder flocks were vaccinated subcutaneously according to the manufacturer's directions with live-attenuated commercially available vaccines as follows: IBDV alone, MDV alone, IBDV + MDV, and unvaccinated control. IBDV was not detected after vaccination by reverse transcriptase/polymerase chain reaction in any of the bursal, thymic, and splenic tissues tested. Serum IBDV antibody levels, as monitored by enzyme-linked immunosorbent assay (ELISA), showed similar rates of decline among the four groups, and, by day 28 postinoculation, serum antibody levels in all groups were below detectable limits. IBDV-specific neutralizing maternal antibody (MA) titers in the IBDV + MDV and control groups, as monitored by the virus neutralization assay (VN), remained higher and declined more slowly throughout the experiment as compared with VN titers in the IBDV- and the MDV-vaccinated groups. In a second experiment, groups of one-day-old broiler chicks were vaccinated as in Experiment 1. Serum IBDV antibody detected by ELISA and VN assay declined parallel to those observed in Experiment 1. Histopathologic lesions characteristic of IBDV were not observed in any of the groups. Vaccination with MDV alone was associated with increased rates of IBDV neutralizing MA decline similar to those caused by vaccination with IBDV alone, whereas concurrent vaccination with IBDV + MDV was not associated with increased rates of IBDV neutralizing MA decline over that of nonvaccinated controls. Results of these studies indicate that IBDV vaccination at 1 day of age does not cause accelerated IBDV-specific MA decline as detected by ELISA but does appear to cause an accelerated decline in neutralizing IBDV-specific MA. Furthermore, vaccination with IBDV at 1 day of age appeared to slow the accelerated rate of IBDV neutralizing antibody decline caused by MDV vaccination.

Correlation of enzyme-linked immunosorbent assay titers with protection against infectious bursal disease virus.

We examined the utility of baculovirus-expressed infectious bursal disease virus (IBDV) proteins to act as antigens in the enzyme-linked immunosorbent assay (ELISA). The three IBDV protein antigens tested included 1) a truncated VP2, 2) whole VP2, and 3) the polyprotein products VP2, VP3, and VP4. Serum samples from 2-wk-old commercially reared broilers were collected and tested in the three ELISAs. Serum samples were obtained from 34 different commercial broiler flocks. An average of 14 serum samples (range = 11-17) were tested for each flock. The ELISA results were compared with the percentage of protection of these birds following challenge with IBDV. Fifty 2-wk-old chicks from each of the 34 broiler flocks were challenged with STC classic virus or Del-E variant virus. At 7 days postchallenge, the bursa from each of the birds was removed and bursa/body weights were recorded. Percentage of protection was determined by the number of birds in each challenge group that had normal relative bursal weights compared with unchallenged controls. No evidence was found of a relationship between ELISA data generated with the polyprotein antigen (VP2, VP3, VP4) and percentage of protection observed in the STC and Del-E challenged birds. A significant relationship was found between ELISA data and percentage of protection to STC and Del-E when the truncated VP2 or whole VP2 antigens were used in the ELISA. The results of this study indicate that predicting the percentage of protection against classic or variant IBDV strains in broilers from vaccinated breeder flocks can be improved when VP2 is used as the only antigen in the ELISA.

Restriction fragment length polymorphisms in the VP2 gene of infectious bursal disease viruses from outside the United States.

Infectious bursal disease viruses (IBDVs) were identified in bursa samples from chickens reared outside the United States. All samples were obtained from commercially reared chicken flocks experiencing signs typical of infectious bursal disease. The reverse transcriptase/polymerase chain reaction (RT/PCR) technique was used to detect the viruses and restriction fragment length polymorphism (RFLP) was used to compare a 743-bp region of the VP2 gene among the viruses detected. The 81 IBDVs detected were from 16 different countries and were assigned to molecular groups on the basis of their RFLP following digestion with the restriction enzymes BstNI and MboI. The presence of an SspI site in the 743-bp RT/PCR fragment was used to predict the very virulent (vv) phenotype. Almost half (49%) of the viruses detected had the same RFLP with the BstNI and MboI enzymes. These viruses were placed into a new molecular group, designated group 6, that was exemplified by the RS593 vaccine strain of IBDV, which also had this molecular RFLP pattern. Some viruses detected had RFLP patterns similar to molecular groups 2, 3, and 4, previously described in our laboratory. Sixteen RFLP patterns were observed that did not match RFLP results we previously obtained for vaccine IBDV strains. The SspI restriction site was found in 46% of the viruses detected, predicting that these viruses have the vvIBDV phenotype. The SspI site was not observed in viruses from Central and South America.

Development of a ssRNA internal control reagent for an infectious bursal disease virus reverse transcription/polymerase chain reaction-restriction fragment length polymorphism diagnostic assay.

Infectious bursal disease virus (IBDV), family Birnaviradae, is the etiologic agent of a commercially important, globally distributed, contagious, immunosuppressive disease of young chickens. A restriction enzyme-compatible ssRNA internal control was developed for an IBDV reverse transcription/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) diagnostic assay. An 841-bp bacteriophage-lambda DNA fragment was directionally ligated to 3' and 5' oligonucleotide linkers containing the IBDV RT/PCR target primer sequences. A pGEM-3Zf (+) transcription vector containing the internal control construct was used in an in vitro transcription reaction to produce ssRNA. After RT and PCR amplification, the transcripts produced an 882-bp cDNA product, larger than, co-amplifiable with, and free of the restriction sites used to prepare RFLP patterns of the 743-bp IBDV cDNA target product. The limit of detection of the transcripts in the RT/PCR test is 3.2 femtograms. With the internal control, a test inhibition rate of 7.7% (20/261) was determined for the IBDV RT/PCR assay. By identifying inhibited tests, the assay was improved through a reduction in the number of false-negative results.

Genetic heterogeneity in the VP2 gene of infectious bursal disease viruses detected in commercially reared chickens.

The genetic heterogeneity of infectious bursal disease virus (IBDV) vaccine strains was compared with IBDV detected in bursa tissue of commercially reared chickens. The vaccine strains tested represented classic viruses from the United States, South Africa, England, and France plus variant viruses from the United States. Bursa tissue samples used for the detection of IBDV from commercially reared chickens were from the United States. Genetic heterogeneity was examined using restriction fragment length polymorphisms (RFLP) of a 743-bp fragment of the VP2 gene that was amplified using reverse transcriptase/polymerase chain reaction (RT/PCR). The RT/PCR products were digested using restriction enzymes BstNI and MboI. On the basis of RFLP profiles, viruses were placed into molecular groups. Thirty-eight vaccine and laboratory strains of IBDV were placed into five molecular groups. Groups 1 and 2 contained variant viruses, groups 3 and 4 contained classic viruses, and group 5 contained Lukert/Edgar strain classic viruses. In contrast to these five molecular groups, 22 molecular groups were observed for 70 IBDV-positive samples from U.S. chicken flocks. Twenty-two of the 70 IBDV-positive samples were placed into molecular groups 1 (2 samples), 2 (18 samples), and 5 (2 samples). Nineteen new RFLP profiles that did not match the five molecular groups observed for vaccine strains were detected. No viruses were observed to have RFLP profiles like molecular groups 3 and 4 observed in the vaccine strains. The genetic heterogeneity was greater among IBDV strains circulating in commercially reared poultry compared with vaccine strains of the virus.

Restriction fragment length polymorphisms in the VP2 gene of infectious bursal disease viruses.

Infectious bursal disease viruses (IBDVs) were examined for restriction fragment length polymorphisms in a fraction of the VP2 gene with the use of the reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay. The restriction enzymes BstNI and Mbol were used to obtain RFLP results. A third enzyme, StyI, was tested, but its utility for differentiation of IBDV strains was limited. Thirteen vaccine viruses and five IBDV strains that were previously characterized were placed into five molecular groups. Two groups contained viruses described as being classic strains, and two groups contained viruses described as being variant strains. The fifth group contained both classic and variant strains. The RFLP observed for the serotype 2 IBDV strain OH was unlike any of the RFLPs observed in viruses in the five molecular groups. Seven IBDV strains from commercially reared chickens in the United States, Mexico, Puerto Rico, and Thailand were tested in the RT/PCR-RFLP assay to determine if they were similar to the commercial IBDV vaccine strains tested. These viruses were selected because they were associated with lesions in the bursa of chickens that should have been protected by maternal antibodies or active immunity. Each of the viruses tested contained a unique RFLP compared with the IBDV strains and vaccine viruses examined in this study and, thus, did not fit into any of the five molecular groups. These viruses also were distinguishable from each other.

Identification and comparison of point mutations associated in classic and variant infectious bursal disease viruses.

Restriction enzymes (RE) were used to identify point mutations in the nucleotide sequences of the infectious bursal disease virus (IBDV) strains Del-E, Del-A, STC, IN, Bursine 2, and Bio-Burs. The point mutations at amino acid sites 222, 254 and 323 were identified using REs BstNI, StyI and MboI, respectively. The nucleotide sequences of STC, IN, Bursine 2 and Bio-Burs were determined at each of these sites. Nucleotide sequence analysis using this data and previously reported data for Del-E and Del-A was used to confirm the point mutation and predict the resulting amino acids. Although there were some exceptions, the BstNI and StyI enzymes detected point mutations in the first base of the 222 and 254 codons, respectively and could be used to predict an amino acid change in the viruses. MboI was detecting a mutation in the third base of codon 323 and could not reliably predict an amino acid change at that position. The results indicated that amino acids 222 and 254 were consistently mutated in the variant viruses examined and that amino acid 323 was not. Furthermore, point mutations resulting in amino acid changes at position 222 suggested that several groups of viruses may be defined by this site alone; proline for classic strains like STC, threonine for Del-E and GLS, serine for IN, Bursine 2, and Bio-Burs and glutamine for Del-A.

Successful management of CMV pneumonia in a mechanically ventilated patient.

We report a case of severe respiratory failure due to cytomegalovirus pneumonitis in a patient who underwent an allogeneic bone marrow transplant, who was successfully treated with the combination of ganciclovir and high-dose intravenous immune globulin. We also reviewed the rationale for the use of combination therapy with an antiviral agent and immunotherapy. Because of the bone marrow toxicity of ganciclovir, an aggressive diagnostic approach, including bronchoalveolar lavage and open lung biopsy, may be necessary to establish a definitive diagnosis prior to institution of therapy.

Effect of the rat liver S9 fraction on the mutagenicity of azathioprine in the Salmonella/mammalian microsome assay.

Azathioprine is a direct acting mutagen in Salmonella typhimurium TA100 and TA1535. Addition of rat liver S9 fraction with or without co-factors, or glutathione, causes a decrease in the mutagenicity of azathioprine in TA100 and an increase in TA1535, indicating the effect of SH groups.