Phylogenetic analysis reveals a correlation between the expansion of very virulent infectious bursal disease virus and reassortment of its genome segment B.
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Infectious bursal disease virus (IBDV) is a birnavirus causing immunosuppressive disease in chickens. Emergence of the very virulent form of IBDV (vvIBDV) in the late 1980s dramatically changed the epidemiology of the disease. In this study, we investigated the phylogenetic origins of its genome segments and estimated the time of emergence of their most recent common ancestors. Moreover, with recently developed coalescence techniques, we reconstructed the past population dynamics of vvIBDV and timed the onset of its expansion to the late 1980s. Our analysis suggests that genome segment A of vvIBDV emerged at least 20 years before its expansion, which argues against the hypothesis that mutation of genome segment A is the major contributing factor in the emergence and expansion of vvIBDV. Alternatively, the phylogeny of genome segment B suggests a possible reassortment event estimated to have taken place around the mid-1980s, which seems to coincide with its expansion within approximately 5 years. We therefore hypothesize that the reassortment of genome segment B initiated vvIBDV expansion in the late 1980s, possibly by enhancing the virulence of the virus synergistically with its existing genome segment A. This report reveals the possible mechanisms leading to the emergence and expansion of vvIBDV, which would certainly provide insights into the scope of surveillance and prevention efforts regarding the disease. (+info)
Comparison of Sybr Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus.
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The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV. (+info)
Impact of coccidial infection on vaccine- and vvIBDV in lymphoid tissues of SPF chickens as detected by RT-PCR.
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BACKGROUND: This study aimed at investigating a potential effect caused by coccidia on the immune response to vaccine- and very virulent infectious bursal disease virus (vvIBDV) in SPF chickens. METHODS: Two groups of three weeks old SPF chickens were vaccinated prior to inoculation with coccidia and challenge with virulent IBDV, all within a period of eight days. Two control groups were similarly treated, except that challenge with field virus was omitted in one group while inoculation with coccidia was omitted in the other group. Clinical signs, lesions in the intestines caused by coccidia, lesions in the bursa of Fabricius caused by IBDV, IBDV-antibody titres, and virus detection by reverse transcription polymerase chain reaction (RT-PCR) were compared among the groups. Lymphoid tissues and swab samples were analysed by general RT-PCR, and positive results were identified by strain specific duplex (DPX) RT-PCR. RESULTS: In the triple-infected groups, vaccine strain IBDV was detected in spleen and thymus tissues, and no field virus was detected in bursa samples, contrary to the double-infected groups. CONCLUSION: The results suggest an enhancing effect on the immune response caused by subclinical coccidiosis and vvIBDV acting in concert. (+info)
Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-gamma on protective immunity by a DNA vaccine against IBDV in chickens.
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This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-a was constructed. Twice at 2-week intervals, two-week-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect. (+info)
Detection of infectious bursal disease virus from formalin-fixed paraffin-embedded tissue by immunohistochemistry and real-time reverse transcription-polymerase chain reaction.
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Formalin fixed paraffin embedded tissues blocks are used routinely to diagnose the economically important immunosuppressive infectious bursal disease virus (IBDV) in chickens. Immunohistochemical detection of viruses in tissue blocks has been done with varying results between laboratories. Extraction of IBDV RNA from tissue blocks allows IBDV strain identification at a molecular level. This allows correlation between virus identity and histological lesions present in the tissue. Experimentally reverse transcription-polymerase chain reaction (RT-PCR) detectable IBDV RNA could always be extracted from tissue blocks with acute +3 or higher histological lesion scores. However, many blocks from diagnostic field cases did not yield detectable IBDV RNA, in spite of having severe IBDV histological lesion scores. The reason for this can be the effect different formalin fixation conditions have on RNA detection from tissue blocks. To study the effect of various fixation parameters on RNA extraction and immunohistochemical detection of IBDV, bursas with maximum histological lesion score of 4 for IBDV were fixed in formalin under various conditions (different pH levels, temperatures, concentrations of formalin, and fixation duration). Only tissues fixed in formalin with a pH of 7.0, concentration of 5 or 10% formaldehyde, storage temperature of 25 degrees C or less, and kept for up to 2 weeks in formalin yielded detectable IBDV RNA upon extraction. No RNA could be detected from tissues fixed under extreme temperature, pH, or formalin concentrations. Optimal fixation conditions for IHC detection of IBDV were 10% formalin concentration, pH 7.0, and temperature of 4 degrees C, where maximum intensity of immunostaining was observed. (+info)
Genetic characteristics of infectious bursal disease viruses from four continents.
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Following the initial discovery of very virulent infectious bursal disease virus (vvIBDV) strains in Europe, these viruses spread to many parts of the world. In this study, we examined the phylogenetic relationship of never-before-published IBDV from 18 countries on four continents. All the samples were collected between 1997 and 2005 and were reported to be from broiler flocks experiencing higher than expected mortality which is often associated with acute very virulent infectious bursal disease. A total of 113 samples were imported into the U.S. and viral genetic material was used to determine the nucleotide sequence of the VP2 gene hypervariable region. Although all the samples were reported to be associated clinically with high mortality, genetic analysis suggests that some were not vvIBDV strains. Two viruses from South Africa were genetically similar to U.S. variant viruses. A majority (71/113) of the viruses examined had the amino acid Alanine at position 222 and sixty-seven of these suspect vvIBDV also had amino acids I242, I256, I294 and S299 which are highly conserved among vvIBDV strains. Phylogenetic analysis placed putative vvIBDV strains from many different countries and geographic regions in a single clade with some minor non-significant branching. (+info)
Nucleotide sequence analysis of VP2 hypervariable domain of infectious bursal disease virus detected in Japan from 1993 to 2004.
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Bursae of Fabricius were collected from 20 chickens diagnosed with infectious bursal disease virus (IBDV) infection from 15 prefectures in 1993 to 2004. Here we report the nucleotide sequence analysis of VP2 hypervariable domain of IBDV genome detected by reverse transcription-polymerase chain reaction from these samples. Ten sequences derived from 10 prefectures in 1996 to 2003 were of the classical type and other 10 sequences derived from 6 prefectures in 1993 to 2004 were of the highly virulent type. Of the classical type sequences, 9 sequences were closely related to the sequence of classical attenuated vaccines used in Japan. Furthermore, two were identical to the sequence of B-Chi5 which represents Vaccine B passaged 5 times in chickens and was reported to be reverted the virulence during the passages. The 10 highly virulent type sequences were classified into four sequences, none of which had been previously detected in Japan. However, the deduced amino acid sequences were identical to each other and to the sequences of highly virulent IBDVs previously detected in Japan. The most common nucleotide sequences, which accounted for 6 of the sequences, were identical to 34 highly virulent type sequences detected in various countries in BLAST search. This is the first report of detection of the sequence in Japan which is identical to highly virulent strains detected in other countries. These findings show the prevalence of classical IBDVs closely related to the attenuated vaccines and highly virulent IBDVs derived from other countries throughout Japan since 1993. (+info)
Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity.
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