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ABSTRACT: In present study, different types of infectious bursal disease virus (IBDV), virulent strain DK01, classic strain F52/70 and vaccine strain D78 were quantified and detected in infected bursa of Fabricius (BF) and cloacal swabs using quantitative real time RT-PCR with SYBR green dye. For selection of a suitable internal control gene, real time PCR parameters were evaluated for three candidate genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA and beta-actin to IBDVs. Based on this beta-actin was selected as an internal control for quantification of IBDVs in BF. All BF samples with D78, DK01 or F52/70 inoculation were detected as virus positive at day 1 post inoculation (p.i.). The D78 viral load peaked at day 4 and day 8 p.i., while the DK01 and F52/70 viral load showed relatively high levels at day 2 p.i. In cloacal swabs, viruses detectable were at day 2 p.i. for DK01 and F52/70, day 8 p.i. for D78. Importantly, the primers set were specific as the D78 primer set gave no amplification of F52/70 and DK01 and the DK01 primer set gave no amplification of D78, thus DK01 and D78 could be quantified simultaneously in dually infected chickens by use of these two set of primers. The method described here is robust and may sever as a useful tool with high capacity for diagnostics as well as in viral pathogenesis studies.
Research in Veterinary Science 03/2007; 82(1):126-33. · 1.65 Impact Factor
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ABSTRACT: Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease in chickens and causes a significant economic loss for the poultry industry. Little is understood about the mechanism involved in the host responses to IBDV infection. For better understanding the IBDV-host interaction, we measured steady-state levels of transcripts from 28 cellular genes of chicken embryo (CE) cell cultures infected with IBDV vaccine stain Bursine-2 during a 7-day infection course by use of the quantitative real-time RT-PCR SYBR green method. Of the genes tested, 21 genes (IRF-1, IFN 1-2 promoter, IFNAR-1, IRF-10, IFN-gamma, 2',5'-OAS, IAP-1, caspase 8, TRAIL-like, STAT-3, IL-6, IL-8, MIP-3 alpha, MHC-I, MHC-II, TVB, GLVR-1, OTF, IL-13R alpha, ST3GAL-VI and PGK) showed an increased expression. The remaining seven genes (IFNAR-2, IFN-alpha, NF-kappaB subunit p65, BLRcp38, DDX1, G6PDH and UB) showed a constant expression or only slight alteration. Apparently, the host genes involved in pro-inflammatory response and apoptosis, interferon-regulated proteins, and the cellular immune response were affected by IBDV infection, indicating involvement in the complex signaling pathways of host responses to the infection. This study thus contributes to the understanding of the pathogenesis of IBD and provides an insight into the virus-host interaction.
Archives of Virology 02/2007; 152(3):463-78. · 2.11 Impact Factor
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ABSTRACT: A system including RNA isolation, primers and two novel oligonucleotide probes (NC22 and VC22) was established to rapidly pathotype Newcastle disease virus (NDV) from infected allantoic fluid; The sequence of the probes is: VC22, 5'-AAGGAGGCAGAAACGCTTTATA-3'; NC22, 5'-GGGGAGACAGG GGCGCCTTATA-3'. Application efficacy of the probes was verified by differentiating NDV that derived from experimentally infected organs. NC22 and VC22 both were labeled with digoxigenin (DIG) and were successful in differentiating NDV strains from the infected allantoic fluid and organs of experimentally infected SPF chickens. The RT-PCR products of NDV isolates and strains were dotted onto nylon membrane and then hybridized with two specific probes separately. The VC22 probe is specific to virulent strains, and the NC22 probe is specific to avirulent strains. The results of hybridization coincide with viral intracerebral pathologenicity index (ICPI). The specificity of two probes and sensitivity of NC22 probe were evaluated. NC22 could detect down to 10(-8) dilution from 10(7.5) EID(50)/ml or 800 fg of viral RNA. The system could be completed within 1 day to conduct experiment from clinical sample to the result of assay, and its potential practical application in clinical assay was discussed basing on specificity, sensitivity and rapidness.
Archives of Virology 07/2004; 149(6):1231-43. · 2.11 Impact Factor
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ABSTRACT: Two infectious bursal disease virus (IBDV) strains were inoculated both intranasally and by eye drop into 5-week-old specific pathogen free chickens. The bursa, the liver, the kidney, the spleen, the thymus, the caecal tonsil and the thigh muscle were harvested at 4, 8, 16, 28, 40, 56, 72, 96 h post-inoculation (p.i.) for IBDV detection by in situ reverse transcriptase-polymerase chain reaction and, at the same time, the pathological changes in these tissues were investigated. A typical positive signal was detected in the liver, the kidney and the spleen of chickens inoculated with the very virulent IBDV H strain at 4 h p.i., but not in the thymus, the caecal tonsil or the thigh muscle until 8 h p.i. Virus was also found in the liver, the spleen, the kidney, the thymus, the caecal tonsils and the muscle of birds inoculated with the cell-adapted Ts strain at 4 h p.i. A positive signal was observed in the bursa later than in the other tissues. The signals increased markedly at 8 h p.i. A decrease in bursal lymphocytes was observed in haematoxylin and eosin stained sections at 28 h p.i. for the H strain and at 40 h p.i. for the Ts strain.
Avian Pathology 01/2003; 31(6):593-7. · 1.71 Impact Factor
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ABSTRACT: In present study, different types of infectious bursal disease virus (IBDV), virulent strain DK01, classic strain F52/70 and vaccine strain D78 were quantified and detected in infected bursa of Fabricius (BF) and cloacal swabs using quantitative real time RT-PCR with SYBR green dye. For selection of a suitable internal control gene, real time PCR parameters were evaluated for three candidate genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA and β-actin to IBDVs. Based on this β-actin was selected as an internal control for quantification of IBDVs in BF. All BF samples with D78, DK01 or F52/70 inoculation were detected as virus positive at day 1 post inoculation (p.i.). The D78 viral load peaked at day 4 and day 8 p.i., while the DK01 and F52/70 viral load showed relatively high levels at day 2 p.i. In cloacal swabs, viruses detectable were at day 2 p.i. for DK01 and F52/70, day 8 p.i. for D78. Importantly, the primers set were specific as the D78 primer set gave no amplification of F52/70 and DK01 and the DK01 primer set gave no amplification of D78, thus DK01 and D78 could be quantified simultaneously in dually infected chickens by use of these two set of primers. The method described here is robust and may sever as a useful tool with high capacity for diagnostics as well as in viral pathogenesis studies.
Research in Veterinary Science.
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ABSTRACT: The VP2 hypervariable region of infectious bursal disease virus (IBDV) from nine Mainland Chinese strains was amplified by reverse transcriptase/nested polymerase chain reaction and cloned into pGEM-T vector. The nine isolates, which were from the center (HN3), the north (Bj-1, B2/28, HD96), the east (JS-18 and AH-2), the northeast (D11-2, C4-2), and the west (Ts) of China, were sequenced and compared with each other and with six reference IBDV sequences. Clustering analysis separated the nine isolate into two groups. The six virulent isolates, propagated in bursae, formed the first group. They revealed only one to three amino acid changes from the very virulent (vv) European and Japanese isolates, suggesting that they might have the same origin as European and Japanese vvIBDV strains. On the basis of their distinct geographic origins, extensive dissemination of vvIBDV in China was indicated. (The other three chicken embryo fibroblast cell cultured isolates with mild pathogenicity were placed in the second group.) Their sequences correlated closely with those of the culture-adapted strains (Cu-1 (4) and Cj-801). None of the nine isolates showed very close sequence relationship with the antigenic variant strains from the USA. Although antigenic variants have been reported in China, the reverse transcriptase/polymerase chain reaction-restriction endonuclease analyses of the nine viruses tested herein were not similar to any U.S.A. variant strains on the basis of computer software analysis. Our results and conclusions agree with a previous molecular study of IBDV isolates from the south of China.
Avian Diseases 42(4):762-9. · 1.46 Impact Factor
