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Tiwari AK Kataria RS Indervesh Prasad N Gupta R 《Comparative immunology, microbiology and infectious diseases》2003,26(1):47-53
In order to differentiate infectious bursal disease virus (IBDV) isolates/strains, a quick method of RT-PCR followed by restriction enzyme analysis of VP1 gene sequence is being reported for the first time. A 480 bp fragment, comprising one of the RNA dependent RNA polymerase motifs of VP1 gene sequence of an Indian classical virus, an attenuated vaccine strain, Georgia and two Indian field isolates, genetically similar to reported very virulent strains of IBDV, was amplified by RT-PCR. Restriction enzyme digestion of PCR products with Taq1 enzyme generated distinct profile for field isolates, different from the classical and attenuated viruses, whereas restriction profile with BstNI restriction enzyme was similar in all the viruses, irrespective of the pathotype. Therefore, the present results suggest that Taq1 digestion can be taken up for the differentiation of field isolates from the classical and vaccine strains. The sequence analysis of VPI gene of reported very virulent IBD viruses from Europe and Japan, using 'MapDraw' programme of Lasergene software, revealed similar restriction enzyme profile as in Indian field isolates. 相似文献
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Rapid detection and differentiation of Newcastle disease virus isolates by a triple one-step RT-PCR 总被引：6，自引：0，他引：6
Wang Z Vreede FT Mitchell JO Viljoen GJ 《The Onderstepoort journal of veterinary research》2001,68(2):131-134
A triple one-step RT-PCR was developed to screen and differentiate virulent from avirulent Newcastle disease virus (NDV) isolates. Three sets of oligonucleotides were designed, each specific for amplifying NDV fusion protein gene-specific RNA from virulent, avirulent or all isolates respectively. The sensitivity of one-step RT-PCR was determined using viral RNA extracted from serially diluted NDV-infected allantoic fluid and found to be 10(-5) HA units. Application of one-step RT-PCR to various NDV samples, including wild-type virulent isolates and avirulent vaccine strains, demonstrated the potential for rapid identification (3-4 h) of NDV isolates as well as the differentiation of virulent from avirulent strains. 相似文献
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为评价SUMO原核表达系统(pHisSUMO Express)对病毒基因的可溶性表达,本研究从人工接种发病的鸡传染性法氏囊病(IBD)的病料组织样品中提取总RNA,通过RT-PCR扩增IBD病毒(IBDV)VP3基因,并将其克隆于pHisSUMO中构建了重组表达质粒pHisSUMO-VP3,转化大肠杆菌Rosetta(DE3)PlysS,经IPTG诱导,得到可溶性表达的融合蛋白SUMO-VP3。结果表明,该融合蛋白表达量占细菌总蛋白35%,经HisTrapTMFF crude column层析柱纯化后的SUMO-VP3蛋白可被SUMO蛋白酶Ⅰ有效切割,获得无标签的VP3蛋白,经western blot鉴定表明该VP3蛋白具有良好的抗原性。本研究表明pHisSUMO Express表达系统是高效可溶表达外源蛋白的有效工具,所表达的病毒蛋白具有良好的抗原性,为病原诊断抗原的研究和制备提供有效表达系统。 相似文献
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Infectious bursal disease virus (IBDV) that had been adapted to grow and was then cloned in chick embryo fibroblast (CEF) cell culture was examined for its physicochemical properties, the cellular site of virus replication, and the nature of its viral RNA. The IBDV was an RNA virus, acid-stable, absolutely resistant to chloroform, and moderately thermolabile. It appeared to replicate only in the cytoplasm, as shown by virus-specific antigens restricted to the cytoplasm of infected cells. The viral RNA was composed of single-stranded RNA, as evidenced by flame-red fluorescence on acridine-orange staining and an absence of specific fluorescence in infected cells on immunofluorescent staining with antiserum specific for double-stranded RNA. The IBDV virion had a hexagonal outline with an average diameter of 62 nm and possessed a single layer of capsid composed of hollow capsomeres without envelope. The buoyant density as determined in a continuous sucrose gradient was 1.178 g/cm3. The IBDV was found to possess morphologial and physicochemical properties different from those of any established RNA virus group. 相似文献
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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. 相似文献
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《Research in veterinary science》2014,96(3):957-964
Infectious bursal disease (IBD) is an acute and highly contagious viral disease of young chickens caused by infectious bursal disease virus (IBDV). An effective way to control IBDV would be to breed chickens with a reduced susceptibility to IBDV infection. In the present work, we used chickens selected for high and low specific responses to sheep red blood cells (SRBC) (H and L, respectively) to assess the susceptibility of differential immune competent animals to IBDV infection. The peripheral blood mononuclear cells (PBMCs) of high SRBC line (HL) and low SRBC line (LL) were infected with IBDV and viral RNA loads were determined at different time post-IBDV infection. Chicken orthologues of the T helper 1 (Th1) cytokines, interferon-γ (IFN-γ) and interleukin-2 (IL-2); a Th2 cytokine, IL-10; a pro inflammatory cytokine, IL-6; the CCL chemokines, chCCLi2, chCCLi4 and chCCLi7; colony stimulating factor, GM-CSF; and a anti-inflammatory cytokine, transforming growth factor β-2 (TGFβ-2) were quantified. The expression of chCCLi2, chCCLi4 and chCCLi7 was significantly higher in L line as compared to H line. However, in H line the viral RNA loads were significantly lower than in L line. Therefore, the upregulated chemokines might be associated with the susceptibility to IBDV. The expression of IFN-γ, IL-2 and IL-6 was significantly higher in H line as compared to L line. We assume that the higher proinflammatory cytokines expression in H line might be related to the rapid clearance of virus from PBMCs. Significantly higher levels of IL-10 and TGFβ-2 mRNAs in L line might be related to the pathogenesis of IBDV. In conclusion, selection for antibody responses appears to influence the expression profiles of chemokines and cytokines against IBDV. Further, the selection for high SRBC response might improve the immuno-competence of chickens against IBDV. 相似文献
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Nanthakumar T Kataria RS Tiwari AK Butchaiah G Kataria JM 《Veterinary research communications》2000,24(4):275-286
The technique of RT-PCR and restriction enzyme analysis was standardized to detect and differentiate Newcastle disease viruses. Digestion of RT-PCR-amplified, F gene sequences encoding for the cleavage activation sites of fusion protein with restriction enzymes AluI, BglI, HaeIII, HinfI, HhaI, RsaI, StyI and TaqI was carried out in order to characterize Newcastle disease viruses of varying pathogenicity. Restriction enzyme digestion of the amplicons by BglI and HhaI could group eight viruses, both field isolates and known vaccine strains, into lentogenic, mesogenic and velogenic pathotypes. By employing this technique directly on a clinical sample, Newcastle disease virus of the lentogenic pathotype could be detected. 相似文献