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Pathogenicity of Chicken Infectious Anemia Virus M Mutant and Susceptibility of DF-1 to It

Author: SunDanDan
Tutor: ShiWanYu；YangBing
School: Agricultural University of Hebei
Course: Clinical Veterinary Medicine
Keywords: Chicken infectious anemia virus The CIAV M mutant Pathogenicity DF-1 cell line Growth adaptability
CLC: S858.31
Type: Master's thesis
Year: 2011
Downloads: 31
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Abstract


Chicken infectious anemia virus (Chicken infectious anemia virus, CIAV) chicken infectious anemia (Chicken infectious anemia, CIA) pathogen is a the circovirus Division (Circoviridae), a single-stranded, negative-sense, covalently bound to the ring like DNA. The CIAV genome produce and a polycistronic characteristics transcripts. Three AUG codon on the transcripts, so that the internal formation of three open reading frames (Open Reading Frames, ORFs), wherein the ORF3 (366bp) located OFR2 (651bp) inside of ORF2 and ORF1 (1350bp) partially overlap. The ORFs encoded separately VP3 (13kD), VP2 (24 kD) and VPl (52kD) three viral proteins. VP1 structural protein, and antigenic sites part; VP2 is the VP1 partner protein or skeletal protein VP1 within the host cell to induce the body to produce neutralizing antibodies; VP3 the CIAV main pathogenic protein can be induce apoptosis. Shift of 39 high-tech laboratory of the Beijing Academy of Agriculture and Forestry Xiayong Heng et al using PCR-directed mutagenesis techniques initiation codon encoding CIAV VP3, resulting in incomplete expression of VP3 successfully build a pTCAVm of positive clone and initial infectious clone. The test is positive recombinant clones from the beginning, continue to lay the foundation for pathogenicity and tropism of the DF-1 cell line, looking for better development of the CIA live attenuated vaccine of program research the CIAV M mutant. CIAV mutant gene itself this experiment to select stored in the -80 ℃ pTCAVm positive clones, mutant strains in vitro cyclization Test Method according to Xia Yongheng etc. will, to re-to rejuvenation pTCAVm recombinant strain, extracting the recombinant plasmid, restriction enzyme digestion, cyclization. Liposomal transfection method itself cyclization mutant transfected MDCC-MSB1 host cell, and IFA and RT-PCR method to detect and CIAV sequencing of viral replication in transfected cells circumstances, test results show This test is the same the the infectious mutant-CIAV M mutant. Intraperitoneal injection method using artificial, the venom inoculation of the the CIAV M mutants in 1-day-old SPF chickens and dynamic tracking the CIAV M mutants in SPF chickens in vivo pathogenicity and in vivo passaged in SPF chickens once the host cell pathogenicity . IFA method, measured the CIAV M mutant the TCID 50 value of 10 4.5 / mL; observed by clinical pathological change and biopsy method: 7,14 characteristic of the CIA at 21 days of age, CIAV M mutants can cause SPF chickens gain weight slowly, T lymphocytes disappear within the thymus and other immune organ, tissue atrophy, the drumstick bone marrow color becomes yellow pink, red blood cell volume values ??significantly decreased lesions; vivo the CIAV antibody level test results show that: the antibody levels showed a weak positive, suggesting that the CIAV M mutant antigenic; measured by flow cytometry infected host cell growth cycle and cell apoptosis, test results show: regression animal the CIAV M test mutant host cell cycle of MDCC-MSB1 no significant blocking effect, but it has induced host cell apoptosis. CUX-1 standard strains peer comparison, the test results show that the: M mutants of CIAV have infection, but significantly reduced its pathogenicity. The lower virus titer in the host cell of the MDCC-MSB1, CIAV not conducive to the production of vaccines CIAV Therefore, this test also tentative the CIAV M mutant strain was inoculated in the DF-1 cell lines, to find a mutant of CIAV M new growth of cell lines. The the CIAV M mutant strain was inoculated in the DF-1 cell line, and after 5-7 days of culture, six repeating group did not all appear fibroblast pull network rounded cytopathic; passaged again, using the methods of the blind passage, inoculum venom repeated freezing and thawing of the previous generation of cells obtained directly scraped from the cell plate after 5-7 days of culture, the six repeating groups still not all cell lesions. The the CIAV M mutants by inoculation in the DF-1 cell line, passage 15 times, each generation of cells charged fluid detected by PCR and IFA and RT-PCR method to verify the PCR results for the positive samples, but the latter two methods was not detected in positive samples CIAV. Infer based on the entire test results: CIAV M mutant growth adaptability can not be on the DF-1, may not have the DF-1 cell line tropism.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Chicken
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