Bovine adenovirus type 3 virions cannot be rescued in vivo after full-length viral genome transfection in the absence of detectable polypeptide IX

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.

Construction of a hexon-chimeric human adenovirus type 3 vector expressing two neutralizing epitopes of hepatitis B surface antigen preS1 and analysis of antigenicity of the chimeric epitopes / 中华微生物学和免疫学杂志

Objective To construct a hexon-chimeric human adenovirus type 3 ( HAd3 ) vector expressing two neutralizing epitopes of hepatitis B surface antigen preS 1 (HBsAg-preS1) and to analyze the antigenicity of the chimeric epitopes .Methods Two neutralizing epitopes of HBsAg-preS1 including KR359 and KR127 were inserted into hypervariable region 1 ( HVR1) and hypervariable region 2 ( HVR2) of HAd3 hexon .Chimeric hexon gene encoding the two epitopes was amplified by overlap PCR and then subcloned in -to shuttle plasmid pBR322-L/R containing the homologous recombination regions .The digested shuttle plas-mid containing chimeric hexon gene was co-transfected into Escherichia coli BJ5183 cells together with back-bone plasmid pBRAdΔE3GFP to construct pBRAdΔE3GFP-preS1 vector.Then pBRAdΔE3GFP-preS1 vector was digested with AsiSⅠand transfected into AD293 cells to construct recombinant virus (rAD3E-preS1). CsCl gradient centrifugation was used for purification .Glutathione S-transferase ( GST ) fusion protein KR359KR127 ( GST-KR359KR127 ) was expressed in Escherichia coli BL21 by using expression vector pGEX-4T3.Female BALB/c mice at age 6-8 weeks were intraperitoneally injected with 1010 virus particles or 80 μg GST fusion protein .Serum samples were collected to analyze the antigenicity of two epitopes by ELISA and Western blot .Results ELISA showed that KR 359 and KR127 were successfully exposed on viral sur-faces by using hexon-chimeric HAd3 vector .The induced polyclonal antibodies in serum samples could rec-ognize GST fusion protein and native HBsAg from patients infected with hepatitis B virus .Conclusion The antigen capsid-incorporation strategy could be used to display epitopes on viral surface .Enhanced antigen-specific responses could be achieved through inserting multiple foreign epitopes into hexon HVRs .This study provided evidence for further application of hexon -chimeric human adenovirus type 3 vector in the developmentof vaccine, especially for the development of multivalent hepatitis B vaccine .

Construction of the recombinant human adenovirus type 3 expressing Norovirus capsid protein gene / 中华微生物学和免疫学杂志

Objective To prepare recombinant human adenovirus type 3 expressing Norovirus cap-sid protein gene(Noro-orf2). Methods The cDNA for Noro-orf2 was amplifed by RT-PCR from stool of in-fantile gastroenteritis and cloned into the adenovirus shuttle vector pBSE3CMV-egfp. The vector pBSE3CMV-Nor was linearized with EeoR Ⅴ and Not Ⅰ, and transformed into E. coil BJ5183 with lined edenovirus ge-nomic DNA pLasmid pBRAdv3 by Rsr Ⅱ. The identification of recombinant adenovirus plasmid pBRAdv3E3dNor was performed by PCR, enzyme digestion and DNA sequencing. Then pBRAdv3E3dNor was digested with AsiS Ⅰ and transfeeted into Hep-2 cells with LipofectAMINETM 2000 to package recombi-nant adenovirus particles. Results Noro-orf2 was successfully inserted into the shuttle vector. The recombi-nant adenoviral plasmid pBRAdv3E3dNor was generated by homologous recombination in E. coil BJ5183 and confirmed by PCR and enzyme digestion. The recombinant adenovirus was successfully packaged and puri-fied. Norovirus eapsid protein gene expression was confirmed in Hep-2 cells by immunecytochemistry assay. Conclusion The recombinant type 3 adenovirus expressing Norovirus eapsid protein gene was successfully constructed. This study laid a foundation for developing vaccine against Norovirus.

Effect of Adenovirus Type 3I,7b on Expressions of mRNA and Protein of Transforming Growth Factor-Beta 1 in Human Embryonic Lung Fibroblast Cells / 实用儿科临床杂志

Objective To study the effect of adenovirus type 3I,7b on the expressions of mRNA and protein of transforming growth factor-beta 1(TGF-?1) in human embryonic lung fibroblast cells.Method The expression of mRNA and protein of TGF-?1 were determined in human embryonic lung fibroblast cells before and after being infected by adenovirus type 3I,7b and in normal fibroblast cells with enzyme-linked immunosorbent assay and in situ hybridization.Results The mRNA and protein of TGF-?1 expression in human embryonic lung fibroblast cells increased siginificantly after being infected by adenovirous type 3I,7b compared with those in normal fibroblast cells(Pa0.05).Conclusion Lung fibroblast cells and TGF-?1 may play some roles in pathophysiological processes of viral pneumonia.

Simultaneous Detection of Adenovirus types 3, 7 and 11 by a Multiplex PCR from Cell Cultured Clinical Specimens of Hospitalized Patients

Adenovirus (AD) is classified into A, B, C, D, E and F subgenera on the basis of neutralization, hemagglutination and DNA homology tests. Six AD subgenera have 51 serotypes. We selected AD types 3, 7, and 11 since they are known to frequently cause respiratory disease, gastrointestinal disease and meningitis, which often require hospitalization of the patients. The purpose of this study was to detect and to evaluate the correlation of AD types 3, 7 and 11 and the diseases. The detection method, used for AD types 3, 7 and 11, was a multiplex polymerase chain reaction (PCR) with HEp-2 cell cultured with clinical samples. The result was as follows: AD 3 (22/62, 35.5%), AD 7 (27/62, 43.5%), and AD 11 (50/62, 80.6%) were detected by the multiplex PCR in 62 clinical samples. AD 3 detection rate in cerebrospinal fluid was relatively higher than in other specimens but its statistical significance was low (p=0.07). AD 11 was detected (50/62, 80.6%) with the highest frequency and appeared to significantly be associated with gastroenteritis, respiratory disease, and meningitis in hospitalized children, and arthritis in adult patients. AD 3, AD 7 and AD 11 types were detected singly in 21/62 (33.9%), doubly in 15/62 (24.2%), and triply in 16/62 (25.8%). The multiplex PCR method using Hep2 cell culture with clinical specimens could detect AD types 3, 7, and 11 in children with meningitis, respiratory infection, or gastroenteritis, and in adults with cancer and arthritis.

Study of the MxA protein induced by adenovirus type 3 and it' s antiviral effect against Ad3 / 中国免疫学杂志

Objective: This study aimed to investigate the induction of MxA protein in PBMC treated with various doses of adenovirus type 3(Ad3) and to test the antiviral effect of MxA protein against Ad3 in vitro.Methods:The content of MxA protein in cytoplasm of the peripheral blood mononuclear cells( PBMC) , which were treated with various dose of Ad3 was detected by flow cytometry. The antiviral effect of MxA protein against Ad3 in Hela cells was studied by the microdose cytopathogenic effect inhibition assay. Results: MxA protein that in all the cell groups that were treated with various dose of Ad3 was higher than that of control. 10 ng/ml MxA protein can resist 20TCID50 Aad3. Conclusion: It was suggest that MxA protein that can be induced by Ad3 in PBMC and recombinant MxA can resist Ad3.