Construction of an infectious clone of human adenovirus type 41.

Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K-expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.

Novel recombinant adenovirus type 41 vector and its biological properties.

BACKGROUND: Human adenovirus serotype 41 (Ad41) is a natural pathogen of the digestive tract and can cause gastroenteritis. There has been interest in reconstructing Ad41 as a gene delivery vector targeting the gastrointestinal tract, which is hampered by its fastidiousness. METHODS: An Ad41 E1B55K-transduced 293 cell line (293E12) was established as the packaging cell line. A backbone plasmid (pAdbone41) and a shuttle plasmid (pSh41-CMV) were constructed based on the Ad41 genome. Replication-defective adenovirus (Ad41-GFP) was rescued in 293E12 after being transfected with the linearized adenoviral plasmid, which was generated by homologous recombination of pAdbone41 and the shuttle plasmid carrying the GFP gene in Escherichia coli strain BJ5183. The packaging ability of 293E12, the stability of the Ad41-GFP genome and the acid-resistant property of Ad41-GFP were all investigated. RESULTS: A 293E12 cell could produce approximately 9000 viral particles of Ad41-GFP, which is close to the amount in the control virus (Ad5-GFP) amplified in one 293 cell. Ad41-GFP contained a genetically stable genome after being passaged eight times in 293E12 cells. More significantly, Ad41-GFP was more resistant to acid exposure than Ad5-GFP. It retained almost complete viability when exposed to hydrochloric acid with a pH value of 2 for 30 min, whereas Ad5-GFP lost 99% of its viability under the same conditions. Ad41-GFP was also more tolerant to treatment with artificial digestive fluid. CONCLUSIONS: An Ad41 vector system was successfully constructed, which consisted of the backbone plasmid, shuttle plasmid and packaging cell line 293E12. This system can be utilized to generate genetically stable and acid-resistant recombinant Ad41 carrying any gene of interest.