Genome typing of mouse adenoviruses

Abstract

Restriction endonuclease cleavage site analysis was used to differentiate between mouse adenovirus (MAV) types 1 and 2 strains. Viral DNA of suitable purity and quantity for multiple enzymatic digestions was obtained from cloned CMT-93 mouse tumor cells infected with each type of MAV. Clear differences between the MAV-1 (FL) and MAV-2 (K87) genomes were observed after cleavage with restriction enzymes such as BglJI, EcoRI, and PaeR7. Fast electrophoresis of DNA fragments in miniature agarose slab gels allowed rapid and unequivocal identification of the MAV strains. This relatively simple and accurate method should be quite useful to determine the different modes of transmission of mouse adenoviruses and their presence in various animal populations. Restriction endonuclease analysis has the potential to detect very small differences between closely related virus strains. The technique has been used extensively for characterizing and comparing adenoviruses of different origins (1, 2, 8, 9, 13, 16, 17, 20, 24, 26). In this paper, we show how digestion of viral DNA molecules with restriction enzymes and comparison of the resultant fragments after electrophoresis can be most useful to rapidly identify mouse adenovirus (MAV) types 1 and 2 strains. These viruses share common antigenic properties (18, 21, 23, 25) and are thus difficult to distinguish from one another by conventional methods.

Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection

“…MAdV-1.IXeGFP (MAV-1 inp903) was created by fusing an eGFP open reading frame (ORF) in frame 3' to the ORF encoding capsid protein IX, using recombineering with a bacterial artificial chromosome containing the genome of MAdV-1 (pKBS2 MAV-1, a kind gift of Silvio Hemmi, University of Zurich) [58]. Wild type MAdV-1 and MAdV-1.IXeGFP were both propagated on CMT-93 mouse rectal carcinoma cells (ATCC CCL-223, a kind gift from Susan Compton, Yale University School of Medicine) [59]. CMT-93 cells were cultured in DMEM supplemented with 10% fetal bovine serum (Sigma-Aldrich), 4 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM nonessential amino acids (complete DMEM).…”