AbstractBackgroundCoxsackievirus A16 (CVA16) is a member of theEnterovirusgenus of thePicornaviridaefamily and it is a major etiological agent of hand, foot, and mouth disease (HFMD), which is a common illness affecting children. CVA16 possesses a single-stranded positive-sense RNA genome containing approximately 7410 bases. Current understanding of the replication, structure and virulence determinants of CVA16 is very limited, partly due to difficulties in directly manipulating its RNA genome.ResultsTwo overlapping cDNA fragments were amplified by RT-PCR from the genome of the shzh05-1 strain of CVA16, encompassing the nucleotide regions 1-4392 and 4381-7410, respectively. These two fragments were then joinedviaa nativeXbaI site to yield a full-length cDNA. A T7 promoter and poly(A) tail were added to the 5' and 3' ends, respectively, forming a full CVA16 cDNA clone. Transfection of RD cellsin vitrowith RNA transcribed directly from the cDNA clone allowed the recovery of infectious virus in culture. The CVA16 virus recovered from these cultures was functionally and genetically identical to its parent strain.ConclusionsWe report the first construction and characterization of an infectious cDNA clone of CVA16. The availability of this infectious clone will greatly enhance future virological investigations and vaccine development for CVA16.