Construction and characterization of an infectious clone of coxsackievirus A16

Two 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 joined via a native XbaI 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 cells in vitro with 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.We 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.Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) are major etiological agents of hand, foot, and mouth disease (HFMD), which is a common illness in children [1-6]. Surveillance data indicate that CVA16 and EV71 often co-circulate during HFMD outbreaks [1-3,5-8]. The illness caused by CVA16 infection is usually mild [9], whereas EV71 infection is often associated with severe complications such as brainstem encephalitis, severe pulmonary edema and shock, and significant mortality [6,10,11]. Therefore, EV71 has been the main focus of virological investigations and vaccine development for HFMD. However, recent reports suggest that humans can be co-infected by CVA16 and EV71, and carry these two viruses simultaneously [12,13]. This co-infection may have contributed to the recently observed recombination between CVA16 and EV71 [14,15], which is believed to have led to the emergence of a recombinant EV71 responsible for the large HFMD outbreak in Fuyang City, China, during 2008 [15]. Furthermore, CVA16 infection is not always benign because fatal cases associated with CVA16 infection have been reported [16-18]. These findings indicate th