Construction and virulence evaluation of nonreplicative vaccinia virus modified strain NTV-C7L

Objective To evaluate the virulence of NTV modified strain NTV-C7L in vitro and in vivo. Methods The non-replicative vaccinia virus modified strain NTV-C7L was constructed by homologous recombination, in order to evaluate the virulence of NTV-C7L by cell diffusion ability and virus replication, intranasal challenge, and scratch of tail of mice. Results The homologous recombination non-replicating vaccinia virus modified strain NTV-C7L was selected by blue-white screening, the purity of NTV-C7L was identified by PCR and Western blot, the C7L gene was successfully inserted and expressed; In human embryonic lung fibroblasts (MRC-5) as well as mouse embryonic fibroblasts (BALB/C3T3), the replication ability of NTV-C7L was recovered compared with that of NTV, but was lower than that of VTT; the result of the intranasal route with 106 PFU vaccinia virus showed that the animals infected with TTV had slightly greater weight loss than NTV or NTV-C7L(t=6.56, P<0.001; t=5.73, P<0.001). Compared with the NTV-C7L group, the weight loss of the NTV group was not statistically significant (t=0.597, P=0.081); the intranasal route with 107 PFU vaccinia virus showed that the weight loss of the NTV group and NTV-C7L group was statistically significant compared with the TTV group (t=12.86, P<0.001; t=10.71, P<0.001). Compared with the NTV-C7L group, the weight loss of the NTV group was statistically significant(t=4.616, P<0.001); the result of skin scratch indicated that mice scarified with TTV (106 PFU) formed more obvious skin redness, swelling and injury on the post infected 5 days, whereas only redness was detected after scarification with a much higher dose (107) of NTV or NTV-C7L. Conclusions The intracellular and mouse virulence of NTV modified strain NTV-C7L was recovered compared with NTV, but was significantly lower than that of TTV, which provided reference for optimization and application of vaccinia virus vector.