Vaccinia virus strain Western Reserve protein B14 is an intracellular virulence factor

characterization of the B14R gene from Vaccinia virus (VACV) strain Western Reserve (WR) is presented. Computational analyses of the B14R gene indicated high conservation in orthopoxviruses but no orthologues outside the Poxviridae. To characterize the B14 protein, the B14R gene was expressed in Escherichia coli and recombinant protein was purified and used to generate a rabbit polyclonal antiserum. This antiserum recognized a 15 kDa cytoplasmic protein in mammalian cells that were transfected with the B14R gene or infected with VACV WR, but not from cells infected with a VACV mutant (v Delta B14) from which the B14R gene was deleted. Compared to wild-type and revertant virus controls, v Delta B14 had normal growth kinetics in cell culture. The virulence of v Delta B14 was assessed in two in vivo models. Mice infected intranasally with v Alpha B14 had similar weight loss compared to the controls, but in C57BL/6 mice infected intradermally v Delta B14 induced a smaller lesion size compared with controls. Moreover, intradermal infection with v Delta B14 caused an increased infiltration of cells into the infected lesion despite the smaller lesion size. Therefore, B14 is an intracellular protein that is non-essential for virus replication in cell culture but contributes to virus virulence in vivo and affects the host response to infection.