Cowpox virus serpin CrmA is necessary but not sufficient for the red pock phenotype on chicken chorioallantoic membranes.

It was previously reported that cowpox virus (CPXV) strain Brighton Red (BR) causes red pocks upon inoculation of chorioallantoic membranes (CAMs) of embryonated chicken eggs. Red pocks are characterized by hemorrhage and reduced numbers of inflammatory cells while white pocks induced by other members of the genus Orthopoxvirus lack hemorrhage and have higher numbers of infiltrating heterophils. Analyses of CPXV BR white pock variants identified the cytokine response modifier A (CrmA) as the factor responsible for the differences in pock phenotype through induction of hemorrhage and inhibition of chemotaxis. In the present study CPXV crmA deletion mutants were generated based on a full-length bacterial artificial chromosome clone of CPXV BR (pBR). Deletion of the first crmA start codon was sufficient to abolish protein expression, whereas modification of a potential second start codon had no impact on CrmA production as shown by Western blot analysis. Immunohistochemistry of CAMs inoculated with crmA-positive BR viruses showed accumulation of viral antigen in endothelial cells, which was consistent with the red pock phenotype. On the other hand, crmA-negative mutants were characterized by the induction of white pocks and the absence of CPXV antigen in endothelia. The introduction of the complete CPXV BR crmA gene into the homologous genome region of the attenuated vaccinia virus strain MVA (modified vaccinia virus Ankara), however, resulted in CrmA production but not the red pock phenotype. We therefore conclude that (i) CPXV CrmA is associated with increased accumulation of virus in endothelial cells and (ii) the poxvirus-encoded serpin is necessary but not sufficient for the red pock phenotype and the anti-chemotactic capabilities on CAMs.