Identification of 10 Cowpox Virus Proteins That Are Necessary for Induction of Hemorrhagic Lesions (Red Pocks) on Chorioallantoic Membranes

Abstract

Cowpox viruses (CPXV) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (CAM) of embryonated chicken eggs, while most other members of the genus Orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). Cytokine response modifier A (CrmA) of CPXV strain Brighton Red (BR) is necessary but not sufficient for the induction of red pocks. To identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene CPXV knockout mutants was screened. We identified 10 proteins that are required for the formation of hemorrhagic lesions, which are encoded by CPXV060, CPXV064, CPXV068, CPXV069, CPXV074, CPXV136, CPXV168, CPXV169, CPXV172, and CPXV199. The genes are the homologues of F12L, F15L, E2L, E3L, E8R, A4L, A33R, A34R, A36R, and B5R of vaccinia virus (VACV). Mutants with deletions in CPXV060, CPXV168, CPXV169, CPXV172, or CPXV199 induced white pocks with a comet-like shape on the CAM. The homologues of these five genes in VACV encode proteins that are involved in the production of extracellular enveloped viruses (EEV) and the repulsion of superinfecting virions by actin tails. The homologue of CPXV068 in VACV is also involved in EEV production but is not related to actin tail induction. The other genes encode immunomodulatory proteins (CPXV069 and crmA) and viral core proteins (CPXV074 and CPXV136), and the function of the product of CPXV064 is unknown. IMPORTANCE It has been known for a long time that cowpox virus induces hemorrhagic lesions on chicken CAM, while most of the other orthopoxviruses produce nonhemorrhagic lesions. Although cowpox virusCrmA has been proved to be responsible for the hemorrhagic phenotype, other proteins causing this phenotype remain unknown. Recently, we generated a complete single-gene knockout bacterial artificial chromosome (BAC) library of cowpox virus Brighton strain. Out of 183 knockout BAC clones, 109 knockout viruses were reconstituted. The knockout library makes possible high-throughput screening for studying poxvirus replication and pathogenesis. In this study, we screened all 109 single-gene knockout viruses and identified 10 proteins necessary for inducing hemorrhagic lesions. The identification of these genes gives a new perspective for studying the hemorrhagic phenotype and may give a better understanding of poxvirus virulence.

Both CD8+and CD4+T Cells Contribute to Corneal Clouding and Viral Clearance following Vaccinia Virus Infection in C57BL/6 Mice

“…Thus, one potential explanation for restricted cell infiltration may be the lack of blood vessels growing into the central cornea of the VACV-infected mice. The explanation for why blood vessel infiltration is restricted is not clear, but it is known that only cowpox and rabbitpox viruses induce hemorrhagic pocks on chorioallantoic membranes and that this is due to the expression of specific viral genes (42). The lack of this capability in strain WR could explain the restricted vascularization of the cornea.…”

Extracellular Virions: The Advance Guard of Poxvirus Infections

“…Recently, Xu and colleagues [ 27 ] have brought new information to bear on this issue. Using a library of 109 single-gene knockout variants of cowpox virus, in addition to the crmA and B5R genes, they identified 10 nonessential genes that are required for the production of red pocks by cowpox virus.…”

“… * Cowpox virus mutants lacking the ability to encode these proteins produced white pocks on the CAM [ 27 ]. **A cowpox virus mutant lacking the ability to encode the F13 protein failed to produce any pocks on the CAM [ 27 ]. …”

“… **A cowpox virus mutant lacking the ability to encode the F13 protein failed to produce any pocks on the CAM [ 27 ]. …”

In Vivo Characterization of a Bank Vole-Derived Cowpox Virus Isolate in Natural Hosts and the Rat Model

“…Phylogenetic analyses demonstrated a nucleotide sequence identity of 99.2% to another German CPXV isolate, Ger2010MKY (CPXV-like 3, [28]), and a clustering together with Ectromelia virus (ECTV), and a separation from other CPXV isolates derived from either common voles or accidental host species. Viral replication on the chorioallantoic membrane (CAM) resulted in hemorrhagic pocks typical for CPXV [33], whereas ECTV develops non-hemorrhagic pocks on the CAM [34]. The taxonomical species definition for other orthopox viruses conceded the presumption that the different clades "CPXV-like 1", "CPXV-like 2", and "CPXV-like 3" would be better classified as different species within the genus Orthopoxvirus.…”