Deletion analysis of two tandemly arranged virulence genes in myxoma virus, M11L and myxoma growth factor

Myxoma virus (MYX) is a leporipoxvirus of rabbits that induces a lethal syndrome characterized by disseminated tumorlike lesions, generalized immunosuppression, and secondary gram-negative bacterial infection. A MYX deletion mutant (vMYX-GF^-ΔM11L) was constructed to remove the entire myxoma growth factor (MGF) coding sequence and that for the C-terminal five amino acids of the partially overlapping upstream gene, M11L. Unexpectedly, this deletion completely abrogates the capacity of MYX to cause the characteristic disease symptoms of myxomatosis. Upon inoculation of rabbits with vMYX-GF^- ΔM11L, recipient animals developed only a benign, localized nodule reminiscent of a Shope fibroma virus-induced tumor in which a single primary lesion appeared at the site of injection and then completely regressed within 14 days, leaving the animals resistant to challenge with wild-type MYX. No evidence of the purulent conjunctivitis and rhinitis that always accompany wild-type MYX infection was observed. To ascertain whether the attenuation observed in vMYX-GF^-ΔM11L was due to a combined effect of the MGF deletion and alteration of the upstream M11L gene, two additional MYX recombinants were constructed: an MGF^- virus (vMYX-GF^-) containing an intact M11L gene and an M11L^- virus (vMYX-M11L^-) containing an intact MGF gene. Infection with vMYX-GF^- resulted in moderated symptoms of myxomatosis, but all clinical stages of the disease were still detectable. In contrast, disruption of M11L alone dramatically reduced the virus virulence, resulting in a nonlethal syndrome whose clinical course was nevertheless distinct from that of vMYX-GF^-ΔM11L. Upon inoculation with vMYX-M11L^-, rabbits developed primary and secondary tumors which were larger and more circumscribed than those of wild-type MYX recipients. Whereas wild-type MYX infection always includes severe, purulent conjunctivitis and rhinitis, vMYX-M11L^- recipients remained healthy and displayed only minimal signs of respiratory distress. By about 30 days after infection, the tumors induced by vMYX-M11L^- had completely regressed and these animals were immune to challenge with wild- type MYX. Histological analysis indicated that tumors induced by vMYX-M11L^- are much more heavily infiltrated with macrophages and heterophils and that the sites of viral replication are more edematous and necrotic than those of wild-type infection, suggesting that the host was able to mount a more vigorous inflammatory response to vMYX-M11L^- infection. Although vMYX-GF^- ΔM11L and vMYX-M11L^- propagated efficiently in vitro in susceptible rabbit and primate fibroblast cell lines, a major defect in the replication of both viruses in primary mixed rabbit spleen cell cultures was noted. We conclude that the M11L gene product is an important virulence determinant for MYX and that the inability of vMYX-GF^-ΔM11L to cause disease was due to the combined disruption of two distinct virulence factors, MGF and M11L.