Truncation of gene F5L partially masks rescue of vaccinia virus strain MVA growth on mammalian cells by restricting plaque size

Modified vaccinia virus Ankara (MVA) is a candidate vaccine vector that is severely attenuated due to mutations acquired during several hundred rounds of serial passage in vitro. A previous study used marker rescue to produce a set of MVA recombinants with improved replication on mammalian cells. Here, we extended the characterization of these rescued MVA strains and identified vaccinia virus (VACV) gene F5L as a determinant of plaque morphology but not replication in vitro. F5 joins a growing group of VACV proteins that influence plaque formation more strongly than virus replication and which are disrupted in MVA. These defective genes in MVA confound the interpretation of marker rescue experiments designed to map mutations responsible for the attenuation of this important VACV strain.

Murine Gammaherpesvirus 68 LANA Is Essential for Virus Reactivation from Splenocytes but Not Long-Term Carriage of Viral Genome

ABSTRACT ORF73, which encodes the latency-associated nuclear antigen (LANA), is a conserved gamma-2-herpesvirus gene. The murine gammaherpesvirus 68 (MHV68) LANA (mLANA) is critical for efficient virus replication and the establishment of latent infection following intranasal inoculation. To test whether the initial host immune response limits the capacity of mLANA-null virus to traffic to and establish latency in the spleen, we infected type I interferon receptor knockout (IFN-α/βR−/−) mice via intranasal inoculation and observed the presence of viral genome-positive splenocytes at day 18 postinfection at approximately 10-fold-lower levels than in the genetically repaired marker rescue-infected mice. However, no mLANA-null virus reactivation from infected IFN-α/βR−/− splenocytes was observed. To more thoroughly define a role of mLANA in MHV68 infection, we evaluated the capacity of an mLANA-null virus to establish and maintain infection apart from restriction in the lungs of immunocompetent mice. At day 18 following intraperitoneal infection of C57BL/6 mice, the mLANA-null virus was able to establish a chronic infection in the spleen albeit at a 5-fold-reduced level. However, as in IFN-α/βR−/− mice, little or no virus reactivation could be detected from mLANA-null virus-infected splenocytes upon explant. An examination of peritoneal exudate cells (PECs) following intraperitoneal inoculation revealed nearly equivalent frequencies of PECs harboring the mLANA-null virus relative to the marker rescue virus. Furthermore, although significantly compromised, mLANA-null virus reactivation from PECs was detected upon explant. Notably, at later times postinfection, the frequency of mLANA-null genome-positive splenocytes was indistinguishable from that of marker rescue virus-infected animals. Analyses of viral genome-positive splenocytes revealed the absence of viral episomes in mLANA-null infected mice, suggesting that the viral genome is integrated or maintained in a linear state. Thus, these data provide the first evidence that a LANA homolog is directly involved in the formation and/or maintenance of an extrachromosomal viral episome in vivo, which is likely required for the reactivation of MHV68.