No abstract

Previously, we described three mengovirus mutants derived from cDNA plasmids, containing shortened poly(C) tracts (C8, C12, and C13UC10), that exhibited strong attenuation for virulence in mice yet grew like wild-type virus in HeLa cells. Thirteen additional mutants hav now been constructed and characterized. Five of these differ only in poly(C) length, including one with a precise deletion of the tract. The other mutants bear deletions into the regions juxtaposing poly(C). Studies with HeLa cells confirm the essential dispensability of mengovirus's poly(C) tract but reveal a subtle, measurable correlation between poly(C) length and plaque diameter. Virulence studies with mice also revealed a strong correlation between poly(C) length and virulence. For the poly(C)-flanking mutations, the 15 bases directly 5' of the tract proved dispensable for virus viability, whereas the 20 to 30 bases 3' of poly(C) were critical for growth, thus implicating this region in the basal replication of the virus.

Subacute sclerosing panencephalitis (SSPE) is a progressive fatal neurodegenerative disease associated with persistent infection of the central nervous system (CNS) by measles virus (MV), biased hypermutations of the viral genome affecting primarily the matrix (M) gene with the conversion of U to C and A to G bases, high titers of antibodies to MV, and infiltration of B cells and T cells into the CNS. Neither the precipitating event nor biology underlying the MV infection is understood, nor is their any satisfactory treatment. We report the creation of a transgenic mouse model that mimics the cardinal features of SSPE. This was achieved by initially infecting mice expressing the MV receptor with lymphocytic choriomeningitis virus Cl 13, a virus that transiently suppressed their immune system. Infection by MV 10 days later resulted in persistent MV infection of neurons. Analysis of brains from infected mice showed the biased U to C hypermutations in the MV M gene and T and B lymphocyte infiltration. These sera contained high titers of antibodies to MV. Thus, a small animal model is now available to both molecularly probe the pathogenesis of SSPE and to test a variety of therapies to treat the disease.

Encephalomyocarditis virus (EMCV) and mengovirus are serotypically related cardioviruses of the picornavirus family. Among the unusual features of their positive-sense singlestranded RNA genomes are long 5Ј untranslated polypyrimidine tracts [poly(C)] with sequences consisting of C 115 UCUC 3 UC 10 and C 44 UC 10 for EMCV (strain R) and mengovirus (strain M), respectively (6, 13). These cardioviruses and their closely related cousins, the foot-and-mouth disease viruses of the Aphthovirus genus, are the only known eukaryotic or prokaryotic genomes to contain such poly(C) tracts, and the specific function of the homopolymer region remains a biological enigma.We have reported the construction of multiple cDNA-derived EMCV and mengovirus strains which differ from each other and from wild-type parental strains only in the lengths of their 5Ј poly(C) tracts (8, 9, 13). The mengovirus panel included nine viruses with poly(C) lengths that ranged from C 44 UC 10 (vMwt) down to a precise deletion of all the cytidine residues (vMC 0 ). The EMCV panel (vEC 20 , vEC 9 , and vEC 4 ) was less extensive, although it contained representative analogues for the best-characterized mengovirus strains. As a definitive phenotype, deletion of the mengovirus poly(C) tract clearly correlates with attenuation of virus virulence in animals. vMC 0 , for example, has a median 50% lethal dose (LD 50 ) of Ͼ2 ϫ 10 9 PFU after intracerebral inoculation of mice. In contrast, the LD 50 of vMwt by equivalent inoculation is only 10 PFU (7). All intermediate-tract mengoviruses show correspondingly diminished virulence. For viruses with poly(C) tracts between 25 and 35 bases, the LD 50 in mice increases about 1 log 10 PFU for every 3 C's that are removed from the tract (13,20). For EMCV strains, the correlation between tract length and murine virulence is weaker, however, and the poly(C) needs to be truncated substantially (i.e., ϽC 9 ) before the attenuation becomes measurable (e.g., the LD 50 of vEC 4 is 3 ϫ 10 3 PFU compared to 1 PFU for EMCV-R) (9). Our EMCV and mengovirus recombinant isolates have been extensively characterized for growth in HeLa cells, and all were found to plaque with equivalent plating efficiencies regardless of the poly(C) tract length. While some strains do exhibit subtle changes in plaque size that correlate with incremental tract deletion (9, 13), none of the isolates show tract-dependent variations in replication kinetics or end-point titers when measured directly in single-step growth experiments. The poly(C) tracts also have no apparent influence on genome translation, virion stability, or growth temperature sensitivity, and it is clear, at least for growth in HeLa cells, that the major vegetative life cycle requirements for EMCV and mengovirus are not strongly vested in this region of the viral RNA.However, in infected animals, especially those receiving mengoviruses, there must be some cellular or tissue determinant that rapidly detects subtle differences in poly(C) genotype and reacts in a manner that clearly means l...

We have shown previously that genetically engineered Mengo viruses with artificial deletions in their 5' noncoding polyribocytidylic acid (poly(C)) tracts are highly attenuated for the natural murine host and also for other animals such as baboons, macaques, and domestic pigs. The present report further characterizes select short poly(C) tract Mengo viruses in the natural murine host. A positive correlation was found between the length of the poly(C) tract and murine virulence, as measured by virus brain titers and brain lesion scores after infection. Histological examination of brain tissue collected from infected animals clearly showed that the short poly(C) tract viruses did not induce the devastating pathological effects characteristic of animals inoculated with wild-type virus. Instead, the short-tract Mengo viruses proved excellent immunological agents. A dose of only 100 plaque-forming units of vMC24 (poly(C) tract: C13UC10), injected subcutaneously, protected 80% of recipient animals against a normally lethal dose of encephalomyocarditis virus. The protection was long-lived, and animals similarly immunized with vMCo virus (poly(C) tract: Co) still had protective neutralizing antibody titers up to 16 months after inoculation. In addition, the short-tract viruses proved genetically stable, in that the vMC24 virus did not yield detectable pathogenic revertants even after multiple, forced passages in 4-week-old mice. These studies suggest that Mengo viruses containing deletions in their poly(C) tracts are biologically safe and potent immunogens and imply that they may have uses as cardiovirus vaccines.