Muller's ratchet is an important concept in population genetics. It predicts that when mutation rates are high and a significant proportion of mutations are deleterious, a kind of irreversible ratchet mechanism will gradually decrease the mean fitness of small populations of asexual organisms. In contrast, sexual recombination may stop or reverse this mutational ratchet by recombinational repair Muller (1) suggested that accumulation of deleterious mutations in asexual (2) organisms occurs by "a kind ofirreversible ratchet mechanism;" in contrast, sexual reproduction allows repair of genetic damage by recombination (2-4). Bell (5) used senescence in protozoa to provide experimental support for Muller's ratchet hypothesis, but some models of Muller's ratchet have been questioned recently (6). Chao (7) recently reported that 40 consecutive plaque-to-plaque (genetic bottleneck) transfers of the tripartite RNA bacteriophage 46 led to a significant decrease in mean fitness. Such clone-to-clone transfers of a virus repeatedly reduce virus population size to one infectious particle and should increase the probability for Muller's ratchet to become manifest if mutation rates are high and sexual recombination is absent. Chao (7) tested fitness of his repeatedly bottlenecked 46 phage clones in paired-growth experiments in which each transferred clone was mixed at a known ratio with a genetically marked parental phage clone. These mixtures were then diluted and plated on lawns of bacterial host cells to form independent plaques during each growth competition transfer. This precluded genetic reassortment of the three genome segments of this tripartite bacteriophage while allowing internally controlled comparisons ofthe relative replication rates of wild-type phage versus repeatedly bottlenecked clones. We have now quantitated the relative fitness of repeatedly bottlenecked clones of vesicular stomatitis virus (VSV) a single-stranded RNA virus of animals, humans, and insect vectors. VSV is the prototype model for negative-stranded animal RNA viruses (which include a large number of medically important human pathogens). Because VSV has only a single, nonsegmented genome, which does not undergo recombination at a detectable level, we are able to quantitate relative fitness directly in mixed-infection competition experiments. We can allow the parental VSV clone and the genetically marked monoclonal antibody (mAb)-resistant (MAR) clones to replicate and compete directly in liquid medium cell cultures (and even in the same cells late during each passage). We previously described quantitative relative fitness assay methods for determining virus fitness during direct replicative competition experiments (8, 9), and these are employed here.RNA viruses generally should be ideal for testing Muller's ratchet theory because most or all of them have extremely high mutation frequencies. RNA viruses apparently lack the mechanisms of proofreading and mismatch repair that are available for high-fidelity DNA replication. Their replic...
We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.
Although vesicular stomatitis virus (VSV) neurovirulence and pathogenicity in rodents have been well studied, little is known about VSV pathogenicity in non-human primates. To address this question, we measured VSV viremia, shedding, and neurovirulence in macaques. Following intranasal inoculation, macaques shed minimal recombinant VSV (rVSV) in nasal washes for 1 day post-inoculation; viremia was not detected. Following intranasal inoculation of macaques, wild type (wt) VSV, rVSV, and two rVSV-HIV vectors showed no evidence of spread to CNS tissues. However, macaques inoculated intrathalamically with wt VSV developed severe neurological disease. One of four macaques receiving rVSV developed clinical and histological signs similar to the wt group, while the remaining three macaques in this group and all of the macaques in the rVSV-HIV vector groups showed no clinical signs of disease and reduced severity of histopathology compared to the wt group. The implications of these findings for rVSV vaccine development are discussed.
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A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their "wild-type" progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD 50 ) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10-to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD 50 of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.Vesicular stomatitis virus (VSV) is a member of the Vesiculovirus genus of the family Rhabdoviridae. The negative-sense virus genome is 11,162 nucleotides long and contains five genes in the order 3Ј N-P-M-G-L 5Ј, encoding the five major viral proteins (1, 3). The bullet-shaped VSV particle (160 nm by 80 nm) contains a ribonucleoprotein core (nucleocapsid) composed of genomic RNA closely associated with N protein and a RNA polymerase composed of a complex of L and P proteins enveloped in a host cell-derived plasma membrane (4,18,19,44,53,56). Following uptake of the virus particle by susceptible cells, nucleocapsid and viral RNA polymerase are released into the cytoplasm and viral mRNA transcription ensues. A 3Ј-5Ј gradient of viral mRNA transcription leads to abundant N protein expression and successively decreasing levels of P, M, G, and L proteins (1,3,15,19,27,57). This gene expression gradient provides virus proteins in a suitable ratio for subsequent viral genome replication and assembly of mature virus particles. Virus replication in cell culture is rapid, and virus progeny are detectable 5 to 6 h postinfection.Since the initial recovery of infectious recombinant VSV (rVSV) from genomic cDNA (39, 61), effort has been directed towards the development of rVSV as a vaccine vector targeting a variety of different human pathogens, including human immu...
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