A hallmark of RNA genomes is the error-prone nature of their replication and retrotranscription. The major biochemical basis of the limited replication fidelity is the absence of proofreading/repair and postreplicative error correction mechanisms that normally operate during replication of cellular DNA. In spite of this unique feature of RNA replicons, the dynamics of viral populations seems to follow the same basic principles that classical population genetics has established for higher organisms. Here we review recent evidence of the profound effects that genetic bottlenecks have in enhancing the deleterious effects of Muller's ratchet during RNA virus evolution. The validity of the Red Queen hypothesis and of the competitive exclusion principle for RNA viruses are viewed as the expected result of the highly variable and adaptable nature of viral quasispecies. Viral fitness, or ability to replicate infectious progeny, can vary a million-fold within short time intervals. Paradoxically, functional and structural studies suggest extreme limitations to virus variation. Adaptability of RNA viruses appears to be based on the occupation of very narrow portions of sequence space at any given time.

The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5 untranslated region (UTR) and IGR and downstream by the S 3 UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase. Lymphocytic choriomeningitis virus (LCMV) is the prototypic member of the family Arenaviridae, which also includes important human pathogens such as Lassa virus and Junin virus.LCMV provides one of the most widely used model systems to study viral persistence and pathogenesis (40,42). However, the investigation of the molecular mechanisms underlying LCMV persistence and its associated disorders has been hampered by the lack of a genetic system to analyze the structure and function of the LCMV genome and its gene products.The LCMV genome consists of two negative-sense singlestranded RNA segments, designated L and S, with approximate sizes of 7.2 and 3.4 kb, respectively (48, 52). Each RNA segment has an ambisense coding strategy, encoding two proteins in opposite orientation, separated by an intergenic region (IGR) (3,4,56). The S RNA directs synthesis of the three major structural proteins: the nucleoprotein, NP (ca. 63 kDa); and two mature virion glycoproteins, GP-1 (40 to 46 kDa) and GP-2 (35 kDa), that are derived by posttranslational cleavage of a precursor polypeptide, GP-C (75 kDa) (47,55,57). Tetramers of GP-1 and GP-2 make up the spikes on the virion envelope. Evidence indicated that GP-1 mediates virus interaction with host cell surface receptor, which has been recently identified as ␣-dystroglycan (7, 11). The L RNA segment encodes a high-molecular-mass protein (L; ca. 200 kDa) which has the characteristic motifs conserved in all the viral RNAdependent RNA polymerases and a small polypeptide Z (ca. 11 kDa) which contains a RING finger motif and whose function is unknown (26, 50, 52).The NP, the most abundant viral protein in virally infected cells, is associated with the viral RNA (vRNA) to form the nucleocapsid (NC) which is the template for the viral RNA polymerase (26). The L protein is thought to be the main viral comp...

The great adaptability shown by RNA viruses is a consequence of their high mutation rates. Here we investigate the kinetics of virus fitness gains during repeated transfers of large virus populations in cell culture. Results always show that fitness increases exponentially. Low fitness clones exhibit regular increases observed as biphasic periods of exponential evolutionary improvement, while neutral clones show monophasic kinetics. These results are significant for RNA virus epidemiology, optimal handling of attenuated live virus vaccines, and routine laboratory procedures.RNA viruses are highly mutable and form complex quasispecies populations as defined by Eigen and colleagues (1-4). Quasispecies or "mutant swarms" of RNA viruses evolve thousands-to millionsfold faster than DNA-based organisms (5-9). This provides an invaluable tool to perform evolutionary studies that, for the latter, would take eons. Evolution of RNA viruses depends upon environmental selective forces and random drift (6,10,11). Examples are human immunodeficiency virus 1 (11), hepatitis C virus (12), and foot-and-mouth disease virus (13), all of which can replicate and evolve rapidly and continuously in infected individuals. Because the behavior of quasispecies populations is important for an understanding of RNA virus disease and epidemiology, quantitative studies of virus populations and population genetics are needed. We have developed a relative fitness assay to enable quantitative analysis of RNA virus population behavior (14). This employs genetically marked mutants that are mixed with wild-type virus (as an internal standard), and these mixed RNA virus quasispecies are allowed to compete during replication in a series of repeated transfers in cell culture. The changing ratios of genetically marked virus to wild-type virus allow determination of relative fitness vectors and relative fitness values (W) per passage. For the wild-type virus employed as the internal control, fitness is assigned a neutral value (W = 1.0) because it is the parental standard virus clone from which all of the genetically marked clones have been derived. The marked clones are monoclonal antibody-resistant mutants (MARMs), and their fitness is measured after replicative competition passages in a constant cell culture environment (14).It was observed (15-18) that whenever selection does not have the opportunity to act, as during repeated genetic bottleneck transfers of a MARM of vesicular stomatitis virus (VSV) or a marked mutant of an RNA bacteriophage, the high mutation rates lead to loss of virus fitness. Genetic bottleneck passages involve repeated transfers of only one or a few virions, and loss of fitness results from gradual stochastic accumulation of deleterious mutations in accord with Muller's ratchet theory (19,20). Muller (19) had predicted that when an asexual population is small and the mutation rate is high, the popu-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby ...

No abstract

No abstract

In this study, we analyzed full-length SARS-CoV-2 genomes from multiple countries to determine early trends in the evolutionary dynamics of the novel COVID-19 pandemic. Results indicated SARS-CoV-2 evolved early into at least three phylogenetic groups, characterized by positive selection at specific residues of the accessory proteins ORF3a and ORF8. Also, we are reporting potential relevant sites under positive selection at specific sites of non-structural proteins nsp6 and helicase. Our analysis of co-evolution showed evidence of epistatic interactions among sites in the genome that may be important in the generation of variants adapted to humans. These observations might impact not only public health but also suggest that more studies are needed to understand the genetic mechanisms that may affect the development of therapeutic and preventive tools, like antivirals and vaccines. Collectively, our results highlight the identification of ongoing selection even in a scenario of conserved sequences collected over the first 3 months of this pandemic.

Most RNA virus populations exhibit extremely high mutation frequencies which generate complex, genetically heterogeneous populations referred to as quasispecies. Previous work has shown that when a large spectrum of the quasispecies is transferred, natural selection operates, leading to elimination of noncompetitive (inferior) genomes and rapid gains in fitness. However, whenever the population is repeatedly reduced to a single virion, variable declines in fitness occur as predicted by the Muller's ratchet hypothesis. Here, we quantitated the fitness of 98 subclones isolated from an RNA virus clonal population. We found a normal distribution around a lower fitness, with the average subclone being less fit than the parental clonal population. This finding demonstrates the phenotypic diversity in RNA virus populations and shows that, as expected, a large fraction of mutations generated during virus replication is deleterious. This clarifies the operation of Muller's ratchet and illustrates why a large number of virions must be transferred for rapid fitness gains to occur. We also found that repeated genetic bottleneck passages can cause irregular stochastic declines in fitness, emphasizing again the phenotypic heterogeneity present in RNA virus populations. Finally, we found that following only 60 h of selection (15 passages in which virus yields were harvested after 4 h), RNA virus populations can undergo a 250% average increase in fitness, even on a host cell type to which they were already well adapted. This is a remarkable ability; in population biology, even a much lower fitness gain (e.g., 1 to 2%) can represent a highly significant reproductive advantage. We discuss the biological implications of these findings for the natural transmission and pathogenesis of RNA viruses.

Genetic bottlenecks are important events in the genetic diversification of organisms and colonization of new ecological niches. Repeated bottlenecking of RNA viruses often leads to fitness losses due to the operation of Muller's ratchet. Herein we use vesicular stomatitis virus to determine the transmission population size which leads to fitness decreases of virus populations. Remarkably, the effective size of a genetic bottleneck associated with fitness loss is greater when the fitness of the parental population increases. For example, for starting virus populations with low fitness, population transfers of five-clone-to-five-clone passages resulted in a fitness increase. However, when a parental population with high fitness was transferred, 30-clone-to-30-clone passages were required simply to maintain fitness values.