key: cord-260554-nao59qx4 authors: Wargo, Andrew R; Kurath, Gael title: Viral fitness: definitions, measurement, and current insights date: 2012-09-15 journal: Curr Opin Virol DOI: 10.1016/j.coviro.2012.07.007 sha: doc_id: 260554 cord_uid: nao59qx4 Viral fitness is an active area of research, with recent work involving an expanded number of human, non-human vertebrate, invertebrate, plant, and bacterial viruses. Many publications deal with RNA viruses associated with major disease emergence events, such as HIV-1, influenza virus, and Dengue virus. Study topics include drug resistance, immune escape, viral emergence, host jumps, mutation effects, quasispecies diversity, and mathematical models of viral fitness. Important recent trends include increasing use of in vivo systems to assess vertebrate virus fitness, and a broadening of research beyond replicative fitness to also investigate transmission fitness and epidemiologic fitness. This is essential for a more integrated understanding of overall viral fitness, with implications for disease management in the future. Fitness is a complex concept at the foundation of all ecology and evolution. For viruses fitness was originally defined as ''the capacity of a virus to produce infectious progeny in a given environment'' [16] . This definition is still in wide use today, referred to more specifically as replicative fitness [15 ] , measured in cultured cells, tissue explants, or within individual hosts. This standard definition is founded in, but does not exactly match, the more general Darwinian definition of overall fitness, which is the amount of genetic material passed on to the next generation. Due to host immune clearance of viruses and finite host lifespan, viruses must be transmitted to new hosts to survive. As such, transmission fitness is an important component of overall fitness. Ultimately, replication and transmission contribute to the prevalence of viral genetic material at a population level in the field over time. The capacity of a virus (a serotype, clade, or variant) to become dominant in the field, relative to other serotypes, clades, or variants of the same virus has been defined as epidemiologic fitness [15 ] . In this review we focus on viral fitness work published from 2009 through early 2012. Literature searches found nearly 750 publications involving viral fitness during this period, and we have focused on a subset of 98 papers selected to represent the current breadth of work in the field. In the interest of brevity these papers are not all cited here, but they were used to discern the general trends described below. The field of viral fitness was originally developed through studies of a relatively small number of bacteriophage, animal, and plant viruses. With increasing recognition of the importance of viral fitness, there is now a wide array of study systems as detailed in Table 1 . The majority of viral fitness study systems are based on RNA viruses, and the highest numbers of publications in recent years involves human pathogens associated with major disease emergence events, such as human immunodeficiency virus-1 (HIV-1), influenza virus, and dengue virus (DENV). During development of the viral fitness field most research has assessed replicative fitness of viral variants within individual hosts (in vivo) or in cultured cells (in vitro) . This trend has continued during recent years, likely due to the fact that replicative fitness is most readily measured in the laboratory. Typically replicative fitness studies compare the replication of two or more virus isolates that are variants of the same viral species. Under the general umbrella of replicative fitness there are many variations that will be described here and in later sections. Replicative fitness is sometimes assessed by comparing viral replication in parallel hosts or cell cultures infected with single viral variants. However, it has long been recognized that assessment of fitness in mixed infections of two viral variants is a more sensitive and valid measure of viral fitness differences [16] . Therefore competitive fitness is often examined in growth competition assays, in which cells or hosts are co-infected with mixtures of two viral variants and the replication of each variant is determined in a competitive environment. Variables in these assays include the use of different input ratios, use of a standard reference strain or head-to-head competitions between test variants, and varied timing for analysis of progeny populations. For example, replicative fitness is sometimes examined at one time point post-infection, but it is often assessed at multiple time points [17, 18, 33, 42, 45, 46, 60] There are a variety of methods used to measure replicative fitness. The traditional method quantifies individual viral variants as plaque forming units (PFU). Recent advances in molecular techniques have led to the use of methodologies that measure viral DNA, RNA, or protein levels, such as quantitative PCR, ELISA, fluorescent probe labeling, and next generation sequencing. These molecular methods estimate viral load, and have the advantage that they typically allow for higher throughput as well as sensitivity, and can be used to distinguish viral genotypes in mixed infections. However, they do not quantify infectious virus as PFU assays do, and the relationships between PFU and molecular quantifications of viral load remain to be defined in many systems. Viral fitness is expressed in various ways, such as comparing viral loads statistically [58 ] , using a fitness parameter equation [1, 48, 53] , or determining the slope of a fitness vector in comparison with a constant reference strain after multiple sampling times or serial passage [37] . In the past fitness data were generally presented as relative fitness values, reported as a fitness ratio between two viral variants. The use of appropriate standards with qRT-PCR quantification has recently made it possible to determine absolute fitness values in terms of average RNA copy numbers per mg of host tissue, for each viral variant [58 ] . A major variable in replicative fitness work is the in vitro, ex vivo, or in vivo nature of the environments used for viral replication (Figure 1 ). For viruses of bacteria, insects, and plants there is a long record of sophisticated in vivo fitness studies using controlled laboratory populations of living hosts, and in vivo work in these systems has remained prolific to date [3 ,6,10,11 ,14,19,25,27,35,36 ,39,41,54] . Viruses of vertebrates have traditionally been studied in cultured cell lines, and this work also continues [1,7,11 ,12,23,24,31,34,37,38,47,49 ,51,61,62] . However, over the last three years there has been an expansion of vertebrate virus fitness studies in vivo using systems such as influenza, DENV, West Nile virus (WNV), Viral fitness Wargo and Kurath 539 Replicative fitness studies typically measure the average fitness of populations of multiple virus particles in populations of in vitro cultured cells or in vivo host tissues. An exciting recent advance has been analysis of the replicative fitness of individual virus particles, defined as the total number of virus progeny produced when one virus infects an individual susceptible cell [62] . Work with vesicular stomatitis virus (VSV) has revealed dramatic variation ranging from 50 to 8000 progeny virus particles per host cell, with additional experiments indicating the host cell cycle stage as a major influence on this variability. Another component of competitive co-infection fitness is superinfection fitness, in which infection with one viral variant is established before exposure to the second variant. Despite the clear relevance of superinfection to natural viral infections in the field, there are few studies of controlled superinfections [60] . In general, viruses isolated from natural superinfections have been analyzed in simultaneous co-infection assays, often indicating that the superinfecting strains have higher fitness [55] . Due to the major role of transmission in overall viral fitness, transmission fitness is a research area that is currently expanding. The majority of work on transmission fitness has been conducted in plant viruses In many cases the ultimate goal of replicative and transmission fitness studies is to understand the epidemiology 540 Virus evolution and population level processes governing viral evolution, emergence, and displacement in the field. As such there has been an increased effort in recent years to quantify epidemiologic fitness. Quantification of epidemiologic fitness is based largely on observational data and examines changes in distribution, prevalence, and composition of viral genotypes over time to infer their relative fitness. This is particularly valuable for systems where a wealth of epidemiological data is available, and the ability to conduct in vivo experimental studies is limited. It is of no surprise therefore that most recent work in this area was conducted in human virus systems such as HIV, In recent years there has been an increased effort to mathematically quantify viral fitness. This includes standard statistical package approaches as well as systemspecific mathematical model development. , and the evolutionary impact of incorporating new traits into the viral genome such as photosynthesis genes in a cyanophage [22] . Parameters in mathematical models are typically defined with in vivo laboratory and field data, and recent growth in these areas has made modeling approaches more tractable. Exciting advances in the field of viral fitness have largely been driven by the rapid expansion in molecular technologies and computing power. For example molecular barcoding microarray technologies have been employed to create quasispecies swarms in the laboratory and simultaneously characterize the fitness of numerous mutants of poliovirus [29] . Next generation sequencing is also being used to rapidly sequence entire viral genomes and determine how genome wide mutation accumulation impacts fitness [37] . Likewise, large scale site directed mutagenesis has recently been used as a tool to determine the molecular interactions that regulate fitness [31] . New bioinformatics tools are being employed to explore large HIV field sequence databases and determine the fitness landscapes [52] , and we have entered the age of purely in silico studies that use advanced agent based mathematical models parameterized from published literature to make inferences about viral evolution [22] . Ultimately these advances have made understanding the genetic regulators of fitness within arms grasp, and not surprisingly, revealed that the story is likely to be more complex than previously assumed [11 ,31]. For vertebrate viruses the recent increase of in vivo virus fitness research is encouraging, but the majority of studies still remain in vitro. This is likely due to the ethical and practical constraints of conducting in vivo research in many vertebrate systems, particularly human viruses. Furthermore in vitro work has numerous advantages, most notably it allows for a higher level of control of variables than in vivo work and is useful for understanding phenomena at a molecular or cellular level [62] . The drawback is that it is unclear how well in vitro results reflect natural phenomena in vivo. The relationship between viral fitness and virulence has been of interest for decades, but surprisingly little has been published recently. Many recent publications assume that viral replicative fitness and virulence are positively correlated and thus use the terms interchangeably. For example, the term 'attenuation' is often used to describe a virus with reduced replicative fitness [46] . In actuality, the relationship between viral fitness and virulence remains poorly characterized in many systems, and both agreement and exception to this assumption have been reported [3 ,5,24,27,40,44,58 ] . Some of the discrepancies in this area may come from the multitude of ways virulence is defined or measured, often beyond the standard definition of morbidity and mortality caused to the host due to infection [3 ,19,25-27,43,50,58 ] . More focus on virulence, and definition of system-specific correlations of virulence with fitness would be a benefit to future work. Viral fitness continues to be an active area of research [7] . Given the increase in issues such as drug resistance evolution, vaccine escape, virulence evolution, viral emergence, and host jumps, the understanding of viral fitness has become essential. For decades viral fitness has been primarily defined by replication capacity in the host. This definition is now broadening as researchers attempt to understand the population level evolutionary implications of overall viral fitness in natural infections. Making inferences about population level processes requires integration of replicative, competitive, transmission, and epidemiologic fitness measures (Figure 1 ). Perhaps the most critical need for the field of viral fitness is the further development of integrative approaches, with the ultimate goals of making accurate predictive inferences and informing long-term management or control of disease. Many the tools to achieve this goal are now available, and we are collectively faced with the task of putting them together. A mathematical framework for estimating pathogen transmission fitness and inoculum size using data from a competitive mixtures animal model. PLoS Computational Biology 2011, 7:11. The authors introduce a mathematical framework for quantitative estimation of the relative transmissibility of two viral types in co-infection in vivo, and the inoculum size associated with transmission events. The model is tested using data from in vivo influenza virus co-infection studies in ferrets, providing one of the first rigorous investigations of transmission fitness for a virus that is directly transmitted between vertebrate hosts (i.e. non-arbovirus). Nguyen AH, Molineux IJ, Springman R, Bull JJ: Multiple genetic pathways to similar fitness limits during viral adaptation to a new host. Evolution 2012, 66:363-374. This study investigates the existence of fitness limits using a bacteriophage of Salmonella being adapted to an E. coli host under varying conditions. Although the nucleotide changes associated with adaptation differed dramatically, four independent lines achieved similar absolute fitness increases, demonstrating a fitness limit that could be attained by multiple genetic pathways. Genomic evolution of vesicular stomatitis virus strains with differences in adaptability Gag determinants of fitness and drug susceptibility in protease inhibitor-resistant human immunodeficiency virus type 1 Abiotic heterogeneity drives parasite local adaptation in coevolving bacteria and phages In vivo fitness correlates with host-specific virulence of Infectious hematopoietic necrosis virus (IHNV) in sockeye salmon and rainbow trout Distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f1 Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus Fitness and virulence of different strains of white spot syndrome virus Escape from human monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus High replication fitness and transmission efficiency of HIV-1 subtype C from India: implications for subtype C predominance Oseltamivir-resistant variants of the 2009 pandemic H1N1 influenza a virus are not attenuated in the guinea pig and ferret transmission models Incongruent fitness landscapes, not tradeoffs, dominate the adaptation of vesicular stomatitis virus to novel host types Mutation T74S in HIV-1 subtype B and C proteases resensitizes them to ritonavir and indinavir and confers fitness advantage The role of evolutionary intermediates in the host adaptation of canine parvovirus The authors explored the mechanism of a documented host switch in canine parvovirus (CPV) by constructing viruses with all possible intermediate genomes and assessing their fitness in feline cells. They show that host adaptation involves complex interactions between mutations and most transition intermediates have lower fitness Fitness-related traits of entomopoxviruses isolated from Adoxophyes honmai (Lepidoptera: Tortricidae) at three localities in Japan Divergent evolution in reverse transcriptase (RT) of HIV-1 group O and M lineages: impact on structure, fitness, and sensitivity to nonnucleoside RT inhibitors Estimating the individualized HIV-1 genetic barrier to resistance using a nelfinavir fitness landscape Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response High-throughput analysis of growth differences among phage strains Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a hostdependent manner in vitro and in vivo In vitro characterization of viral fitness of therapy-resistant hepatitis B variants This is the first study to quantify how viral infection cycle traits correlate with viral fitness and virulence, using a fish rhabdovirus in rainbow trout as an in vivo system. Although within-host replication had the largest impact on fitness, host entry, competitive fitness, and shedding also contributed A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B Macaque long-term nonprogressors resist superinfection with multiple CD8(+) T cell escape variants of simian immunodeficiency virus Decreased infectivity of a neutralization-resistant equine infectious anemia virus variant can be overcome by efficient cell-to-cell spread Growth of an RNA virus in single cells reveals a broad fitness distribution Mixed infections and the competitive fitness of faster-acting genetically modified viruses Papers of particular interest, published within the period of review, have been highlighted as:of special interest of outstanding interest 1. Abraha A, Nankya IL, Gibson R, Demers K, Tebit 92:1930-1938 . This paper provides an example of the sophistication of plant virus fitness work, using two groups of ten plant viral satellite RNAs that differ in fitness and virulence on a tomato host. On a melon host these satellite RNAs differ in multiple measures of viral fitness including replication in single or mixed infections and aphid transmissibility, but they do not differ in virulence, demonstrating both host-specific fitness traits and a lack of correlation between replicative fitness and virulence. da Silva J, Coetzer M, Nedellec R, Pastore C, Mosier DE: Fitness epistasis and constraints on adaptation in a human immunodeficiency virus type 1 protein region. Genetics 2010, 185 293-U430. Fitness epistasis was investigated here by creating seven mutations, singly and in combination, in HIV-1 glycoprotein and testing effects on viral infectivity. Epistatic effects were found to be common, complex, and often very strong, providing insights into the barriers and probable pathways of evolution of co-receptor usage.