Viral evolution and the emergence of SARS coronavirus

The recent appearance of severe acute respiratory syndrome coronavirus (SARS-CoV) highlights the continual threat to human health posed by emerging viruses. However, the central processes in the evolution of emerging viruses are unclear, particularly the selection pressures faced by viruses in new host species. We outline some of the key evolutionary genetic aspects of viral emergence. We emphasize that, although the high mutation rates of RNA viruses provide them with great adaptability and explain why they are the main cause of emerging diseases, their limited genome size means that they are also subject to major evolutionary constraints. Understanding the mechanistic basis of these constraints, particularly the roles played by epistasis and pleiotropy, is likely to be central in explaining why some RNA viruses are more able than others to cross species boundaries. Viral genetic factors have also been implicated in the emergence of SARS-CoV, with the suggestion that this virus is a recombinant between mammalian and avian coronaviruses. We show, however, that the phylogenetic patterns cited as evidence for recombination are more probably caused by a variation in substitution rate among lineages and that recombination is unlikely to explain the appearance of SARS in humans.     

Decreased virus population diversity in p53-null mice infected with weakly oncogenic Abelson virus

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53-/- tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.     

Frequent coinfection of cells explains functional in vivo complementation between cytomegalovirus variants in the multiply infected host

In contrast to many other virus infections, primary cytomegalovirus (CMV) infection does not fully protect against reinfection. Accordingly, clinical data have revealed a coexistence of multiple human CMV variants/strains in individual patients. Notably, the phenomenon of multiple infection was found to correlate with increased virus load and severity of CMV disease. Although of obvious medical relevance, the mechanism underlying this correlation is unknown. A weak immune response in an individual could be responsible for a more severe disease and for multiple infections. Alternatively, synergistic contributions of variants that differ in their biological properties can lead to qualitative changes in viral fitness by direct interactions such as genetic recombination or functional complementation within coinfected host cells. We have addressed this important question paradigmatically with the murine model by differently designed combinations of two viruses employed for experimental coinfection of mice. Specifically, a murine cytomegalovirus (MCMV) mutant expressing Cre recombinase was combined for coinfection with a mutant carrying Cre-inducible green fluorescent protein gene, and attenuated mutants were combined for coinfection with wild-type virus followed by two-color in situ hybridization studies visualizing the replication of the two viruses in infected host organs. These different approaches concurred in the conclusion that coinfection of host cells is more frequent than statistically predicted and that this coinfection alters virus fitness by functional trans-complementation rather than by genetic recombination. The reported findings make a major contribution to our molecular understanding of enhanced CMV pathogenicity in the multiply infected host.     

Differential selection of baculovirus genotypes mediated by different species of host food plant

Recent studies have demonstrated high levels of genotypic and phenotypic variation in populations of parasites, even within individual hosts. Several genetic, immunological and epidemiological mechanisms have been postulated as promoters of such variation, but little empirical work has addressed the role of host ecology. A nucleopolyhedrovirus that attacks larvae of the pine beauty moth, Panolis flammea , exists as a complex mixture of genotypes within individual host larvae. We demonstrate that the food plant species eaten by the host (Scots pine vs. lodgepole pine) differentially affects the pathogenicity and productivity of two virus genotypes originally purified from a single host individual. We hypothesize that such food plant-mediated differential selection will promote genotypic variation between baculovirus populations, and that subsequent remixing of virus genotypes could maintain genotypic variation within individual hosts. Our results provide a tritrophic explanation for the genotypic and phenotypic complexity of host–parasite interactions with complex ecologies.     

Emerging geminivirus problems: A serious threat to crop production

Geminiviruses form the second largest family of plant viruses, the Geminiviridae, represented by four genera: Mastrevirus, Curtovirus, Topocuvirus and Begomovirus. During the last two decades these viruses have emerged as devastating pathogens, particularly in the tropics and subtropics, causing huge economic losses and threatening crop production. Epidemics caused by re‐emerging and newly emerging geminiviruses are becoming frequent even in regions that were earlier free from these viruses. Compared to mastreviruses and curtoviruses, begomoviruses have emerged as more serious problems in a variety of crops, for example, cassava, cotton, grain legumes and vegetables. Major contributory factors for the emergence and spread of new geminivirus diseases are the evolution of variants of the viruses, the appearance of the whitefly ‘B’ biotype and the increase in the vector population. Variability in geminiviruses has arisen through mutations, recombination and pseudorecombination. Genomic recombination in geminiviruses, not only between the variants of the same virus but also between species and even between genera, has resulted in rapid diversification. From the disease point of view, most virulent variants have developed through recombination of viral genomes such as those associated with cassava mosaic, cotton leaf curl, and tomato leaf curl diseases. Heterologous recombinants containing parts of the host genome and/or sequences from satellite‐like molecules associated with monopartite begomoviruses provide unlimited evolutionary opportunities. Human activity has also played an important role in the emergence of serious geminivirus diseases across the globe, like the changes in cropping systems, the introduction of new crops, the movement of infected planting materials and the introduction of host susceptibility genes through the exchange of germplasm.     

Multiple V1/V2 env variants are frequently present during primary infection with human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.     

How RNA viruses exchange their genetic material.

One of the most unusual features of RNA viruses is their enormous genetic variability. Among the different processes contributing to the continuous generation of new viral variants RNA recombination is of special importance. This process has been observed for human, animal, plant and bacterial viruses. The collected data reveal a great susceptibility of RNA viruses to recombination. They also indicate that genetic RNA recombination (especially the nonhomologous one) is a major factor responsible for the emergence of new viral strains or species. Although the formation and accumulation of viral recombinants was observed in numerous RNA viruses, the molecular basis of this phenomenon was studied in only a few viral species. Among them, brome mosaic virus (BMV), a model (+)RNA virus offers the best opportunities to investigate various aspects of genetic RNA recombination in vivo. Unlike any other, the BMV-based system enables homologous and nonhomologous recombination studies at both the protein and RNA levels. As a consequence, BMV is the virus for which the structural requirements for genetic RNA recombination have been most precisely established. Nevertheless, the previously proposed model of genetic recombination in BMV still had one weakness: it could not really explain the role of RNA structure in nonhomologous recombination. Recent discoveries concerning the latter problem give us a chance to fill this gap. That is why in this review we present and thoroughly discuss all results concerning nonhomologous recombination in BMV that have been obtained until now.     

Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.     

Metagenomic analyses of novel viruses and plasmids from a cultured environmental sample of hyperthermophilic neutrophiles

Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double‐stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb), and derive apparently from archaeal viruses HAV1 and HAV2. Genomic DNA was obtained from samples enriched in filamentous and tadpole‐shaped virus‐like particles respectively. They yielded few significant matches in public sequence databases reinforcing, further, the wide diversity of archaeal viruses. Several variants of HAV1 exhibit major genomic alterations, presumed to arise from viral adaptation to different hosts. They include insertions up to 350 bp, deletions up to 1.5 kb, and genes with extensively altered sequences. Some result from recombination events occurring at low complexity direct repeats distributed along the genome. In addition, a 33.8 kb archaeal plasmid pHA1 was characterized, encoding a possible conjugative apparatus, as well as three cryptic plasmids of thermophilic bacterial origin, pHB1 of 2.1 kb and two closely related variants pHB2a and pHB2b, of 5.2 and 4.8 kb respectively. Strategies are considered for assembling genomes of smaller genetic elements from complex environmental samples, and for establishing possible host identities on the basis of sequence similarity to host CRISPR immune systems.     

Genotypic variation among wild isolates of two nuclear polyhedrosis viruses isolated from Spodoptera littoralis

Diseased Spodoptera littoralis larvae were collected from 21 different regions of Israel. Nuclear polyhedrosis viruses were isolated from these larvae, and viral DNAs were compared by restriction endonuclease analysis. Two distinct virus types, represented with approximately equal frequency, were found. Several wild isolates of each virus type exhibited minor restriction pattern differences. Plaque purification of the wild isolates revealed the presence of additional genotypic variants.     

Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination

We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.     

Virus adaptation by manipulation of host's gene expression

Viruses adapt to their hosts by evading defense mechanisms and taking over cellular metabolism for their own benefit. Alterations in cell metabolism as well as side-effects of antiviral responses contribute to symptoms development and virulence. Sometimes, a virus may spill over from its usual host species into a novel one, where usually will fail to successfully infect and further transmit to new host. However, in some cases, the virus transmits and persists after fixing beneficial mutations that allow for a better exploitation of the new host. This situation would represent a case for a new emerging virus. Here we report results from an evolution experiment in which a plant virus was allowed to infect and evolve on a na?ve host. After 17 serial passages, the viral genome has accumulated only five changes, three of which were non-synonymous. An amino acid substitution in the viral VPg protein was responsible for the appearance of symptoms, whereas one substitution in the viral P3 protein the epistatically contributed to exacerbate severity. DNA microarray analyses show that the evolved and ancestral viruses affect the global patterns of host gene expression in radically different ways. A major difference is that genes involved in stress and pathogen response are not activated upon infection with the evolved virus, suggesting that selection has favored viral strategies to escape from host defenses.     

Ecological connectivity shapes quasispecies structure of RNA viruses in an Antarctic lake

RNA viruses exist as complex mixtures of genotypes, known as quasispecies, where the evolution potential resides in the whole community of related genotypes. Quasispecies structure and dynamics have been studied in detail for virus infecting animals and plants but remain unexplored for those infecting micro‐organisms in environmental samples. We report the first metagenomic study of RNA viruses in an Antarctic lake (Lake Limnopolar, Livingston Island). Similar to low‐latitude aquatic environments, this lake harbours an RNA virome dominated by positive single‐strand RNA viruses from the order Picornavirales probably infecting micro‐organisms. Antarctic picorna‐like virus 1 (APLV1), one of the most abundant viruses in the lake, does not incorporate any mutation in the consensus sequence from 2006 to 2010 and shows stable quasispecies with low‐complexity indexes. By contrast, APLV2‐APLV3 are detected in the lake water exclusively in summer samples and are major constituents of surrounding cyanobacterial mats. Their quasispecies exhibit low complexity in cyanobacterial mat, but their run‐off‐mediated transfer to the lake results in a remarkable increase of complexity that may reflect the convergence of different viral quasispecies from the catchment area or replication in a more diverse host community. This is the first example of viral quasispecies from natural aquatic ecosystems and points to ecological connectivity as a modulating factor of quasispecies complexity.     

Multiple infection dynamics has pronounced effects on the fitness of RNA viruses

Several factors play a role during the replication and transmission of RNA viruses. First, as a consequence of their enormous mutation rate, complex mixtures of genomes are generated immediately after infection of a new host. Secondly, differences in growth and competition rates drive the selection of certain genetic variants within an infected host. Thirdly, but not less important, a random sampling occurs at the moment of viral infectious passage from an infected to a healthy host. In addition, the availability of hosts also influences the fate of a given viral genotype. When new hosts are scarce, different viral genotypes might infect the same host, adding an extra complexity to the competition among genetic variants. We have employed a two‐fold approach to analyse the role played by each of these factors in the evolution of RNA viruses. First, we have derived a model that takes into account all the preceding factors. This model employs the classic Lotka‐Volterra competition equations but it also incorporates the effect of mutation during RNA replication, the effect of the stochastic sampling at the moment of infectious passage among hosts and, the effect of the type of infection (single, coinfection or superinfection). Secondly, the predictions of the model have been tested in an in vitro evolution experiment. Both theoretical and experimental results show that in infection passages with coinfection viral fitness increased more than in single infections. In contrast, infection passages with superinfection did not differ from the single infection. The coinfection frequency also affected the outcome: the larger the proportion of viruses coinfecting a host, the larger increase in fitness observed.     

Virus reactivation: a panoramic view in human infections

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is 'quiescent' (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi's sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus.     

Mutability Dynamics of an Emergent Single Stranded DNA Virus in a Na?ve Host

Quasispecies variants and recombination were studied longitudinally in an emergent outbreak of beak and feather disease virus (BFDV) infection in the orange-bellied parrot (Neophema chrysogaster). Detailed health monitoring and the small population size (<300 individuals) of this critically endangered bird provided an opportunity to longitudinally track viral replication and mutation events occurring in a circular, single-stranded DNA virus over a period of four years within a novel bottleneck population. Optimized PCR was used with different combinations of primers, primer walking, direct amplicon sequencing and sequencing of cloned amplicons to analyze BFDV genome variants. Analysis of complete viral genomes (n = 16) and Rep gene sequences (n = 35) revealed that the outbreak was associated with mutations in functionally important regions of the normally conserved Rep gene and immunogenic capsid (Cap) gene with a high evolutionary rate (3.41×10⁻³ subs/site/year) approaching that for RNA viruses; simultaneously we observed significant evidence of recombination hotspots between two distinct progenitor genotypes within orange-bellied parrots indicating early cross-transmission of BFDV in the population. Multiple quasispecies variants were also demonstrated with at least 13 genotypic variants identified in four different individual birds, with one containing up to seven genetic variants. Preferential PCR amplification of variants was also detected. Our findings suggest that the high degree of genetic variation within the BFDV species as a whole is reflected in evolutionary dynamics within individually infected birds as quasispecies variation, particularly when BFDV jumps from one host species to another.     

Insect transmission of plant viruses: a constraint on virus variability

Genetic diversity in viruses is shaped by high rates of recombination and is constrained by host defenses and the requirements of transmission. Recent studies of insect-transmitted plant viruses demonstrate highly conserved molecular motifs in viral genomes that regulate the specificity of insect transmission. In contrast, advances in our understanding of host plant response to virus infection reveal some generalized patterns of host defense to a diversity of viruses.     

Why do RNA viruses recombine?

Recombination occurs in many RNA viruses and can be of major evolutionary significance. However, rates of recombination vary dramatically among RNA viruses, which can range from clonal to highly recombinogenic. Here, we review the factors that might explain this variation in recombination frequency and show that there is little evidence that recombination is favoured by natural selection to create advantageous genotypes or purge deleterious mutations, as predicted if recombination functions as a form of sexual reproduction. Rather, recombination rates seemingly reflect larger-scale patterns of viral genome organization, such that recombination may be a mechanistic by-product of the evolutionary pressures acting on other aspects of virus biology.