DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice.

We have determined the complete nucleotide sequence of the DNA of the immunosuppressive variant of the parvovirus minute virus of mice (MVMi) and compared it to the published sequence (12) of the fibroblast-specific strain (MVMp). We have found 175 differences between the two viruses, most of which affect single nucleotides. Despite these differences, the genomic organization of MVMp and MVMi is identical. There are 29 amino-acid changes between the putative viral gene products of MVMi and MVMp, 16 of which are conservative. We discuss the possibility that the differential tissue-specificity of the two variants is linked to differences within the non-transcribed region near the 5' end of the viral genomes.     

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment.

This paper reports the establishment of a model for hantavirus host adaptation. Wild-type (wt) (bank vole-passaged) and Vero E6 cell-cultured variants of Puumala virus strain Kazan were analyzed for their virologic and genetic properties. The wt variant was well adapted for reproduction in bank voles but not in cell culture, while the Vero E6 strains replicated to much higher efficiency in cell culture but did not reproducibly infect bank voles. Comparison of the consensus sequences of the respective viral genomes revealed no differences in the coding region of the S gene. However, the noncoding regions of the S gene were found to be different at positions 26 and 1577. In one additional and independent adaptation experiment, all analyzed cDNA clones from the Vero E6-adapted variant were found to carry substitutions at position 1580 of the S segment, just 3 nucleotides downstream of the mutation observed in the first adaptation. No differences were found in the consensus sequences of the entire M segments from the wt and the Vero E6-adapted variants. The results indicated different impacts of the S and the M genomic segments for the adaptation process and selective advantages for the variants that carried altered noncoding sequences of the S segment. We conclude that the isolation in cell culture resulted in a phenotypically and genotypically altered hantavirus.     

Route of simian immunodeficiency virus inoculation determines the complexity but not the identity of viral variant populations that infect rhesus macaques

A better understanding of the host and viral factors associated with human immunodeficiency virus (HIV) transmission is essential to developing effective strategies to curb the global HIV epidemic. Here we used the rhesus macaque-simian immunodeficiency virus (SIV) animal model of HIV infection to study the range of viral genotypes that are transmitted by different routes of inoculation and by different types of viral inocula. Analysis of transmitted variants was undertaken in outbred rhesus macaques inoculated intravenously (IV) or intravaginally (IVAG) with a genetically heterogeneous SIVmac251 stock derived from a well-characterized rhesus macaque viral isolate. In addition, we performed serial IV and IVAG passage experiments using plasma from SIV-infected macaques as the inoculum. We analyzed the V1-V2 region of the SIV envelope gene from virion-associated RNA in plasma from infected animals by the heteroduplex mobility assay (HMA) and by DNA sequence analysis. We found that a more diverse population of SIV genetic variants was present in the earliest virus-positive plasma samples from all five IV SIVmac251-inoculated monkeys and from two of five IVAG SIVmac251-inoculated monkeys. In contrast, we found a relatively homogeneous population of SIV envelope variants in three of five monkeys inoculated IVAG with SIVmac251 stock and in two monkeys infected after IVAG inoculation with plasma from an SIV-infected animal. In some IVAG-inoculated animals, the transmitted SIV variant was the most common variant in the inoculum. However, a specific viral variant in the SIVmac251 stock was not consistently transmitted by IVAG inoculation. Thus, it is likely that host factors or stochastic processes determine the specific viral variants that infect an animal after IVAG SIV exposure. In addition, our results clearly demonstrate that the route of inoculation is associated with the extent and breadth of the genetic complexity of the viral variant population in the earliest stages of systemic infection.     

Rare and common regulatory variation in population-scale sequenced human genomes

Population-scale genome sequencing allows the characterization of functional effects of a broad spectrum of genetic variants underlying human phenotypic variation. Here, we investigate the influence of rare and common genetic variants on gene expression patterns, using variants identified from sequencing data from the 1000 genomes project in an African and European population sample and gene expression data from lymphoblastoid cell lines. We detect comparable numbers of expression quantitative trait loci (eQTLs) when compared to genotypes obtained from HapMap 3, but as many as 80% of the top expression quantitative trait variants (eQTVs) discovered from 1000 genomes data are novel. The properties of the newly discovered variants suggest that mapping common causal regulatory variants is challenging even with full resequencing data; however, we observe significant enrichment of regulatory effects in splice-site and nonsense variants. Using RNA sequencing data, we show that 46.2% of nonsynonymous variants are differentially expressed in at least one individual in our sample, creating widespread potential for interactions between functional protein-coding and regulatory variants. We also use allele-specific expression to identify putative rare causal regulatory variants. Furthermore, we demonstrate that outlier expression values can be due to rare variant effects, and we approximate the number of such effects harboured in an individual by effect size. Our results demonstrate that integration of genomic and RNA sequencing analyses allows for the joint assessment of genome sequence and genome function.     

Mapping of the fibrotropic and lymphotropic host range determinants of the parvovirus minute virus of mice.

The fibrotropic and lymphotropic strains of minute virus of mice are each unable to grow lytically in the differentiated host cell type of the other strain. To map the viral sequence responsible for the target cell specificities of the two strains, we constructed chimeric viral genomes in vitro from infectious genomic clones. The phenotypes of viral progeny derived from the chimeric genomes were tested by transfecting the plasmids into fibroblast monolayers and assaying plaque formation and by testing stocks of the recombinant viruses for cytotoxicity in fibroblast and lymphocyte cultures. Both the fibrotropic and lymphotropic determinants mapped to the same 237-nucleotide sequence within the coding region of the virus structural gene. A second sequence, near the viral promoter at map unit 38, was also shown to affect viral growth in fibroblast host cells profoundly.     

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies.

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.     

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.     

Hierarchical spatial structure of genetically variable nucleopolyhedroviruses infecting cyclic populations of western tent caterpillars

The cyclic population dynamics of western tent caterpillars, Malacosoma californicum pluviale, are associated with epizootics of a nucleopolyhedrovirus, McplNPV. Given the dynamic fluctuations in host abundance and levels of viral infection, host resistance and virus virulence might be expected to change during different phases of the cycle. As a first step in determining if McplNPV virulence and population structure change with host density, we used restriction fragment length polymorphism (RFLP) analysis to examine the genetic diversity of McplNPV infecting western tent caterpillar populations at different spatial scales. Thirteen dominant genetic variants were identified in 39 virus isolates (individual larvae) collected from field populations during one year of low host density, and another distinct variant was discovered among nine additional isolates in two subsequent years of declining host density. The distribution of these genetic variants was not random and indicated that the McplNPV population was structured at several spatial levels. A high proportion of the variation could be explained by family grouping, which suggested that isolates collected within a family were more likely to be the same than isolates compared among populations. Additionally, virus variants from within populations (sites) were more likely to be the same than isolates collected from tent caterpillar populations on different islands. This may indicate that there is limited mixing of virus among tent caterpillar families and populations when host population density is low. Thus there is potential for the virus to become locally adapted to western tent caterpillar populations in different sites. However, no dominant genotype was observed at any site. Whether and how selection acts on the genetically diverse nucleopolyhedrovirus populations as host density changes will be investigated over the next cycle of tent caterpillar populations.     

Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice

Viral and cellular factors responsible for parvovirus target cell specificity have been examined for two serologically indistinguishable strains of the minute virus of mice which infect mouse cells of dissimilar differentiated phenotype. Both the prototype strain and the immunosuppressive strain grow in and form plaques on monolayers of simian virus 40-transformed human fibroblasts, a finding that has allowed the comparison of several aspects of their virus-host cell interactions. Although closely related by antigenic and genomic criteria, both the prototype strain and the immunosuppressive strain are restricted for lytic growth in each other's murine host cell, that is, in T cells and fibroblasts, respectively. The host range of each virus variant appears to be specified by a genetic determinant that is stably inherited in the absence of selection. In the restrictive virus-host interaction lytic growth is limited to a small or, in some cases, undetectable subset of the host cell population, and the majority of the infected cells remain viable, continuing to grow at the normal rate without expressing viral antigens. The susceptible host cell phenotype is dominant in T lymphocyte x fibroblast fusion hybrids, implying that different cell types express different developmentally regulated virus helper functions that can only be exploited by the virus variant that carries the appropriate strain-specific determinant.     

Dynamic interactions between RNA viruses and human hosts unravelled by a decade of next generation sequencing

Background

Next generation sequencing (NGS) methods have significantly contributed to a paradigm shift in genomic research for nearly a decade now. These methods have been useful in studying the dynamic interactions between RNA viruses and human hosts.

Emergence of simian virus 40 variants during serial passage of plaque isolates

Three serial passage series of simian virus 40 (SV40) in CV-1 cells were initiated by infection directly from the same wild-type plaque isolate, three series were initiated by infection with another plaque isolate, and two series were initiated with each of two other plaque isolates. Aberrant SV40 genomes were not detected in any of the passage series until after the fifty undiluted passage, and each series generated a different array of variant genomes. The results show that the variants were not present in the original plaque isolates but, instead, were randomly generated during subsequent high-input multiplicity passages. Although many of the aberrant viral genomes in each passage series contained reiterations of the SV40 origin of replication and some also contained host cell sequences, there was no indication that SV40 is predisposed toward generating any particular variant.     

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

SV40 recombinants carrying a d(CT.GA)22 sequence show increased genomic instability.

Repetitive d(CT.GA)n sequences are commonly found in eukaryotic genomic DNA. They are frequently located in sites involved in genetic recombination or in promoter regions. To test for their possible biological function, a d(CT.GA)22 synthetic sequence was introduced into the genome of SV40, since it constitutes an appropriate model system for eukaryotic chromatin. When SV40 infects permissive cells, it proliferates in the form of a minichromosome. The simple repetitive sequence indicated above was inserted at the unique HpaII site of SV40 (at nt 346), and the genomic stability of SV40 recombinants carrying the d(CT.GA)22 sequence (SV/CT22 viruses) was analyzed. Upon serial passage through permissive CV1 cells, SV/CT22 recombinants show an increased production of defective viruses. Generation of SV/CT22 variants is likely to take place via recombination between and within viral molecules. The enhancement of the rate of recombination induced by the repetitive sequence is likely to be related to its known propensity to form triple-stranded structures. Many different variants coexist in the same viral population indicating that the mechanism by which they are produced is not unique. One variant (SV/X), showing a replicative advantage, was characterized in detail. Variant SV/X accounts for a large proportion of the total viral population. Its genomic organization corresponds to a tandem duplication of an early SV40 DNA fragment spanning from approx. nt 3200-nt 160. Variant SV/X contains a duplicated SV40 ori.     

Genomic Evolution of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Isolates Revealed by Deep Sequencing

Most studies on PRRSV evolution have been limited to a particular region of the viral genome. A thorough genome-wide understanding of the impact of different mechanisms on shaping PRRSV genetic diversity is still lacking. To this end, deep sequencing was used to obtain genomic sequences of a diverse set of 16 isolates from a region of Hong Kong with a complex PRRSV epidemiological record. Genome assemblies and phylogenetic typing indicated the co-circulation of strains of both genotypes (type 1and type 2) with varying Nsp2 deletion patterns and distinct evolutionary lineages (“High Fever”-like and local endemic type). Recombination analyses revealed genomic breakpoints in structural and non-structural regions of genomes of both genotypes with evidence of many recombination events originating from common ancestors. Additionally, the high fold of coverage per nucleotide allowed the characterization of minor variants arising from the quasispecies of each strain. Overall, 0.56–2.83% of sites were found to be polymorphic with respect to cognate consensus genomes. The distribution of minor variants across each genome was not uniform indicating the influence of selective forces. Proportion of variants capable of causing an amino acid change in their respective codons ranged between 25–67% with many predicted to be non-deleterious. Low frequency deletion variants were also detected providing one possible mechanism for their sudden emergence as cited in previous reports.     

Fitness increase of memory genomes in a viral quasispecies

Viral quasispecies may contain a subset of minority genomes that reflect those genomic sequences that were dominant at an early phase of quasispecies evolution. Such minority genomes are referred to as memory in viral quasispecies. A memory marker previously characterized in foot-and-mouth disease virus (FMDV) is an internal oligoadenylate tract of variable length that became dominant upon serial plaque-to-plaque transfers of FMDV clones. During large population passages, genomes with internal oligoadenylate were outcompeted by wild-type revertants but remained in the mutant spectra as memory genomes. Here, we report a quantification of relative fitness of several FMDV clones, harboring internal oligoadenylate tracts of different length, and that were retrieved at early or late times (passage number) after implementation of memory. The results show that for any given length range of the oligoadenylate, maintenance in memory resulted in an increase in relative fitness, comparable to the increase undergone by the entire population. The fitness increase is in agreement with the Red Queen hypothesis, and implies a replicative memory mechanism. Thus, permanence of memory genomes may be a source of high fitness variants despite their initial low fitness, and despite having remained hidden in mutant spectra. This reinforces the interest of diagnosing minority genomes during chronic human and animal viral infections.     

Rapid in vivo exploration of a 5S rRNA neutral network

A partial knockout compensation method to screen 5S ribosomal RNA sequence variants in vivo is described. The system utilizes an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction with a plasmid which is compensatory when carrying a functionally active 5S rRNA. The partial knockout strain is transformed with a population of potentially compensatory plasmids each carrying a randomly generated 5S rRNA gene variant. a The ability to compensate the slow growth rate of the knockout strain is used in conjunction with sequencing to rapidly identify variant 5S rRNAs that are functional as well as those that likely are not. The assay is validated by showing that the growth rate of 15 variants separately expressed in the partial knockout strain can be accurately correlated with in vivo assessments of the potential validity of the same variants. A region of 5S rRNA was mutagenized with this approach and nine novel variants were recovered and characterized. Unlike a complete knockout system, the method allows recovery of both deleterious and functional variants.. The method can be used to study variants of any 5S rRNA in the E. coli context including those of E. coli.     

‘Nothing is permanent but change’† – antigenic variation in persistent bacterial pathogens

Pathogens persist in immunocompetent mammalian hosts using various strategies, including evasion of immune effectors by antigenic variation. Among highly antigenically variant bacteria, gene conversion is used to generate novel expressed variants from otherwise silent donor sequences. Recombination using oligonucleotide segments from multiple donors is a combinatorial mechanism that tremendously expands the variant repertoire, allowing thousands of variants to be generated from a relatively small donor pool. Three bacterial pathogens, each encoded by a small genome (< 1.2 Mb), illustrate this variant generating capacity and its role in persistent infection. Borrelia burgdorferi VlsE diversity is encoded and expressed on a linear plasmid required for persistence and recent experiments have demonstrated that VlsE recombination is necessary for persistence in the immunocompetent host. In contrast, both Treponema pallidum TprK and Anaplasma marginale Msp2 expression sites and donors are chromosomally encoded. Both T. pallidum and A. marginale generate antigenic variants in vivo in individual hosts and studies at the population level reveal marked strain diversity in the variant repertoire that may underlie pathogen strain structure and the capacity for re‐infection and heterologous strain superinfection. Here, we review gene conversion in bacterial antigenic variation and discuss the short‐ and long‐term selective pressures that shape the variant repertoire.     

Functional splice sites in a zebrafish LINE and their influence on zebrafish gene expression

Long interspersed elements (LINEs) are transposable elements that exist in many kinds of eukaryotic genomes, where they have a large effect on genome evolution. There are several thousands to hundreds of thousands of LINE copies in each eukaryotic genome. LINE elements are amplified by a mechanism called retrotransposition, in which a LINE-encoded protein reverse transcribes (copies) its own RNA. We previously isolated two retrotransposition-competent LINEs, ZfL2-1 and ZfL2-2, from zebrafish. Although it has generally been thought that LINEs do not have ‘introns’ (because the LINE RNA is used as the template during retrotransposition), we now show that these two LINEs contain multiple putative functional splice sites. We further show that at least one pair of these splice sites is actually functional in zebrafish cells. Moreover, some of these splice sites are coupled with the splicing signal of a host endogenous gene, thereby generating a new chimeric spliced mRNA variant for this gene. Our results suggest the possible role of these LINE splice sites in modulating retrotransposition and host gene expression.     

Structure of a hemoglobin gene cluster and nucleotide sequence of three hemoglobin genes from the midge Chironomus thummi piger (Diptera, Insecta)

The aquatic larvae of the genus Chironomus (Diptera, Insecta) contain at least 12 different hemoglobin (Hb) variants in their hemolymph. In the present study we have analysed the structure and part of the nucleotide sequence of a Hb gene cluster cloned from the genomic DNA of Chironomus thummi piger. The cluster contains probably 6 different genes, separated by intergenic regions of various lengths. The nucleotide sequence of three putative Hb genes including the intergenic regions is presented. The inferred amino-acid sequences show clearly that two of these putative genes code for subvariants of the Hb variant VIIB. The third gene codes for a so far unknown Hb protein. As known already for other chironomid Hb genes, there are no intron sequences present in the coding regions.     

Genomics of Divergence along a Continuum of Parapatric Population Differentiation

The patterns of genomic divergence during ecological speciation are shaped by a combination of evolutionary forces. Processes such as genetic drift, local reduction of gene flow around genes causing reproductive isolation, hitchhiking around selected variants, variation in recombination and mutation rates are all factors that can contribute to the heterogeneity of genomic divergence. On the basis of 60 fully sequenced three-spined stickleback genomes, we explore these different mechanisms explaining the heterogeneity of genomic divergence across five parapatric lake and river population pairs varying in their degree of genetic differentiation. We find that divergent regions of the genome are mostly specific for each population pair, while their size and abundance are not correlated with the extent of genome-wide population differentiation. In each pair-wise comparison, an analysis of allele frequency spectra reveals that 25–55% of the divergent regions are consistent with a local restriction of gene flow. Another large proportion of divergent regions (38–75%) appears to be mainly shaped by hitchhiking effects around positively selected variants. We provide empirical evidence that alternative mechanisms determining the evolution of genomic patterns of divergence are not mutually exclusive, but rather act in concert to shape the genome during population differentiation, a first necessary step towards ecological speciation.