Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function^1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution^4-7.     

RNA Viral Metagenome of Whiteflies Leads to the Discovery and Characterization of a Whitefly-Transmitted Carlavirus in North America

Whiteflies from the Bemisia tabaci species complex have the ability to transmit a large number of plant viruses and are some of the most detrimental pests in agriculture. Although whiteflies are known to transmit both DNA and RNA viruses, most of the diversity has been recorded for the former, specifically for the Begomovirus genus. This study investigated the total diversity of DNA and RNA viruses found in whiteflies collected from a single site in Florida to evaluate if there are additional, previously undetected viral types within the B. tabaci vector. Metagenomic analysis of viral DNA extracted from the whiteflies only resulted in the detection of begomoviruses. In contrast, whiteflies contained sequences similar to RNA viruses from divergent groups, with a diversity that extends beyond currently described viruses. The metagenomic analysis of whiteflies also led to the first report of a whitefly-transmitted RNA virus similar to Cowpea mild mottle virus (CpMMV Florida) (genus Carlavirus) in North America. Further investigation resulted in the detection of CpMMV Florida in native and cultivated plants growing near the original field site of whitefly collection and determination of its experimental host range. Analysis of complete CpMMV Florida genomes recovered from whiteflies and plants suggests that the current classification criteria for carlaviruses need to be reevaluated. Overall, metagenomic analysis supports that DNA plant viruses carried by B. tabaci are dominated by begomoviruses, whereas significantly less is known about RNA viruses present in this damaging insect vector.     

Rat C-Type Virus induced in Rat Sarcoma Cells by 5-Bromodeoxyuridine

HALOGENATED derivatives of uridine are highly effective inducers of latent C-type RNA viruses^1,2 and have been successfully used to induce viruses identical to, or similar to, the C-type RNA tumour viruses in mouse, rat and human cells^3–6. In previous experiments we used 5-bromodeoxyuridine (BrUdR) for induction of focus-forming virus in non-productive rat cells that have been transformed by mouse sarcoma virus². We describe here the induction of a C-type RNA virus in the cells of the rat tumour cell line XC, which contains the Rous sarcoma virus genome⁷. The induced virus possesses the group specific (gs) antigens of rat C-type viruses but not those of chicken C-type viruses.     

The Evolutionary and Epidemiological Dynamics of the Paramyxoviridae

Paramyxoviruses are responsible for considerable disease burden in human and wildlife populations: measles and mumps continue to affect the health of children worldwide, while canine distemper virus causes serious morbidity and mortality in a wide range of mammalian species. Although these viruses have been studied extensively at both the epidemiological and the phylogenetic scales, little has been done to integrate these two types of data. Using a Bayesian coalescent approach, we infer the evolutionary and epidemiological dynamics of measles, mumps and canine distemper viruses. Our analysis yielded data on viral substitution rates, the time to common ancestry, and elements of their demographic history. Estimates of rates of evolutionary change were similar to those observed in other RNA viruses, ranging from 6.585 to 11.350 × 10⁻⁴ nucleotide substitutions per site, per year. Strikingly, the mean Time to the Most Recent Common Ancestor (TMRCA) was both similar and very recent among the viruses studied, ranging from only 58 to 91 years (1908 to 1943). Worldwide, the paramyxoviruses studied here have maintained a relatively constant level of genetic diversity. However, detailed heterchronous samples illustrate more complex dynamics in some epidemic populations, and the relatively low levels of genetic diversity (population size) in all three viruses is likely to reflect the population bottlenecks that follow recurrent outbreaks.     

Separation of Single-stranded DNA,Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth^1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes ³. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes ^4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates ¹⁰. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community’s genetic signature. Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s¹¹. By exploiting the charge interaction between the positively-charged Ca²⁺ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing ¹². Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.Download video file.^{(86M, mov)}     

Nature of Ribonuclease-resistant Nucleic Acid of Chick Embryo Cells transformed by Schmidt–Ruppin Strain of Rous Sarcoma Virus

THE mode of replication of RNA or RNA-containing tumour viruses is not understood. The recent studies on Rous sarcoma and other RNA-containing oncogenic viruses suggest that the replicative cycle of the RNA of these viruses might not be associated with ribonuclease-resistant structures (double stranded RNAs), but might involve the synthesis of a DNA intermediate specific to viral RNA^1–3. Two groups of workers, however, presented evidence for the presence of a double stranded RNA in 78 Al cell line of rat embryo fibroblasts which had been transformed and chronically infected with the murine sarcoma-leukaemia virus complex (MSV-MLV)^4,5 and it was suggested that the mode of replication of oncogenic viral RNAs was the same as that of non-oncogenic viral RNAs⁴. This apparent discrepancy prompted me to look for ribonuclease-resistant RNA structures in the chick embryo cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV).     

RNA viruses in the sea

Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.     

Caprine Arthritis-Encephalitis Virus Is Distinct from Visna and Progressive Pneumonia Viruses as Measured by Genome Sequence Homology

Competitive inhibition of hybridization between ¹²⁵I-labeled caprine arthritis-encephalitis viral RNA and homologous cDNA by heterologous viral RNA shows that the caprine retrovirus shares <20% genome sequence homology with visna and progressive pneumonia viruses. These viruses, however, are indistinguishable in immunodiffusion reactions involving the major structural protein (p28).     

RNA and protein components of maize streak and cassava latent viruses

Polyacrylamide gel electrophoresis indicated that maize streak (MSV) and cassava latent (CLV) viruses each contain one species of protein and two of RNA. The estimated protein mol. wt is 28000 for MSV and 34000 for CLV. The mol. wts obtained for the two RNA species using formamide-containing gels were the same for the two viruses: 17×10⁸ and 1–3 × 10⁶. It is suggested that the viruses have a two-part genome and that the tendency of their nucleoprotein particles to form pairs favours the delivery of complete genomes to sites of infection.     

DNA Polymerase Activity associated with Purified Kilham Rat Virus

RNA tumour viruses contain an enzyme which can transcribe DNA from an RNA template^1,2, an endonuclease and a DNA-dependent DNA polymerase activity^3,4. RNA polymerase has been reported in vaccinia virus^5,6, reovirus^7,8 and cytoplasmic polyhidrosis virus⁹. I wish to describe a DNA polymerase activity associated with a highly purified preparation of the parvovirus, Kilham rat virus (KRV), which is thus the first report of a DNA polymerase associated with a DNA virus. KRV, a small virus first isolated from a rat sarcoma¹⁰, is antigenically related to the H viruses isolated from human transplantable tumours¹¹. Those parvoviruses which have been characterized all contain single stranded DNA with molecular weights of 1.5 to 2.5 × 10⁶ (refs. 12,13 and 14).     

Growth of Pseudotypes of Vesicular Stomatitis Virus with N-Tropic Murine Leukemia Virus Coats in Cells Resistant to N-Tropic Viruses

Formation of pseudotypes between murine RNA tumor viruses and vesicular stomatitis virus (VSV) has been confirmed. Pseudotypes of VSV genomes coated by the surface envelope from an N-tropic tumor virus grew equally well in cells homozygous for either the Fv-1ⁿ or Fv-1^b alleles. Therefore, the product of the Fv-1 locus, which restricts growth of murine RNA tumor viruses, must act on an intracellular aspect of tumor virus replication, a step after attachment and penetration.     

Nucleic acid of flacherie viruses of the silkworm, Bombyx mori

It was reported previously that two spherical flacherie viruses of silkworm, FVS I and FVS II, had been isolated from flacherie silkworm larvae and the nucleic acid of FVS II was RNA as suggested by the experiments of incorporation of [³H]-uracil. In this paper, it has been confirmed by biochemical methods that the nucleic acid of FVS I and FVS II is RNA. FVS I and FVS II were labeled with ³²P in flacherie silkworms, and the viruses were analyzed by sucrose density gradient centrifugation. When the ³²P-labeled compound in the viruses was treated with 0.5 n KOH, the acid-insoluble ³²P-labeled compound changed to acid-soluble compounds. It was determined by paper chromatography and ion-exchange column chromatography that the alkali-decomposed compounds included four ribonucleotides. Therefore, the viral nucleic acid of FVS I and FVS II was determined to be RNA. The correlations between FVS I and FVS II particles were discussed, and it was suggested that FVS I and FVS II might be closely related or were the same viral species.     

Abundance and Diversity of Viruses in Six Delaware Soils

The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 × 10⁹ to 4.17 × 10⁹ g⁻¹ dry weight) than in agricultural soils (8.7 × 10⁸ to 1.1 × 10⁹ g⁻¹ dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.     

Upper-limit mutation rate estimation for a plant RNA virus

It is generally accepted that mutation rates of RNA viruses are inherently high due to the lack of proofreading mechanisms. However, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. Here, based on the analysis of in vivo mutation frequencies in tobacco etch virus, we calculate an upper-bound mutation rate estimation of 3×10⁻⁵ per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. Nonetheless, the value is barely on the lower side of the range accepted for RNA viruses, although in good agreement with the only direct estimate obtained for other plant viruses. These observations suggest that, perhaps, differences in the selective pressures operating during plant virus evolution may have driven their mutation rates towards values lower than those characteristic of other RNA viruses infecting bacteria or animals.     

病毒生态学研究进展

病毒是目前所知的最简单的生命单元,通常由外壳蛋白和包裹在外壳蛋白内的核酸两部分组成。病毒本身缺乏完整的酶系统及能量转化系统,当游离于环境中时,它只是一个有机大分子,只有侵染宿主后才具有生命特征,进行复制。病毒也是地球上最丰富的生物实体,是微生物群落和功能的重要影响因素。尽管病毒在生态系统中发挥着重要的作用,但因病毒间缺少通用的标记基因,病毒生态学的研究远远滞后于细菌和真核生物。近年来高通量测序技术的发展应用帮助人们发现和认识了许多未知的新病毒及其基因,极大地丰富了病毒基因数据库,直接推动了病毒生态学的发展。从生态学角度对病毒的结构与分类、病毒生态学研究方法、病毒的生态功能及土壤病毒生态学研究进展作一简要综述,并提出今后土壤病毒生态学研究的重点。     

Morphological Transformation of Human Astrocytes by Visna Virus with Complete Virus Production

VISNA, a medium-sized RNA virus, is comparable in mode of maturation and a number of other properties with the RNA tumour viruses^1,2. The virion also carries RNA-dependent DNA polymerase^3–5. This enzyme was first discovered in RNA tumour viruses and thought perhaps unique to them^6–9; but it was subsequently found in a very few other RNA viruses^3,10, where tumorigenicity was not a known attribute. The question whether RNA-dependent DNA polymerase is a hallmark of malignancy therefore remains entirely open.