Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.     

Isolation of viral double-stranded RNAs using a LiCl fractionation procedure

A general procedure for the isolation of virus-specific double-stranded RNA (ds-RNA) is discribed. The procedure is based on the differential solubility of different types of nucleic acids in LiCl. Principal advantages over conventional methods are simplicity, avoidance of enzymatic treatment, and relatively good yields of undegraded ds-RNA while permitting separation of several main groups of cellular and viral nucleic acids from the same batch of tissue. The method has been successfully applied in tissues infected by several representative plant RNA viruses. The virus-specific ds-RNAs obtained have been identified by their resistance to ribonuclease and comparison of their electrophoretic mobilities with those of the corresponding single-stranded RNA (ss-RNA) in polyacrylamide gels. The molecular weights of the ds-RNAs of tobacco mosaic virus, turnip yellow mosaic virus, alfalfa mosaic virus, and peanut stunt virus fit the curved log molecular weight-migration relationship constructed from a set of known marker ds-RNAs.     

Sugar pucker and phosphodiester conformations in viral genomes of filamentous bacteriophages: fd, If1, IKe, Pf1, Xf, and Pf3

The laser Raman spectra of filamentous viruses contain discrete bands which are assignable to molecular vibrations of the encapsidated, single-stranded DNA genomes and which are informative of their molecular conformations. Discrimination between Raman bands of the DNA and those of the coat proteins is facilitated by analysis of viruses containing deuterium-labeled amino acids. Specific DNA vibrational assignments are based upon previous studies of A-, B-, and Z-DNA oligonucleotide crystals of known structure [Thomas, G.J., Jr., & Wang, A.H.-J. (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., & Lilley, D.M.J., Eds.) Vol. 2, Springer-Verlag, Berlin]. The present results show that canonical DNA structures are absent from six filamentous viruses: fd, If1, IKe, Pfl, Xf, and Pf3. The DNAs in three viruses of symmetry class I (fd, If1, IKe) contain very similar nucleoside sugar puckers and glycosyl torsions, deduced to be C3'-endo/anti. However, nucleoside conformations are not the same among the three class II viruses examined: Pf1 and Xf DNAs contain similar conformers, deduced to be C2'-endo/anti, whereas Pf3 DNA exhibits bands usually associated with C3'-endo/anti conformers. Conformation-sensitive Raman bands of the DNA 3'-C-O-P-O-C-5' groups show that in all class I viruses and in Pf1 the ssDNA backbones do not contain regularly ordered phosphodiester group geometries, like those found in ordered single- and double-stranded nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence sensing of intermolecular interactions and development of direct molecular biosensors

Molecular biosensors are devices of molecular size that are designed for sensing different analytes on the basis of biospecific recognition. They should provide two coupled functions - the recognition (specific binding) of the target and the transduction of information about the recognition event into a measurable signal. The present review highlights the achievements and prospects in design and operation of molecular biosensors for which the transduction mechanism is based on fluorescence. We focus on the general strategy of fluorescent molecular sensing, construction of sensor elements, based on natural and designed biopolymers (proteins and nucleic acids). Particular attention is given to the coupling of sensing elements with fluorescent reporter dyes and to the methods for producing efficient fluorescence responses.     

Various molecular aspects of cytology and embryology

Our present knowledge of the cell structure, which is largely based on electron microscopy, is compared with what was known a few decades ago, when only light microscopy was available to the cytologist. The importance of cytochemical methods for the detection and localization of macromolecules (nucleic acids, proteins) is stressed. But it is pointed out that further analysis, with biochemical techniques, was required in order to understand the actual mechanisms of macromolecule synthesis in the cell (in particular, the relationships existing between nucleic acids and protein synthesis). The importance of genetical analysis in simple systems such as viruses and bacteria for the development of ‘molecular’ biology is then emphasized: in particular, the work of Avery identifying the ‘transforming principle’ with DNA, of Beadle leading to the ‘one gene, one enzyme’ theory, of the virologists who demonstrated that it is the nucleic acid component of viruses which carries the genetical information, have been of fundamental importance for the development of modern biology. No less important has been the work of the X-ray crystallographers (Crick and Watson, Perutz, Kendrew, etc.) who established the fine structure of nucleic acids and of proteins. A brief review and a schematic representation of present ideas regarding the control exerted by DNA on the synthesis of specific proteins are then given: the main characteristics of the different kinds of RNA's, their interactions for the formation of polysomes, the role of the latter in protein synthesis, the main principles of the genetic codes, are briefly summarized. But cells are, in many respects, more complicated than bacteria. The concepts of molecular biology cannot be applied to cell differentiation without a recognition of the greater complexity of animal and plant cells. They represent, however, a most useful and powerful guide for research in that area: for instance, many aspects of morphogenesis in the unicellular alga Acetabularia and in amphibian eggs can be explained on the assumption that messenger RNA's are produced by the nucleus and stored, in a stabilized form, in the cytoplasm during days or even weeks. This stability of messenger RNA's in eggs and algae is at variance with their short life in bacteria. The behaviour of non-nucleate fragments of Acetabularia is surprising in many respects: they are the site, not only of the synthesis of specific proteins, but even of RNA and DNA net synthesis. Such a synthesis of macromolecules, in the absence of the nucleus is probably linked to the presence of the chloroplasts in this alga: they contain DNA, can synthesize RNA and proteins, and can even increase in number in the absence of the nucleus. The presence of large amounts of DNA in the cytoplasm of many animal eggs raises a number of questions and might account for the extremely important role of the cytoplasm in the very early stages of embryonic development. It is concluded that none of the great problems of cell biology will be solved without the help of the techniques and the theoretical ideas which have been so fruitful for the simpler systems used by the molecular biologists.     

Tick‐borne viruses: Current trends in large‐scale viral surveillance

Ticks are ectoparasites that transmit pathogens, such as tick‐borne viruses, to their hosts. Tick‐borne viruses are diverse: they can be categorized into two orders, nine families, and at least 12 genera. Almost 25% of these viruses are infectious to humans and some are a serious threat to public health. The global rise in tick‐borne virus diseases has been linked to climate change which has reduced tick mortality in the winter and extended their active period. The spread of tick‐borne viral diseases to humans has received significant interest due to the increased threat to human life; epidemiological monitoring of tick‐borne viruses using molecular, immunological, and environmental methods is now a priority. Nevertheless, many tick‐borne diseases remain undiagnosed, which poses a challenge to public administration and health care officials. This review discusses three major tick‐borne RNA viruses that cause serious infection in humans: severe fever with thrombocytopenia syndrome (SFTS) virus, tick‐borne encephalitis (TBE), and Crimean–Congo hemorrhagic fever (CCHF) virus. Specifically, we discuss the epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants related to these viruses. Furthermore, we review the current surveillance of these tick‐borne viruses with a specific focus on diagnostic approaches that employ molecular interventions such as viral nucleic acid isolation, PCR‐based diagnostics, and high‐throughput sequencing technologies.     

Are we missing half of the viruses in the ocean?

Viruses are abundant in the ocean and a major driving force in plankton ecology and evolution. It has been assumed that most of the viruses in seawater contain DNA and infect bacteria, but RNA-containing viruses in the ocean, which almost exclusively infect eukaryotes, have never been quantified. We compared the total mass of RNA and DNA in the viral fraction harvested from seawater and using data on the mass of nucleic acid per RNA- or DNA-containing virion, estimated the abundances of each. Our data suggest that the abundance of RNA viruses rivaled or exceeded that of DNA viruses in samples of coastal seawater. The dominant RNA viruses in the samples were marine picorna-like viruses, which have small genomes and are at or below the detection limit of common fluorescence-based counting methods. If our results are typical, this means that counts of viruses and the rate measurements that depend on them, such as viral production, are significantly underestimated by current practices. As these RNA viruses infect eukaryotes, our data imply that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance. This conclusion is a departure from the prevailing view of viruses in the ocean, but is consistent with earlier theoretical predictions.     

Viruses as environmental mutagenic factors

Injections into Drosophila melanogaster males of 9 DNA- and RNA-containing viruses non-infectious for this insect, proved that all of them are highly mutagenic, inducing numerous gene mutations or microdeletions but no gross chromosome rearrangements. Relatively few specific loci are affected, they vary per virus, and the mutation rate for such a locus is extraordinarily high. These peculiarities of the mutagenic action of viruses closely resemble those of the mutagenic action of exogenous non-viral DNA earlier studied by the author and his co-workers. It was shown that the mutagenic element of a virus is its nucleic acid; viral proteins completely lack mutagenic properties. Virus-induced gene mutations are probably due to insertions of fragments of viral DNA (or cDNA) into the host chromosomes; at least some of these mutations are capable of transpositions and reversions. On the other hand, chromosome aberrations which arise in virus-infected cells are evidently produced secondarily by pathological disturbances of metabolic processes in the infected cells. Our results and literature data lead to the conclusion that a significant share of hereditary defects in man might be caused by viruses, even by those apathogenic for humans, particularly by attenuated live viral vaccines. It is, therefore, important to work on the creation of purely proteinaceous viral vaccines free from nucleic acids and thus genetically safe. The hazards of viral mutagenesis should also be kept in mind when preparing recombinant DNA molecules used in genetic engineering.     

A decade with nucleic acid‐based microbiological methods in safety control of foods

In the last decade, nucleic acid‐based methods gradually started to replace or complement the culture‐based methods and immunochemical assays in routine laboratories involved in food control. In particular, real‐time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry‐over contamination. Basic advantages provided by nucleic acid‐based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid‐based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.     

The molecular diagnostics of viruses

For many years data of cancer research indicated that viruses can cause cancer. Virus infections induce cancer by different mechanisms. To predict the significance of a viral DNA fragment in human cells we have to be aware of the changes the particular virus is able to induce there.However, no matter which mechanisms of viral carcinogenesis are utilized, generally other factors (environmental, chemical, immunodeficiency, etc.) are also needed to induce invasive cancer in human. Before the introduction of nucleic acid based detection technique virus identification was a long and cumbersome process. This has been eliminated by the invention of recombinant gene technology and polymerase chain reaction. Virus nucleic acid can be detected without amplification using Southern, Northern and in situ hybridization. Techniques for target (polymerase chain reaction)or signal (hybrid capture, tyramine) amplification improved the sensitivity of detection. In the meantime, for the successful use of the arsenal of new methods we have to consider the characteristic feature of molecular virus research. A major achievement of molecular virus detection is that it proved the pathological significance of viruses in human cancers even in those where this was not expected. Hopefully these informations will increase the effort for elimination of oncogene virus infections.     

Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses,Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.     

Methods for long-term virus preservation

Viruses exhibit a wide variety of structural and chemical differences, but, in general, their infectivity may be destroyed by degradative enzymes that destroy nucleic acids, by detergents that solubilize the lipid-containing envelopes thus exposing the nucleic acid, by temperatures higher than about 50 degrees C, or by chemicals that breakdown capsid proteins. Preserving the viruses at low or ultra-low temperatures, and/or in the absence of water, slows down these destructive processes sufficiently to increase significantly the length of time that the virus can be stored as infectious material. Supplements such as serum are presumed to stabilize the environmental conditions and to block degradative processes. The methods by which viruses may be preserved for long periods of time are similar to those employed for other microorganisms and are relatively simple. Nevertheless, attention to detail, good laboratory practice, aseptic technique, meticulous recordkeeping, and regular monitoring of the stored materials will increase the success rate and reduce problems of contamination or loss in the storage containers, where many different viruses may be stored for posterity! This article describes some of the simplest and most reliable storage procedures for viruses, but the author recognizes that everyone will have a favorite method to suit his or her own particular virus.     

Photoactivatable Reagents Based on Aryl(trifluoromethyl)diazirines: Synthesis and Application for Studying Nucleic Acid–Protein Interactions

Recent data on the synthesis of photoactivatable derivatives of nucleic acids and proteins on the basis of aryl(trifluoromethyl)diazirines—analogs of nucleosides, nucleotides, oligonucleotides, as well as amino acids and peptides—are reviewed. The synthesis of bi- and polyfunctional photoactivatable reagents, including those containing a cleavable function, designed for postsynthetic modification of biopolymers is described. Data are given on the use of the photoactivatable derivatives for studying nucleic acid–protein interactions by the method of photoaffinity labeling. Special consideration is paid to the results obtained by the authors" team in cooperation with other researchers as well as graduate students of the Chemistry of Natural Products Chair, Chemical Faculty, Moscow State University and destined to solve various scientific tasks in the domain of nucleic acid–protein recognition with the use of photoaffinity crosslinking.     

Roles for host factors in plant viral pathogenicity

The simple, obligate nature of viruses requires them to usurp or divert cellular resources, including host factors, away from their normal functions. The characterization of host proteins, membranes, and nucleic acids that are implicated in viral infection cycles, together with other recent discoveries, is providing fundamental clues about the molecular bases of viral susceptibility. As viruses invade susceptible plants, they create conditions that favor systemic infections by suppressing multiple layers of innate host defenses. When viruses meddle in these defense mechanisms, which are interlinked with basic cellular functions, phenotypic changes can result that contribute to disease symptoms.     

富含半胱氨酸病毒蛋白的研究进展

病毒编码的富含半胱氨酸的小分子量蛋白(CRPs)在植物和动物病毒中均有发现。动物病毒中研究较多的是反转录病毒的核蛋白(NC)。在植物病毒中由hordei,tobra,furoandcarlaviruses编码的CRPs的分子生物学研究近年来才开展起来。动物和植物病毒的CRPs共有的典型特征是均有锌指结构和碱性氨基酸丰富区,这使它们在核酸结合特性上有共同特征。动物病毒CRPs的结构和功能方面的研究已有很好的进展。相反,植物病毒的CRPs的研究进展较为缓慢。本文对病毒的CRPs的最新进展进行了综述。对动物和植物病毒的CRPs的比较分析有助于将来这类蛋白功能的阐明。     

Utilization of adenovirus vectors for multiple gene transfer applications

Mammalian viruses have evolved over millions of years to achieve a single goal, namely to rapidly enter a host mammalian cell, in order to achieve virus propagation. In so doing, these biologic parasites have acquired the molecular tools to rapidly and efficiently deliver their own nucleic acids into the nucleus of the host cell. The human adenovirus is one of the best studied of these parasites. As such the adenovirus has been re-engineered to allow it to be used as a tool to allow researchers to deliver desired nucleic acid sequences into a large variety of cell targets, both in tissue culture systems, as well as directly into living animals. Adenovirus based gene transfer systems can overcome most of the problems inherent to high efficiency gene transfer, and perform in a fashion that in many ways cannot be matched by most other currently utilized gene transfer systems. This article will attempt to summarize the multiple attributes of this widely utilized gene delivery system.     

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.     

Discovery of Novel Rhabdoviruses in the Blood of Healthy Individuals from West Africa

Next-generation sequencing (NGS) has the potential to transform the discovery of viruses causing unexplained acute febrile illness (UAFI) because it does not depend on culturing the pathogen or a priori knowledge of the pathogen’s nucleic acid sequence. More generally, it has the potential to elucidate the complete human virome, including viruses that cause no overt symptoms of disease, but may have unrecognized immunological or developmental consequences. We have used NGS to identify RNA viruses in the blood of 195 patients with UAFI and compared them with those found in 328 apparently healthy (i.e., no overt signs of illness) control individuals, all from communities in southeastern Nigeria. Among UAFI patients, we identified the presence of nucleic acids from several well-characterized pathogenic viruses, such as HIV-1, hepatitis, and Lassa virus. In our cohort of healthy individuals, however, we detected the nucleic acids of two novel rhabdoviruses. These viruses, which we call Ekpoma virus-1 (EKV-1) and Ekpoma virus-2 (EKV-2), are highly divergent, with little identity to each other or other known viruses. The most closely related rhabdoviruses are members of the genus Tibrovirus and Bas-Congo virus (BASV), which was recently identified in an individual with symptoms resembling hemorrhagic fever. Furthermore, by conducting a serosurvey of our study cohort, we find evidence for remarkably high exposure rates to the identified rhabdoviruses. The recent discoveries of novel rhabdoviruses by multiple research groups suggest that human infection with rhabdoviruses might be common. While the prevalence and clinical significance of these viruses are currently unknown, these viruses could have previously unrecognized impacts on human health; further research to understand the immunological and developmental impact of these viruses should be explored. More generally, the identification of similar novel viruses in individuals with and without overt symptoms of disease highlights the need for a broader understanding of the human virome as efforts for viral detection and discovery advance.     

A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions

The widespread exploitation of biosensors in the analysis of molecular recognition has its origins in the mid-1990s following the release of commercial systems based on surface plasmon resonance (SPR). More recently, platforms based on piezoelectric acoustic sensors (principally 'bulk acoustic wave' (BAW), 'thickness shear mode' (TSM) sensors or 'quartz crystal microbalances' (QCM)), have been released that are driving the publication of a large number of papers analysing binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights salient theoretical and practical aspects of the technologies that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells and lipidic and polymeric interfaces. Key differentiators between optical and acoustic sensing modalities are also reviewed.