Virus-specific Ribonucleic Acid Research Articles

A microfluidic system integrated with buried optical fibers for detection of Phalaenopsis orchid pathogens

Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Fast, accurate, and on-site detection of pathogens in these orchids is therefore of critical importance in order to prevent or suppress costly disease outbreaks. Traditional pathogen detection methods are time-consuming, require well-equipped laboratories with highly trained personnel, and cannot be conducted in situ. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by a pre-treatment incubation with magnetic beads, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. A buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip itself by measuring the change in the optical signals caused by the turbidity change associated with a positive amplification. The limit of detection for this system was found to be 25fg, which is of similar sensitivity to existing, more laborious methods. Therefore, by using the integrated microfluidic system, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65min.

Bats and other reservoir hosts of Filoviridae. Danger of epidemic on the African continent?--a deductive literature analysis

ZusammenfassungDie Familie der Filoviridae beinhaltet das Ebola- und das Marburgvirus, die hämorrhagisches Fieber in sub-Sahara Afrika auslösen können. Diese viralen Erkrankungen weisen eine hohe Letalitätsrate und ein plötzliches, epidemisches Auftreten auf. In der Literatur gibt es zahlreiche Hinweise, dass die Ordnung der Fledertiere (Chiroptera) einen potentiellen Reservoirwirt darstellen könnte. Signifikante Hinweise lassen ein hypothetisches Ökosystem der Filoviridae mit Beteiligung von Chiroptera skizzieren. Während der Ebola-Ausbrüche zwischen 2001 und 2005 in Gabun und der Republik Kongo konnten in Hammerkopf- (Hypsignathus monstrosus), Epauletten- (Epomops franqueti) und Schmalkragenflughunden (Myonycteris torquata) IgG-spezifische Ebola-Zaire-Antikörper nachgewiesen werden. Die Entdeckung von IgG-spezifischen Marburgvirus-Antikörpern und virusspezifischer Ribonukleinsäure in Nilflughunden (Rousettus aegyptiacus) gilt als weiteres Indiz in der Erforschung des Reservoirwirtes. In der Kitaka-Mine (Uganda) gelang 2007 erstmals eine Marburgvirus-Isolation aus asymptomatischen, natürlich infizierten Nilflughunden (Rousettus aegyptiacus). Anhand einer chronologischen Aufarbeitung und Interpretation der einzelnen Epidemiefälle am afrikanischen Kontinent und der Suche nach den Infektionsgründen der Indexfälle, lassen sich menschliche Risikogruppen abgrenzen. Neben der Verwertung von Wildtierkadavern, stellen die touristische Besichtigung von Höhlen und der berufliche Alltag in Minen nicht zu vernachlässigende Risiken für eine Infektion dar. Im Vordergrund dieser Arbeit steht die Darstellung dieser Risikoprofile im Zusammenhang mit einer Exposition gegenüber Chiroptera und anderen möglichen Reservoirwirten.

Polyadenylic Acid [Poly(A)] Sequences Associated with Measles Virus Intracellular Ribonucleic Acid (RNA) Species

The kinetics of 3H-uridine incorporation into measles-infected Vero cells demonstrated that maximum virus-specific RNA synthesis occurred between 16 and 20 h after infection. Sedimentation analysis on sucrose gradients revealed the presence of four species of RNA having sedimentation coefficients 4S, 12 to 26S, 28 to 36S and 50S. Annealing studies showed that RNA sedimenting in the 12 to 36S regions was 100% complementary in base sequence to nucleocapsid 50S RNA, and at least 96% of the 50S genomic RNA was transcribed during virus replication. Polynucleotide binding experiments ane ribonuclease treatment indicated that poly(A) sequences were associated with the intracellular 12 to 26S, 28 to 36S and 50S RNAs. Denaturation of intracellular 50S RNA followed by sucrose gadient centrifugation demonstrated that this was a mixture of genomic 50S and heterogeneous RNAs which sedimented at 4 to 40S. The genomic RNA did not contain poly(A) sequences, and these are presumably associated with the heterogeneously sedimenting RNAs. The size of poly(A) sequences present on the 12 to 36S RNAs was estimated to be in the range of 70 to 140 nucleotides. Treatment of the 12 to 36S RNAs and their poly(A) sequences with polynucleotide phosphorylase indicated that the poly(A) was located on the 3' end of the RNAs, but that under the experimental conditions used this was protected by the secondary structure of the molecules.

Measurement of dengue virus-specific double-stranded ribonucleic acid in infected primate cell extracts by microquantitative complement-fixation methods.

Microquantitative complement-fixation methods have been applied to the detection and quantitation of double-stranded RNA (dsRNA) in cytoplasmic extracts of uninfected and dengue virus-infected primate cell lines. LLC-MK2 and PGLC-33H cells exhibited increased levels of dsRNA following dengue virus infection. Cytoplasmic extracts of infected and uninfected LLC-MK2 cells subjected to density gradient analysis showed that increased levels of dsRNA were associated with structures believed to be virus-specific. Primate cell dsRNA and dengue dsRNA preferentially reacted with rabbit anti-rIn-rCn, while reovirus dsRNA and rAn-containing synthetic duplexes preferentially reacted with anti-rAn-rUn.

Identification of self-complementary virus-specific ribonucleic acid in chick kidney cells infected with chicken embryo lethal orphan virus.

RNA was extracted from primary chicken embryo kidney (CEK) cells infected with chicken embryo lethal orphan (CELO) virus and exposed to a pulse of (5-3H)-uridine late in infection. When this RNA was self-annealed, 4.5% became resistant to pancreatic ribonuclease digestion. The ribonuclease-resistant RNA was isolated by chromatography on Sephadex G-100, and the RNA was found to have the characteristics of a double-stranded molecule of sedimentation coefficient 8S. Half of the column-isolated RNA hybridized to CELO DNA with equal amounts of virus RNA binding to the heavy or light stands of the CELO DNA, indicating the presence of complementary RNA species late in the infectious cycle of CELO.

The presence and expression of RNA tumor virus genes in normal and infected cells: detection by molecular hybridization.

Three molecular probes have been used to detect genes of the Rous sarcoma virus and mouse mammary tumor virus in normal and infected cells: the 70S ribonucleic acid (RNA) of the viral genomes, and the single- and double-stranded deoxyribonucleic acids (DNA) transcribed from viral RNA by RNA-directed DNA polymerase. The findings indicate that multiple copies of viral genes may be present in the DNA of ostensibly normal cells. However, infection and transformation of mammalian (mouse and rat) cells by Rous sarcoma virus is accompanied by the appearance of virus-specific nucleotide sequences in nuclear DNA of the host cells. The expression of Rous sarcoma virus and mouse mammary tumor virus genes in normal and infected cells has been measured by RNA–DNA hybridization. Virus-specific RNA is present in both normal and transformed cells which produce neither virus nor detectable viral proteins. These data present the possibility that the expression of viral genes is not controlled exclusively by regulation of transcripton.

Quantitative determination and location of newly synthesized virus-specific ribonucleic acid in chicken cells infected with Rous sarcoma virus.

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.

Intracellular components associated with Chikungunya virus-specific ribonucleic acids in infected BHK21 cells.

Cytoplasmic extracts of BHK21 cells infected with Chikungunya virus were analyzed by sucrose gradient sedimentation. Besides 140 S nucleocapsid, 65 S component associated with 26 S single-stranded ribonucleic acid was labeled with 3H-uridine in actinomycin-treated infected cells. The 65 S component, demonstrable in ethylenediaminetetraacetic acid-containing buffers, appeared to accumulate when protein synthesis was inhibited. After glutaraldehyde fixation, this component had a density of 1.48–1.50 g/ml in CsCl. Pulse-chase experiments did not indicate that the 65 S component was a precursor of the 140 S nucleocapsid.

Measles virus ribonucleic acid and protein synthesis: effects of 6-azauridine and cycloheximide on viral replication.

Cycloheximide and 6-azauridine were employed to study the time course of measles virus protein and nucleic acid syntheses in AV3 cells. Synthesis of ribonucleic acid (RNA) essential for infectivity was first detected at 6 hr and increased concurrently with the formation of essential protein. Maximum levels of virus-specific RNA and protein were present by 18 hr, a time when only 5% of progeny virus was detected. Essential RNA and protein syntheses preceded the formation of infectious virus by at least 10 to 12 hr. The time course of RNA and protein syntheses essential for the formation of complement-fixing (CF) antigen and salt-dependent agglutinin (SDA) was also determined. RNA synthesis essential for the formation of SDA was first detected at 2 hr and was present maximally by 6 hr, whereas SDA-protein increased concurrently with the protein essential for infectivity. This suggested that the last protein essential for infectivity may be SDA. RNA synthesis essential for the formation of CF antigen was first detected at 4 hr, while CF-protein increased at 5 hr and preceded SDA-protein and protein essential for infectivity by approximately 3 hr. Reversal of inhibition of protein synthesis by cycloheximide indicated that early protein synthesis (1 to 3 hr) was required for the formation of infectious virus. The data suggest that the relatively long eclipse period observed with measles virus is related to a long maturation period rather than to late formation of early proteins, viral RNA, or structural proteins.

Transcription and transport of virus-specific ribonucleic acids in African green monkey kidney cells abortively infected with type 2 adenovirus.

The techniques of deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization and immunological precipitation were used to compare the synthesis of adenovirus-specific macromolecules in African green monkey kidney (AGMK) cells infected with adenovirus, an abortive infection, and coinfected with both adenovirus and simian virus 40 (SV40), which renders the cells permissive for adenovirus replication. When viral protein synthesis was proceeding at its maximum rate, the incorporation of (14)C-amino acids into adenovirus structural proteins was about 90 times greater in the doubly infected cells than in cells infected only with adenovirus. However, the rates of synthesis of virus-specific ribonucleic acid appeared to be comparable in the two infections at all times measured. A time-dependent increase in the rate of RNA synthesis observed late in the abortive infection was dependent upon the prior replication of viral DNA. Moreover, all virus-specific RNA species that are normally made late in a productive adenovirus infection (i.e., the true late and class II early RNA species) were also detected in the abortive infection. Adenovirus-specific RNA was detected by molecular hybridization in both the cytoplasm and nuclei of abortively infected cells. Comparable amounts of viral RNA were found in the cytoplasmic fractions of AGMK cells infected either with adenovirus or with both adenovirus and SV40. The results of hybridization-inhibition experiments clearly showed that there was a class of virus-specific RNA molecules, representing about 30% of the total, in the nucleus that was not transported to the cytoplasm. This class of RNA was also identified in similar amounts in productively infected human KB cells. The difference in the abilities of cytoplasmic and nuclear RNA to inhibit the hybridization of virus-specific RNA from whole cells was shown not to be due to a difference in the molecular size of the RNA species from the two cell fractions or to the specific loss of a cytoplasmic species during RNA extraction procedures.

Mechanism of Mengo Virus-Induced Cell Injury in L Cells: Use of Inhibitors of Protein Synthesis to Dissociate Virus-Specific Events

L cells were infected with Mengo virus in the presence of varying concentrations of protein synthesis inhibitors (azetidine-2-carboxylic acid, p-fluorophenylalanine, puromycin), and examined with respect to the effects of the inhibitors on several features of virus-induced cell injury. The virus-specific events in the cells could be dissociated into three groups, based on their sensitivity to the inhibitors: (i) viral ribonucleic acid (RNA) synthesis, bulk viral protein synthesis, and infectious particle production, all of which were prevented by low inhibitor concentrations; (ii) the cytopathic effect (CPE) and stimulation of phosphatidylcholine synthesis, which were sensitive to intermediate concentrations of the inhibitors; and (iii) the virus-induced inhibitions of host RNA and protein synthesis, which were resistart to the inhibitors of protein synthesis except at very high concentrations. It is concluded from this that the virus-induced CPE and stimulation of phosphatidylcholine synthesis are not consequences of the inhibition of cellular RNA or protein synthesis. Analysis of the virus-specific protein and RNA synthesized at several concentrations of azetidine and puromycin suggests that the CPE may be induced by a viral protein precursor. Virus-induced inhibition of host RNA and protein synthesis occurred at azetidine concentrations which blocked the synthesis of over 99.7% of the total viral RNA and over 99% of the viral double-stranded RNA (dsRNA). Calculations show that this would correspond to less than 150 dsRNA molecules per infected cell, resulting in a dsRNA-polysome ratio of less than 1:1,000; this indicates that host protein synthesis cannot be inhibited by an irreversible binding of dsRNA to polysomes.

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.

Identification of double-stranded virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus

Using agarose gel chromatography, RNA with characteristics of double-stranded molecules was isolated from KB cells infected with type 2 adenovirus. Results of studies measuring its susceptibility to a number of nuclease activities were consistent with its identification as double-stranded RNA. The material was resistant to digestion by both pancreatic and T1 ribonuclease and by pancreatic deoxyribonuclease; and after heating at 100°C, it was much more sensitive to ribonuclease hydrolysis. Moreover, nearly 30% of the RNA formed a ribonuclease-resistant hybrid when denatured and incubated with purified type 2 adenovirus DNA, indicating that at least a portion of the double-stranded RNA in infected KB cells was transcribed from the viral genome.

Virus-specific ribonucleic acid in cells producing rous sarcoma virus: detection and characterization.

Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts.

Transcription of the adenovirus genome by an -amanitine-sensitive ribonucleic acid polymerase in HeLa cells.

The properties of the ribonucleic acid (RNA) polymerase activity which transcribes the major portion of the adenovirus genome were studied. Nuclei were prepared from infected cells and incubated in vitro. Virus-specific RNA was determined by hybridization to adenovirus deoxyribonucleic acid (DNA). Adenovirus DNA is transcribed principally by an activity which resembles closely polymerase II of the host cell. This activity is inhibited by alpha-amanitine and stimulated by (NH(4))(2)SO(4). Its product is high-molecular-weight heterogeneous RNA. The polymerase activity measured early in infection (3 to 5 hr) resembles that found late in infection (16 to 18 hr).

State of adenovirus 2 deoxyribonucleic acid in the nucleus and its mode of transcription: studies with isolated viral deoxyribonucleic acid-protein complexes and isolated nuclei.

Newly replicated adenovirus 2 deoxyribonucleic acid (DNA) can be isolated from the nucleus of HeLa cells by a gentle lysis procedure as a fairly homogeneous complex with a sedimentation of 73S. The viral DNA complex can be prepared completely free from host cell DNA. The viral complex is slightly active in ribonucleic acid (RNA) synthesis in vitro. Treatment of the complex with Pronase and sodium dodecyl sulfate converts the DNA to a form which sediments at 43S. Nuclei isolated from adeno-infected cells synthesize high-molecular-weight virus-specific RNA in vitro. Optimal RNA synthesis requires a divalent cation, preferentially manganese, and relatively high salt concentrations. The synthesis of virus-specific RNA by the isolated nuclei is strongly inhibited by low doses of alpha-amanitine. The latter experimental result is discussed in terms of the polymerase used to transcribe the adenovirus DNA in vivo.

Temperature-sensitive mutants isolated from L cells persistently infected with Newcastle disease virus.

Virus mutants (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus have been previously reported by this laboratory to differ from the wild-type virus (NDV(o)) in several physical and biological properties. It has now been determined that, in addition to these differences, the NDV(pi) mutants are also spontaneously selected temperature-sensitive mutants. The temperature sensitivity of 10 NDV(pi) clones was confirmed by temperature inhibition, plaquing efficiency, and single-cycle yield experiments. The cut-off temperature, at which more than 90% of virus replication is inhibited was between 41 and 42 C. All 10 NDV(pi) clones were also found to be defective in virus-specific ribonucleic acid (RNA) synthesis in infected chick embryo cells at 42 C and are tentatively classified as RNA(-). The possible relationships of the temperature sensitivity, the other NDV(pi) properties, and the maintenance of the persistently infected state are discussed.

Use of miracil D to suppress bacterial ribonucleic acid and protein synthesis during bacteriophage MS2 infection.

Under certain culture conditions, Miracil (35 mug/ml) halts the growth of uninfected Escherichia coli. Cellular ribonucleic acid (RNA) synthesis is almost completely suppressed, whereas deoxyribonucleic acid and protein synthesis are inhibited to a lesser extent. When the drug is added to host bacteria prior to infection with bacteriophage MS2, the phage adsorb to the cells, but penetration of the viral RNA is inhibited. Penetration may be achieved without further viral development by infection in the presence of chloramphenicol. If the bacteria are infected with MS2 in the presence of chloramphenicol, subsequently washed to remove the chloramphenicol, and then treated with Miracil at any time between 0 and 20 min postinfection, a second viral function is inhibited and the yield of progeny phage is reduced. Addition of the drug after 20 min postinfection does not inhibit the infection process. When Miracil is present from early times in infection, only a limited synthesis of both double- and single-stranded virus-specific RNA is observed. The viral RNA species thus produced do not appear to differ from those made in the absence of the drug. A comparison of the activities of the viral RNA synthetase produced during the course of infection in the presence and in the absence of Miracil suggests that a possible cause of the inhibition is the synthesis of an unstable enzyme in the presence of the drug.

Structure and development of viruses as observed in the electron microscope. XI. Entry and uncoating of herpes simplex virus.

Two morphologically distinct types of capsids are described. The dense capsid appeared to be disrupted near the cellular membrane with release of core material. The light capsid was more stable and was frequently encountered close to the nucleus, where empty capsids were also found. Pretreatment of cells before infection with either puromycin or actinomycin D markedly decreased the percentage of empty capsids. It is suggested that the two types of capsids play different roles in the process of initiating infection. One (the dense capsid) releases deoxyribonucleic acid (DNA) shortly after entry. This DNA is transcribed into a virus-specific ribonucleic acid, which codes for an enzyme capable of altering the permeability of the second type of capsid (the light capsid). In proximity to the nucleus, the infectious DNA then escapes without gross disruption of the capsid.

Ribonuclease-sensitive deoxyribonucleic acid polymerase activity in uninfected rat cells and rat cells infected with Rous sarcoma virus.

Rat cells infected with the B77 strain of avian sarcoma virus [R(B77) cells] produced no virus-like particles but contained information for the production of infectious B77 virus. (3)H-labeled deoxyribonucleic acid (DNA) product of the B77 virus endogenous DNA polymerase system was used to determine the relative amounts of B77 virus-specific ribonucleic acid (RNA) in B77 virus-infected chicken and R(B77) cells. R(B77) cells were found to contain much less B77 virus RNA than did B77 virus-infected chicken cells. Ribonuclease-sensitive DNA polymerase activity was present in high-speed pellet fractions from Nonidet extracts of B77 virus-infected rat cells. Similar preparations from some uninfected rat cells contained lesser amounts of a similar ribonuclease-sensitive DNA polymerase activity. The endogenous template for the DNA polymerase activity in high-speed pellet fractions from R(B77) cells was not related to B77 virus RNA or to RNA of a rat C-type virus. The DNA product of the endogenous DNA polymerase in high-speed pellet fractions of R(B77) cells hybridized to a small extent with RNA from the same fraction and to a similar extent with RNA from uninfected rat cells.