bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of a commercial kit for detection of PCR products to quantification of human immunodeficiency virus type 1 RNA and proviral DNA
Authors: Lin H J , Haywood M , Hollinger F B
Published: January 2020

Quantitative tests for human immunodeficiency virus type 1 (HIV-1) RNA in plasma and proviral DNA in peripheral blood mononuclear cells (PBMC) provide valuable information on the status of HIV-1 infection. This paper describes tests that were carried out with commercially available materials and an enzyme-linked immunosorbent assay reader for detecting spectrophotometric changes. Samples consisted of 100 microliters of plasma or 200,000 PBMC. The procedure involved sample preparation, PCR-based amplification with the primer pair SK39 (biotinylated at the 5' end) and SK38, hybridization of the cDNA PCR product to an RNA probe, capture of the RNA-DNA hybrid on a solid phase by means of strepavidin, binding to an alkaline phosphatase-conjugated antibody directed against RNA-DNA hybrids, and incubation with p-nitrophenylphosphate. Spectrophotometric changes were recorded at four intervals over a period of 20 h. The inclusion of HIV-1 RNA or proviral DNA standards in each run was an integral part of the procedure. The dynamic ranges afforded by these assays--500 to 1 million RNA copies per ml and 10 to 5,000 proviral DNA copies per 10(6) PBMC--were applicable to most plasma specimens and to all PBMC specimens from HIV-1-infected patients. Correlations of log-transformed HIV-1 RNA and proviral DNA concentrations with those found by reference methods were, respectively, 0.88 and 0.80. The between-run coefficients of variation for the detection method were < or = 25% (range, 9.1 to 24.7) and < or = 15% (range, 10.9 to 15.1), respectively, for HIV-1 RNA and proviral DNA. The reproducibility of the overall procedure for HIV-1 RNA in plasma (including sample preparation, amplification, and detection) was given by a duplicate standard deviation of log10 copies per ml of 0.11. Thus, the method was sufficiently precise to allow the detection of fourfold changes in plasma HIV-1 RNA concentrations, with a power of 0.95.
Molecular analysis of microflora associated with dentoalveolar abscesses
Authors: Dymock D , Weightman A J , Scully C , Wade W G
Published: January 2020

The microflora associated with three dentoalveolar abscesses was determined by cultural and molecular methods. 16S rRNA genes were randomly amplified by means of conserved eubacterial primers and cloned. Restriction fragment length polymorphism analysis of the clones and amplified genes encoding 16S rRNA from the cultured bacteria was used to detect putative unculturable bacteria. Clones representative of five predominant groups of uncultured organisms were sequenced. Two were identified as Porphyromonas gingivalis and Prevotella oris, and one was found to be closely related to Peptostreptococcus micros. The remaining two clones did not correspond to known, previously sequenced organisms. One was related to Zoogloea ramigera, a species of aerobic waterborne organisms, while the other was distantly related to the genus Prevotella. This study has demonstrated the possibility of the characterization of microflora associated with human infection by molecular methods without the inherent biases of culture.
Rapid antigen detection assay for identification of Burkholderia (Pseudomonas) pseudomallei infection
Authors: Anuntagool A , Intachote P , Naigowit P , Sirisinha S
Published: January 2020

A simple antigen detection test was developed for rapid diagnosis of melioidosis caused by Burkholderia (Pseudomonas) pseudomallei infection. The method was based on the use of a specific monoclonal antibody in a sandwich enzyme-linked immunosorbent assay to capture antigen in clinical specimens culture positive for B. pseudomallei. The results showed the sensitivity and specificity of the test to be 75 and 98%, respectively.
Detection of hepatitis E virus RNA in stools and serum by reverse transcription-PCR
Authors: Turkoglu S , Lazizi Y , Meng H , Kordosi A , Dubreuil P , Crescenzo B , Benjelloun S , Nordmann P , Pillot J
Published: January 2020

Stools and sera collected during an experimental hepatitis E virus (HEV) infection in monkeys and collected from humans with acute HEV infections during epidemic and sporadic cases were analyzed by reverse transcription-PCR. Two methods for RNA purification were compared. Proteinase K digestion and phenolchloroform extraction were more efficient than guanidinium isothiocyanate extraction in improving the sensitivity and specificity for the detection of HEV genomes.
Continuous monitoring of oxygen concentrations in several systems for cultivation of anaerobic bacteria
Authors: Imhof A , Heinzer I
Published: January 2020

Anaerobic chambers and jars are the two conventional methods used in clinical microbiology laboratories to produce an anaerobic atmosphere. The evacuation-replacement method, the Oxoid AnaeroGen, the Merck Anaerocult A, the BBL GasPak, the BBL GasPakPlus, the Adams Scientific GasGendicator, the Difco Anaerobic, and the bioMérieux Generbox anaer systems were compared for the timed decrease in the oxygen concentration in an anaerobic jar. The experiment was repeated 10 times for each system. The oxygen concentration was measured with an oxygen analyzer series 3600 instrument (Orbisphere Laboratories, Neuchâtel-Geneva Switzerland). The BBL GasPak, the BBL GasPakPlus, the bioMérieux Generbox, the Adams Scientific GasGendicator, and the Difco Anaerobic systems contain sodium borohydride, which liberates hydrogen. The Anaerocult A system contains iron powder which binds the oxygen. The active ingredient of the AnaeroGen system is ascorbic acid. The times to reach an O2 concentration of 0.5% were 8 to 15 min for the evacuation-replacement method, 26 to 41 min for the AnaeroGen system, 60 to 93 min for the Anaerocult A system, and 22 to 419 min for the sodium borohydride systems. The AnaeroGen system, the Anaerocult A system, and the evacuation-replacement method never failed to produce an anaerobic atmosphere. The sodium borohydride systems failed in 10 of 70 runs. These results suggest that the evacuation-replacement method or the Oxoid AnaeroGen system should be used to produce an anaerobic atmosphere. The Anaerocult A system showed a good reproducibility, but the length of time required to reach an appropriately low level of oxygen was too long. Because of the high failure rate, the borohydride systems cannot be recommended.
New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a
Authors: Ohno O , Mizokami M , Wu R R , Saleh M G , Ohba K , Orito E , Mukaide M , Williams R , Lau J Y
Published: January 2020

Recent studies have focused on whether different hepatitis C virus (HCV) genotypes are associated with different profiles of pathogenicity, infectivity, and response to antiviral therapy. The establishment of a simple and precise genotyping system for HCV is essential to address these issues. A new genotyping system based on PCR of the core region with genotype-specific PCR primers for the determination of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a was developed. A total of 607 samples (379 from Japan, 63 from the United States, 53 from Korea, 35 from Taiwan, 32 from China, 20 from Hong Kong, 15 from Australia, 6 from Egypt, 3 from Bangladesh, and 1 from South Africa) were tested by both the assay of Okamoto et al. (H. Okamoto, Y. Sugiyama, S. Okada, K. Kurai, Y. Akahane, Y. Sugai, T. Tanaka, K. Sato, F. Tsuda, Y. Miyamura, and M. Mayumi, J. Gen. Virol. 73:673-679, 1992) and this new genotyping system. Comparison of the results showed concordant results for 539 samples (88.8%). Of the 68 samples with discordant results, the nucleotide sequences of the HCV isolates were determined in 23, and their genotypes were determined by molecular evolutionary analysis. In all 23 samples, the assignment of genotype by our new genotyping system was correct. This genotyping system may be useful for large-scale determination of HCV genotypes in clinical studies.
Interspecies sharing of two distinct nonstructural protein 1 alleles among human and animal rotaviruses as revealed by dot blot hybridization
Authors: Fujiwara Y , Nakagomi O
Published: January 2020

The distribution of the nonstructural protein 1 (NSP1) alleles from human strain AU-1 and canine strain K9 among rotaviruses of human, feline, canine, bovine, and simian origin was studied by a dot blot hybridization assay. Human and feline strains belonging to the AU-1 genogroup had the same NSP1 allele, while canine and feline strains belonging to the canine-feline genogroup shared another NSP1 allele. This canine-feline NSP1 allele had a significant level of homology with the NSP1 of rhesus rotavirus strain MMU18006.
Human salmonellosis associated with exotic pets
Authors: Woodward D L , Khakhria R , Johnson W M
Published: January 2020

During the period from 1994 to 1996, an increase in the number of laboratory-confirmed cases of human salmonellosis associated with exposure to exotic pets including iguanas, pet turtles, sugar gliders, and hedgehogs was observed in Canada. Pet turtle-associated salmonellosis was recognized as a serious public health problem in the 1960s and 1970s, and in February 1975 legislation banning the importation of turtles into Canada was enacted by Agriculture Canada. Reptile-associated salmonellosis is once again being recognized as a resurgent disease. From 1993 to 1995, there were more than 20,000 laboratory-confirmed human cases of salmonellosis in Canada. The major source of Salmonella infection is food; however, an estimated 3 to 5% of all cases of salmonellosis in humans are associated with exposure to exotic pets. Among the isolates from these patients with salmonellosis, a variety of Salmonella serotypes were also associated with exotic pets and included the following: S. java, S. stanley, S. poona, S. jangwani, S. tilene, S. litchfield, S. manhattan, S. pomona, S. miami, S. rubislaw, S. marina subsp. IV, and S. wassenaar subsp. IV.
Use of serum-specific immunoglobulins A and G for detection of Helicobacter pylori infection in patients with chronic gastritis by immunoblot analysis
Authors: Karvar S , Karch H , Frosch M , Burghardt W , Gross U
Published: January 2020

Multiple invasive and noninvasive tests for detecting Helicobacter pylori infection are available. The current "gold standard" for the diagnosis of H. pylori infection requires histology and the rapid urease test. Our aim was to test the performance of immunoglobulin A (IgA) and IgG immunoblot assays in comparison with that of the gold standard tests for the diagnosis of H. pylori infection. Ninety patients who underwent gastroscopy were analyzed in a prospective study. Fifty-nine of them were defined to be H. pylori positive by the gold standard tests. The IgA and IgG immunoblot assays correctly identified H. pylori infection in 17 and 58 of these patients, respectively, indicating that determination of IgA antibodies seems to be of low diagnostic value for H. pylori infection. In contrast, the sensitivity and specificity of the IgG immunoblot assay were 98 and 71%, respectively, with positive and negative predictive values of 87 and 96%, respectively. Therefore, the IgG immunoblot assay proved to be a sensitive and useful, noninvasive test for the diagnosis of H. pylori infection.
Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis
Authors: Carrière C , Riska P F , Zimhony O , Kriakov J , Bardarov S , Burns J , Chan J , Jacobs W R
Published: January 2020

TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.
Cutaneous hyalohyphomycosis caused by Fusarium solani in a loggerhead sea turtle (Caretta caretta L.)
Authors: Cabañes F J , Alonso J M , Castellá G , Alegre F , Domingo M , Pont S
Published: January 2020

Fusarium solani was reported as the agent of a cutaneous infection in an injured sea turtle collected in the Mediterranean Sea. The turtle was treated with both a topical 10% solution of iodine in alcohol and ketoconazole. The source of the causal agent was traced to the sand in the tank in which the turtle was maintained. The strain was only sensitive in vitro to amphotericin B and was resistant to 5-fluorocytosine, fluconazole, itraconazole, and ketoconazole.
A novel Western blot test containing both viral and recombinant proteins for anticytomegalovirus immunoglobulin M detection
Authors: Lazzarotto T , Maine G T , Dal Monte P , Ripalti A , Landini M P
Published: January 2020

We devised a novel Western blot (WB) test for anti-human cytomegalovirus (HCMV) immunoglobulin M (IgM) detection which contains viral structural polypeptides, significant portions of recombinant p150 (ppUL32), and a significant portion of the most immunogenic nonstructural protein p52 (ppUL44). This new test was evaluated in latently infected blood donors, pregnant women, and transplant recipients with ongoing HCMV infection and shown to be more sensitive and specific than traditional WB and conventional enzyme immunoassay for the detection of HCMV-specific IgM.
Specific PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients
Authors: Ombrouck C , Ciceron L , Biligui S , Brown S , Marechal P , van Gool T , Datry A , Danis M , Desportes-Livage I
Published: January 2020

A routine assay based on the PCR was developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples. Two oligonucleotide primer pairs from a conserved region in the small-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and SI500) were used to amplify microsporidian DNA. We achieved specific amplification of a 382-bp DNA fragment in E. intestinalis and a 353-bp DNA fragment in E. bieneusi. Boiling of the samples appeared to be most effective for DNA extraction. Fecal samples containing fewer than 10 microsporidia gave a positive result in the PCR assay. Fecal specimens from 30 human immunodeficiency virus-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by the PCR assay. The PCR assay was validated against standard staining methods (the Uvitex 2B and Chromotrope 2R staining methods) and immunofluorescence assay specific for E. intestinalis. This comparative study has shown that PCR improved species determination and can thus be considered a fast and reliable method for the detection and identification of each intestinal species.
Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis
Authors: Morace G , Sanguinetti M , Posteraro B , Lo Cascio G , Fadda G
Published: January 2020

A single primer pair amplifying a cytochrome P-450 lanosterol-14 alpha-demethylase (L1A1) gene fragment that encodes a highly conserved region was used to detect yeast DNA in clinical specimens. Positive PCR products were obtained from genomic DNAs of Candida albicans, C. parapsilosis, C. tropicalis, C. guilliermondii, C. krusei, C. (Torulopsis) glabrata, and C. kefyr. No human, bacterial, or parasitic DNA was amplified. The sensitivity was evaluated for C. albicans genomic DNA by using various DNA concentrations (200 pg to 2 fg). The amplified DNAs of Candida species with unknown P-450 L1A1 gene sequences were subcloned and sequenced. Identification at the species level was achieved by digestion of the PCR products with different restriction enzymes. A specific restriction enzyme analysis pattern was determined for each species investigated. Subsequently, we used PCR to detect specific yeast DNA directly with clinical specimens such as blood and bronchoalveolar lavage specimens. After appropriate treatment, the specimens were processed by PCR and the results were compared with those obtained by traditional diagnostic procedures such as cultures and serology. Although preliminary, the PCR results seem to correlate well, at least for blood, with those of antigen detection assays and traditional blood cultures, with a better and earlier detection of candidemia.
Temporal study of immunoglobin M seroreactivity to Borrelia burgdorferi in patients treated for Lyme borreliosis
Authors: Hilton E , Tramontano A , DeVoti J , Sood S K
Published: January 2020

Forty-six patients with late Lyme disease who were considered improved or cured following treatment were monitored by immunoglobin M (IgM) immunoblotting (mean monitoring period, 27.6 months). There was a persistent IgM response in 32 (97%) of 33 initially positive patients. All but three showed a consistent number, type, and intensity of IgM bands over the entire follow-up period. IgM immunoblotting may not be useful for monitoring the response to treatment of Lyme borreliosis.
Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria
Authors: Cattani P , Rossolini G M , Cresti S , Santangelo R , Burton D R , Williamson R A , Sanna P P , Fadda G
Published: January 2020

Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSV-specific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes.
Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR
Authors: Albert M J , Islam D , Nahar S , Qadri F , Falklind S , Weintraub A
Published: January 2020

In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V. cholerae O139 Bengal. PCR with the same primer pair was used to screen 180 diarrheal stool specimens. All the 67 V. cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR.
Simple color tests based on an alanyl peptidase reaction which differentiate Listeria monocytogenes from other Listeria species
Authors: Clark A G , McLaughlin J
Published: January 2020

The hydrolysis of DL-alanine-beta-naphthylamide and D-alanine-p-nitroanilide for identification of Listeria spp. has been studied with 227 cultures. All species of Listeria, except L. monocytogenes, hydrolyzed these substrates. The reactions were detected by simple chromogenic reactions and could substitute for the CAMP test.
Temperature-sensitive Steps in the Biosynthesis of Venezuelan Equine Encephalitis Virus
Authors: Zebovitz Eugene , Brown Arthur
Published: January 2020

In contrast to Eastern equine encephalitis virus, the replication of Venezuelan equine encephalitis (VEE) virus was strongly inhibited at 44 C in chick embryo cells. The inhibited steps were analyzed by shifting the incubating temperatures up or down, and by determining during the shifts the rate and extent of infectious ribonucleic acid (RNA) synthesis, intact virus synthesis, and formation of complement-fixing antigen or of antigen detectable by a direct fluorescent-antibody technique. The inhibition appeared to be due to two temperature-sensitive steps involved in the synthesis of VEE virus in chick embryo cells. The first step of inhibition at 44 C occurred early in virus replication and could be completely reversed simply by transferring cultures to 37 C. The inhibition appeared to take place at some point between the time when the virus entered the cell and was uncoated and the beginning of viral RNA synthesis. The second temperature-sensitive step in VEE virus synthesis was irreversible; it occurred at a point after the synthesis of viral RNA, and before the formation of virus protein measured as complement-fixing antigen or as antigen that could be stained with fluorescent antibody.
Immuno-Electron Microscopy of the Morphogenesis of Mumps Virus
Authors: Duc-Nguyen Huu , Rosenblum Edith N.
Published: January 2020

The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy.
Characteristics of a Lytic Enzyme Induced by Bacteriophage Infection of
Micrococcus lysodeikticus
Authors: Goepfert J. M. , Naylor H. B.
Published: January 2020

A lytic enzyme induced in
Micrococcus lysodeikticus
strain 1 by infection with N1 bacteriophage was purified 45- to 50-fold by ammonium sulfate precipitation, acid precipitation, and selective adsorption of contaminating proteins with calcium phosphate gel. The optimal
p
H for activity of the enzyme was 6.5 to 7.0. Maximal activity occurred at 45 to 50 C and at an ionic strength of 0.06. The enzyme had a limited specificity and lysed cell walls of
M. lysodeikticus
with the release of dinitrofluorobenzene reactive groups. Living cells were lysed in the absence of phage; however, the rate of lysis increased when phage was present in excess of 10 particles per bacterial cell. Young cells were most sensitive, and the sensitivity decreased to a minimum with stationary-phase cells. Acting synergistically, lysozyme and the N1-induced lysin caused lysis of cells which were resistant to either enzyme acting independently. The N1 lysin did not exhibit proteolytic activity.
Nonproducing State of Rous Sarcoma Cells: Its Contagiousness in Chicken Cell Cultures
Authors: Vogt Peter K.
Published: January 2020

Nonproducing Rous sarcoma cells of the chicken were capable of transmitting the Rous sarcoma virus genome to neighboring chick embryo fibroblasts. This transfer required close proximity of sarcoma and normal cells and may have been mediated by a subcellular infectious agent which was found to be released from nonproducing cells.
Intracellular Uncoating of Type 5 Adenovirus Deoxyribonucleic Acid
Authors: Lawrence William C. , Ginsberg Harold S.
Published: January 2020

Highly purified,
32
P-labeled type 5 adenovirus was employed to study “uncoating” of viral deoxyribonucleic acid (DNA)—defined as the development of sensitivity to deoxyribonuclease. Viral infectivity and radioactivity adsorbed to KB cells at the same rate, and significant amounts of
32
P did not elute from cells throughout the eclipse period. Kinetic studies of viral penetration, eclipse of infectivity, and uncoating of viral DNA indicated that the three events were closely related temporally, that the rates of each were similar, and that they were completed within 60 to 90 min after infection. Viral penetration, eclipse, and uncoating proceeded normally under conditions which blocked protein synthesis, but they did not occur at 0 to 4 C. Neither viral DNA nor viral protein was degraded to acid-soluble material during the eclipse period. The nature of adenovirus DNA was studied after it was converted intracellularly from deoxyribonuclease-resistant to deoxyribonuclease-susceptible. Intact virions centrifuged in sucrose gradients had a sedimentation coefficient of approximately 800, and viral DNA sedimented as a particle of about 30
S
. Infection of KB cells with purified
32
P-labeled virus yielded deoxyribonuclease-susceptible viral nucleic acid which was in particles with sedimentation coefficients of 350 to 450
S
, i.e., greater than 10 times faster than DNA obtained from purified virions which had been disrupted by exposure to
p
H 10.5. When the DNA from disrupted virions was mixed with cell lysates, its sedimentation characteristics were essentially unchanged by the presence of cellular material.
Separation and Characterization of Soluble Adenovirus Type 9 Components
Authors: Norrby E. , Nyberg B. , Skaaret P. , Lengyel A.
Published: January 2020

Four different soluble components of adenovirus type 9 (Rosen's group II) were identified. These were a complete hemagglutinin (HA), an incomplete HA, components carrying group-specific complement-fixing (CF) antigen, and components identified only by their hemagglutination-inhibition (HI) antibody consuming capacity and antigen activity in CF tests with an antiserum against complete HA. The complete HA sedimented relatively rapidly. It was composed of 12 pentons (vertex capsomers plus projections) aggregated into the form of a pentagonal dodecahedron. The length of the projections was about 12 to 13 mμ. Thus they appeared longer than the corresponding structures of types 3 and 11, but shorter than those of types 4 and 5. The rate of sedimentation of complete HA of type 9 was intermediate to those of the complete HA of types 3 and 11. The incomplete HA sedimented together with components carrying group-specific CF antigen, but could be separated from those by anion-exchange chromatography. Two different antigens were present in incomplete HA. One could absorb a group-specific hemagglutination-enhancing antibody, and was sensitive to treatment with trypsin. The other antigen could absorb the type-specific HI antibody and was not destroyed by trypsin. In addition to the incomplete HA, a separate population of more slowly sedimenting components showed a capacity to absorb HI antibody. These components could also be identified in CF tests when an antiserum against complete HA was applied. The incomplete HA, group-specific CF antigen, and slowly sedimenting HI antibody absorbing components are suggested to represent isolated penton, hexon, and fiber components, respectively.
Interferon Production in Mice by Cell Wall Mutants of
Salmonella typhimurium
Authors: Youngner Julius S. , Feingold David S.
Published: January 2020

The interferon response elicited by
Salmonella typhimurium
mutants in mice is not dependent on the presence of a complete cell wall lipopolysaccharide. In fact, a mutant (G30/C21) which has lost all the polysaccharide side chains and sugars of the O antigen and contains only 2-keto-3-deoxyoctonate and lipid is indistinguishable in its interferon-stimulating ability from the wild type which possesses a complete O antigen with polysaccharide side chains.
Morphology and Nucleic Acid Composition of the
Azotobacter
Bacteriophages
Authors: Knovicka James , Pope Leodocia , Wyss Orville
Published: January 2020

Long-tailed azotophages of two different serological groups have deoxyribonucleic acid species whose densities correspond to a molar percentage of guanine plus cytosine of 52 to 53. Similar measurements on the short-tailed phages gave values of 56 to 57 moles percent. The azotophages include representatives of the three morphological types classified by Bradley as groups A, B, and C.
Filamentous Bacterial Viruses V. Asymmetric Replication of fd Duplex Deoxyribonucleic Acid
Authors: Tseng B. Y. , Marvin D. A.
Published: January 2020

Short pulses (30 sec at 32 C) of
3
H-thymidine were found primarily in the viral strands of replicating fd deoxyribonucleic acid (DNA), even at a time when most DNA being synthesized was duplex DNA. Much of the labeled viral strand DNA was longer than unit length, but some was shorter than unit length. Most of the corresponding complementary-strand DNA was recovered in closed supercoiled duplex molecules, even for short pulses; the remainder of the complementary-strand DNA was found in replicative intermediates in pieces shorter than unit length. Some of the viral strands in open replicating DNA lacked a corresponding complementary strand.
Adenylate-Rich Sequences in Vesicular Stomatitis Virus Messenger Ribonucleic Acid
Authors: Ehrenfeld Ellie , Summers Donald F.
Published: January 2020

Vesicular stomatitis virus (VSV)-specific messenger ribonucleic acid (mRNA) species contain sequences of adenylate-rich RNA which are more heterogeneous in their migration through sodium dodecyl sulfate-polyacrylamide gels than the corresponding fractions from HeLa cell mRNA. VSV virion RNA contains no adenylaterich sequences. The possible role of such sequences in the mRNA species of a cytoplasmically replicating virus is discussed.
Cleavage of Viral Precursor Proteins In Vivo and In Vitro
Authors: Korant B. D.
Published: January 2020

The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.
Host-Bacteriophage Interaction in
Agrobacterium tumefaciens
I. Characterization of Bacteriophage R4 and Physiological Changes in the Infected Host Cell
Authors: Adler Richard , Pootjes Christine
Published: January 2020

Infection of
Agrobacterium tumefaciens
B6, a tumor-producing plant pathogen, by bacteriophage R4, does not immediately shut off host deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis. Viral DNA synthesis begins soon after infection, but the host DNA is not shut off until after 35 min; net RNA and protein synthesis are not inhibited until 30 min after infection. The pattern of synthesis of phage particles was confirmed by electron microscopy of thin sections during the infection cycle. The phage particle consists of a polyhedral head, 65 nm in diameter, and a long flexible tail 210 nm long and 10 nm wide with helically arranged subunits. By gel electrophoresis, four major protein components with the following molecular weights were found in the capsid: 72,000, 45,000, 28,000, and 14,500. The phage DNA has a molecular weight of 30 million and a guanine-cytosine content of 59%.
Electron Microscopical and Biochemical Characterization of Infectious Pancreatic Necrosis Virus
Authors: Kelly Russell K. , Loh Philip C.
Published: January 2020

An electron microscopical and biochemical examination of the properties of infectious pancreatic necrosis virus (IPN) and of its ribonucleic acid (RNA) was made. The buoyant density of IPN in CsCl was found to be 1.33 g/cm
3
. Electron microscopical examination of the banded virus revealed structures similar in size (74 nm) and shape to reoviruses but lacking a characteristic inner capsid structure. Polyacrylamide gel electrophoretic analysis of IPN-RNA revealed a single non-segmented component of molecular weight 3.2 × 10
6
. Its susceptibility to ribonuclease, base composition, and resistance to thermal denaturation indicated a single-stranded RNA structure. However, its sedimentation behavior (16
S
) independent of ionic strength in sucrose gradients, partial solubility in 2
m
LiCl, and ribonuclease resistance in the presence of Mg
2+
suggest an unusual secondary structure of unknown nature. The accumulated data indicate that IPN virus does not belong to either the picornavirus or reovirus groups and may represent a new group of viruses.
Ligase-Defective Bacteriophage T4 II. Physiological Studies
Authors: Koch Robert E.
Published: January 2020

The timing of the suppression of gene 30 (deoxyribonucleic acid ligase) mutations by
rII
mutations was studied by temperature shift-down experiments with a temperature-sensitive
rII
mutation. The
rII
function must remain inactivated for about 5 to 8 min at 37 C for suppression to occur, thus making suppression an early function. This result is in agreement with the timing of expression of other
rII
functions. A gene 30 defect can also be overcome by replacing the Na
+
cation in the growth medium with the Mg
2+
cation, a result similar to the relief of the lethality of
rII
mutations in lambda lysogens. Prior infection with bacteriophages T3 or T7, which produce their own deoxyribonucleic acid ligases, can also partially overcome the lethality of gene 30 mutations.
Patterns of Simian Virus 40 Deoxyribonucleic Acid Transcription II. In Transformed Cells
Authors: Khoury George , Byrne Janet C. , Takemoto Kenneth K. , Martin Malcolm A.
Published: January 2020

The pattern of simian virus 40 (SV40) deoxyribonucleic acid transcription has been examined in 11 SV40-transformed cell lines. In all cases, substantial regions of the minus strand (35-75%) appeared to be transcribed. In the lines tested, these regions included the “early” gene sequences. The SV40-specific ribonucleic acid from at least two of the transformed cell lines represented significantly greater portions of the minus strand than are represented in “early” lytic ribonucleic acid. Small regions of the plus strand appeared to be transcribed in only two of the transformed cell lines.
Characterization of Nucleic Acid of Pichinde Virus
Authors: Carter Michael F. , Biswal Nilambar , Rawls William E.
Published: January 2020

The nucleic acid of Pichinde virus was found to be single-stranded ribonucleic acid (RNA) as determined by sensitivity to ribonuclease, by alkaline degradation, by buoyant density in cesium sulfate, and by analysis of the base composition. The RNA of the virion could be separated into five components which had sedimentation coefficients corresponding to 31
S
, 28
S
, 22
S
, 18
S
and 4 to 6
S.
The 28
S
, 18
S
, and possibly the 4 to 6
S
RNAs appear to be derived from host cell components incorporated into the virion, whereas the 31
S
and 22
S
components appear to represent the genome of the virus.
Replication of Simian Virus 40 Deoxyribonucleic Acid: Analysis of the One-Step Growth Cycle
Authors: Manteuil Simone , Pages Jacqueline , Stehelin Dominique , Girard Marc
Published: January 2020

The time course of replication of simian virus 40 deoxyribonucleic acid (DNA) was investigated in growing monolayer cultures of subcloned CV1 cells. At multiplicities of infection of 30 to 60 plaque-forming units (PFU)/cell, first progeny DNA molecules (component 1) were detected by 10 hr after infection. During the following 10 to 12 hr, accumulation of virus DNA proceeded at ever increasing rates, albeit in a non-exponential fashion. The rate of synthesis then remained constant, until approximately the 40th hour postinfection, when DNA replication stopped. Under these conditions, the duration of the virus growth cycle was approximately 50 hr. The time needed for the synthesis of one DNA molecule was found to be approximately 15 min. At multiplicities of infection of 1 or less than 1 PFU/cell, the onset of the linear phase of DNA accumulation was delayed, but the final rate of DNA synthesis was the same, independent of the input multiplicity. This was taken as a proof that templates for the synthesis of viral DNA multiply in the cell during the early phase of replication. However, the probability for every replicated DNA molecule to become in turn replicative decreased constantly during that phase. This could be accounted for by assuming a limited number of replication sites in the infected cell.
Inhibition in the Joining of DNA Intermediates to Growing Simian Virus 40 Chains
Authors: Salzman Norman P. , Thoren Marilyn M.
Published: January 2020

Viral DNA synthesis was inhibited for 1 h by the addition of 5-fluorodeoxyuridine (FUdR) to simian virus 40 (SV40)-infected cultures at 28 to 30 h postinfection. The subsequent addition of
3
H-thymidine to the inhibited cultures reverses the effect of the inhibitor, and during a 1-min labeling period there is rapid synthesis of SV40 DNA. By alkaline sedimentation analysis, it is observed that in FUdR-treated cultures there is synthesis of 4
S
SV40 DNA intermediates but there is a block in the joining of these intermediates to growing SV40 chain cultures. In addition to 4
S
fragments that are associated with replicating SV40 molecules, there is accumulation of SV40 DNA in the 6 to 8
S
region which is observed in neutral sucrose gradients. In an inhibited culture that is pulsed for 1 min with
3
H-thymidine and then chased for 10 min, accumulation of a Component II (Comp. II)-like material is observed. This Comp. II has the same neutral sedimentation characteristics and yields the same R
I
restriction endonuclease product as does authentic Comp. II. However, in alkali it is seen that it is composed of fragmented SV40 DNA. The basis for the failure of 4
S
fragments to join to growing SV40 chains is discussed. A model in which there is a requirement for two DNA polymerases and a ligase to permit SV40 DNA chain growth is proposed which is consistent with the data presented.
RNA Synthesis in Cells Infected with Herpes Simplex Virus VII. Control of Transcription and of Transcript Abundancies of Unique and Common Sequences of Herpes Simplex Virus 1 and 2
Authors: Frenkel Niza , Silverstein Saul , Cassai Enzo , Roizman Bernard
Published: January 2020

Analysis of the kinetics of hybridization in liquid of labeled herpes simplex virus (HSV) 1 and 2 DNAs with excess unlabeled RNA extracted at 2 (early) and 8 (late) h postinfection revealed the following. (i) The RNA transcripts present in the HSV-1-infected cells at 2 and 8 h postinfection are complementary to 44 and 48% of HSV-1 DNA. The RNA transcripts present in the HSV-2-infected cells at 2 and 8 h postinfection are complementary to 21 and 50% of HSV-2 DNA. (ii) The transcripts present in 2-h HSV-1- or HSV-2-infected cells treated with cycloheximide are complementary to 44 and 45% of the respective DNAs. (iii) The RNA transcripts present in the HSV-1-infected cells at 2 h postinfection and in HSV-2-infected cells at 8 h postinfection form 2 classes, abundant and scarce, differing in molar concentrations. The RNA transcripts present in the HSV-2-infected cells at 2 h postinfection form only one abundance class. (iv) The transcripts present in the HSV-1-infected cells at 8 h postinfection are complementary to 24% of HSV-2 DNA and therefore 50% of the transcribed HSV-1 sequences are shared by the two viruses. Of the RNA sequences complementary to HSV-2 DNA, 13% arise from HSV-1 templates specifying abundant RNA and 11% arise from HSV-1 templates specifying scarce RNA. Thus, the DNA sequences shared in common by HSV-1 and HSV-2 DNAs constitute 71% of the HSV-1 templates specifying abundant RNA and 39% of sequences specifying scarce RNA.
Comparison of the Lipids of Intracellular and Extracellular Rabies Viruses
Authors: Schlesinger Harvey R. , Wells Henry J. , Hummeler Klaus
Published: January 2020

The lipid composition of both intracellular and extracellular forms of the ERA strain of rabies virus grown in BHK/21 cells was determined. The lipids from purified preparations of both intracellular and extracellular virus yielded 57 and 58% neutral lipid, respectively. The phospholipids of the intracellular and extracellular virus constituted 43 and 42%, respectively. Triglyceride and cholesterol appear to be the major neutral lipids, whereas sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine comprise the major bulk of phospholipid in both virus types. The molar ratio of cholesterol to phospholipid was 0.87 (intracellular) and 0.92 (extracellular). On the basis of the data presented, it is reasonable to assume that the lipids of both intracellular and extracellular rabies virus are similar.
Replication of Bacteriophage SH-133 in the Facultative Autotroph
Hydrogenomonas facilis
I. Bacteriophage Synthesis Under Heterotrophic, Autotrophic, and Mixotrophic Growth Conditions
Authors: Aron Gary M. , Pootjes Christine F.
Published: January 2020

The ability of bacteriophage SH-133 to replicate in heterotrophically (H-) and autotrophically (A-) grown
Hydrogenomonas facilis
was examined. Both the synthesis of infectious phage particles and the efficiency of plating (EOP) were reduced by 90% in A-grown cells. Adsorption of phage and lethal effects on
H. facilis
were identical in both systems. One-step growth experiments showed that cell lysis preceded the appearance of infectious particles in A-grown cells. Burst size studies with mixotrophically grown cells did not indicate the presence of an inhibitor of phage synthesis indigenous to autotrophic metabolism. DNA synthesis was identical in H- and A-grown infected cells; however, protein synthesis was significantly reduced in A-grown infected cells when compared with protein synthesis in H-grown infected cells. The data suggest that the reduction in EOP and phage synthesis in A-grown cells is caused by a defect in viral protein synthesis which results in the limited production of an essential viral protein at the time of cell lysis.
Isolation and Characterization of a Bacteriophage for
Thiobacillus novellus
Authors: Johnson K. , Chow C. T. , Lyric R. M. , Van Caeseele L.
Published: January 2020

A DNA-containing bacteriophage for
Thiobacillus novellus
has been isolated from sewage and purified by ammonium sulfate precipitation, differential centrifugation, and cesium chloride equilibrium centrifugation. The buoyant density of this phage in CsCl is 1.51 g/cm
3
. Electron microscopy studies have revealed a polyhedral head about 60 nm in diameter and a tail surrounded by a number of fine filaments. It has an adsorption rate constant of 1.1 × 10
−9
ml/min, a latent period of 45 min, and an average burst size of 150. The mole guanine and cytosine content in its DNA has been estimated to be 57 to 58%. Five structural proteins with molecular weights of 62,000, 42,500, 30,500, 17,750, and 13,500 have been detected by polyacrylamide gel electrophoresis techniques.
Isolation and Preliminary Characterization of the RNA-Containing R-Type, Virus-Like Particle of BHK-21 Cells
Authors: Albu Evelyn , Holmes Kathryn V.
Published: January 2020

An R-type virus-like particle (VLP) has been isolated from the medium of BHK-21-F cells by ultracentrifugation and polyethylene glycol precipitation. The R-type VLP contains RNA which sediments at 60 to 70
S
in sucrose density gradients and has a molecular weight of approximately 10
7
, as estimated by gel electrophoresis. The R-type VLP can be labeled with
3
H-uridine in the presence of actinomycin D. On the basis of morphology, site of maturation, and preliminary biochemical characterization, the R-type VLP does not appear to fit into any of the major groups of animal viruses.
Effects of Polyadenylic Acids on Functions of Murine RNA Tumor Viruses
Authors: Tennant Raymond W. , Farrelly James G. , Ihle James N. , Pal B. C. , Kenney Francis T. , Brown Arthur
Published: January 2020

Single-stranded polyribonucleotides, which competitively inhibit murine RNA tumor virus reverse transcriptase in vitro, were tested as inhibitors of various virus functions in cell culture. The compounds had two concentration-dependent effects. At high concentrations (100 μg/ml), both poly(adenylic acid) [poly(A)] and poly(2′-
O
-methyladenylic acid) [poly(Am)] inhibited the uptake of radioactively labeled leukemia virus by Swiss mouse embryo cells, but neither had a similar effect on Sindbis virus adsorption. At low concentrations (10 μg/ml), poly(Am) did not inhibit the uptake of leukemia virus but did inhibit virus replication by 85%; in contrast, the replication of Sendai virus and Sindbis virus was not inhibited significantly at this concentration. Both compounds were effective only when added prior to or early during virus infection. Poly(Am) was a much more effective inhibitor than poly(A), probably due to the nuclease resistance of the former compound. Poly(Am) at 5 μg/ml also inhibited transformation of 3T3 cells by Moloney sarcoma virus. However, neither poly(A) nor poly(Am) at high concentrations inhibited the activation of endogenous leukemia virus by iododeoxyuridine in AKR mouse embryo cells. These results suggest that virus reverse transcriptase plays an essential role in both the replication of exogenous murine leukemia viruses and the transformation of cells by murine sarcoma viruses but probably has no role in the activation of endogenous leukemia virus.