bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of synthetic antigens improves detection by enzyme-linked immunosorbent assay of antibodies against abortigenic Chlamydia psittaci in ruminants
Authors: Kaltenboeck B , Heard D , DeGraves F J , Schmeer N
Published: January 2020

Synthetic peptide antigens were prepared for use in enzyme-linked immunosorbent assays (ELISAs) to detect serum antibodies against abortigenic strains of Chlamydia psittaci in livestock. Peptide antigens were identified with C. psittaci B577-immune sera by solid-phase scanning of overlapping octapeptides of variable domains (VDs) of the major outer membrane protein of C. psittaci serovar 1 (omp1 type C. psittaci B577). Two VD 4 regions and one VD 2 region were strongly reactive with all C. psittaci B577 antisera. Peptides encompassing these regions were synthesized with biotin and a serine-glycine-serine-glycine spacer at the N terminus and were attached to streptavidin-coated microtiter plates. In direct ELISAs with these plates, the synthetic peptides reacted with C. psittaci B577 antisera, but not with sera from specific-pathogen-free animals. Serum specimens from 40 sheep and 40 cattle, obtained from herds with abortion problems, were screened for antibodies by these C. psittaci B577 peptide ELISAs and an ELISA with recombinant, genus-specific Chlamydia lipopolysaccharide (LPS) antigen. Results from these newly developed ELISAs were compared to those from the reference C. psittaci B577 elementary body (EB) ELISA and the Chlamydia complement fixation test (CFT). The C. psittaci B577 peptide ELISAs, the LPS ELISA, and the EB ELISA correctly identified the presence or absence of antibodies against chlamydiae in all sheep and bovine sera. The Chlamydia CFT, which is the most widely accepted serodiagnostic method for chlamydial infections in animals, correctly identified the presence or absence of antibodies against chlamydiae in only 78 and 4.9% of sheep and bovine sera, respectively. These results suggest that the C. psittaci B577-peptide and Chlamydia LPS ELISAs are superior for the serodiagnosis of ruminant infections with abortigenic chlamydiae, since they are more sensitive than the CFT, they are easy to standardize, and they use readily available synthetic antigens instead of organism-derived CFT antigen.
Molecular epidemiology and antibiotic susceptibility of enterococci in Cincinnati, Ohio: a prospective citywide survey
Authors: Perlada D E , Smulian A G , Cushion M T
Published: January 2020

To determine patterns of antimicrobial susceptibility among enterococci and to assess molecular characteristics of vancomycin-resistant enterococci, 157 clinical blood isolates of enterococci from 10 hospitals in Cincinnati, Ohio, were prospectively collected during a 6-month period from February to July 1995. The isolates included 108 (69%) E. faecalis isolates, 46 (29%) E. faecium isolates, and 1 isolate each of E. avium, E. durans, and E. gallinarum. The E. faecalis and E. faecium isolates differed in their susceptibilities to ampicillin (100 versus 20%), ampicillin-sulbactam (100 versus 13%), vancomycin (100 versus 57%), imipenem (94 versus 2%), and high levels of gentamicin (59 versus 83%). Supplemental susceptibility testing of the 21 vancomycin-resistant isolates showed that 21 (100%) were susceptible to chloramphenicol and that only 7 (33%) were susceptible to doxycycline. Nineteen (90%) of the vancomycin-resistant E. faecium isolates were of the VanB phenotype, with vanB resistance genes detected by PCR and hybridization with gene-specific probes; and the E. gallinarum isolates demonstrated the VanC phenotype with the vanC1 gene. One vancomycin-resistant E. faecium isolate was highly resistant to both teicoplanin and vancomycin, corresponding to the VanA phenotype; however, it was found to have the vanB gene. Pulsed-field gel electrophoresis (PFGE) revealed that all of the 19 E. faecium isolates with the VanB phenotype had identical to closely related banding patterns. Hybridization of restriction enzyme-digested DNA separated by PFGE with a vanB gene probe demonstrated differences in the locations of vanB genes that corresponded closely to the PFGE banding patterns. Our study has documented that the emerging vancomycin resistance in our city was mainly due to the clonal dissemination of a single strain of E. faecium VanB.
Identification of clinical isolates of indole-positive Klebsiella spp., including Klebsiella planticola, and a genetic and molecular analysis of their beta-lactamases
Authors: Liu Y , Mee B J , Mulgrave L
Published: January 2020

In a collection of 43 indole-positive Klebsiella clinical isolates, which were initially identified as Klebsiella oxytoca, there were 18 isolates which exhibited a pattern characteristic of extended-spectrum beta-lactamase (ESBL) resistance. This study aimed to confirm their identity by biochemical tests and by PCR and to determine the genetic basis for their resistance to the beta-lactams and broad-spectrum cephalosporins. Chromosomal beta-lactamase genes were analyzed by PCR, and plasmid-mediated beta-lactamase genes were analyzed by conjugation and transformation. There were 39 isolates which grew on melezitose but failed to grow on 3-hydroxybutyrate, confirming them as K. oxytoca. PCR analysis of their beta-lactamase genes divided these isolates into two groups, the bla(OXY-1) group and the bla(OXY-2) group. Each group had beta-lactamases with different isoelectric points; the bla(OXY-1) group had beta-lactamases with isoelectric points at 7.2, 7.8, 8.2, and 8.8, and the more common bla(OXY-2) group had beta-lactamases with pIs at 5.2, 5.4 (TEM-1), 5.7, 5.9, 6.4, and 6.8. A pI of 5.2 was the most frequently detected and accounted for 59% of all the bla(OXY-2) beta-lactamases. Hyperproduction of clavulanate-inhibited chromosomal beta-lactamases was detected in 17 K. oxytoca isolates, resulting in an ESBL phenotype. K. oxytoca isolates having a plasmid-mediated genetic basis for their ESBL phenotype were not found, confirming that, in K. oxytoca, plasmids are rarely involved in conferring resistance to the newer cephalosporins. Four isolates proved to be isolates of K. planticola in which the beta-lactamase genes failed to react with the primers used in the PCR. One K. planticola isolate contained a transferable plasmid harboring the SHV-5 beta-lactamase gene and showed an ESBL phenotype, while the other non-ESBL K. planticola isolates contained chromosomal beta-lactamases with isoelectric points at 7.2, 7.7, and 7.9 plus 7.2.
Study of Norwalk virus and Mexico virus infections at Ga-Rankuwa Hospital, Ga-Rankuwa, South Africa
Authors: Smit T K , Steele A D , Peenze I , Jiang X , Estes M K
Published: January 2020

Small round-structured viruses (SRSVs) or Norwalk-like viruses have been implicated as an important causative agent of gastroenteritis outbreaks. We used the relatively newly developed recombinant enzyme immunoassays (EIAs) to determine the seroprevalence of Norwalk virus (NV) and Mexico virus (MxV) in a family-based cohort and an antenatal clinic cohort at Ga-Rankuwa Hospital, Ga-Rankuwa, South Africa. High prevalences (96 to 99%) of anti-NV and anti-MxV antibodies were detected in both cohorts. We also investigated the pattern of antibody acquisition in a cohort of infants and young children without gastroenteritis and found that by 48 months of age all children had acquired adult antibody levels to both these viruses. Lastly, we tested 276 stool specimens collected from infants and young children with gastroenteritis for the presence of NV or MxV antigen by recombinant EIAs to each virus, by electron microscopy (EM), and by reverse transcription (RT)-PCR. NV and MxV antigens were present in 1.8 and 4.3% of the stool specimens, respectively, by the recombinant EIAs; 9.2% were positive for SRSVs by EM, and 25% of these SRSVs gave a positive result by RT-PCR for primer pair 35-36 directed to a region of the RNA-dependent RNA polymerase gene. The seroprevalence studies indicate a high level of exposure to these viruses in both children and adults. Although the viral antigens are not highly prevalent in diarrheal stools, it was determined by the two assays for NV and MxV that children are, nevertheless, infected early in life.
Tampon sampling for diagnosis of bacterial vaginosis: a potentially useful way to detect genital infections?
Authors: Wilkinson D , Ndovela N , Kharsany A , Connolly C , Sturm A W
Published: January 2020

Genital tract infections are important causes of ill health in developing countries, but diagnosis is difficult. Bacterial vaginosis (BV) was correctly diagnosed by using a vaginal specimen obtained by tampon sampling in 22 of 24 women (91.6%) for whom BV was diagnosed by Gram staining. The yield for other vaginal infections was higher (28% for Trichomonas vaginalis and 32.7% for Candida albicans) than it was for cervical infections (0% for Neisseria gonorrhoeae and 30% for Chlamydia trachomatis). Tampon sampling was acceptable to patients and may facilitate diagnosis of genital infections in developing countries.
Evaluation of a commercial ligase chain reaction kit (Abbott LCx) for direct detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens
Authors: Tortoli E , Lavinia F , Simonetti M T
Published: January 2020

Direct detection of Mycobacterium tuberculosis by means of a commercial ligase chain reaction DNA amplification method (LCx M. tuberculosis; Abbott Diagnostics Division, Abbott Park, Ill.) was investigated with 511 (including 147 extrarespiratory) specimens collected from 358 patients. LCx results were compared with standard microbiological data, and conflicting cases were resolved according to the final clinical diagnosis. M. tuberculosis was detected in 45 of 358 subjects by means of the LCx test. The test was negative for all 30 specimens with mycobacteria other than M. tuberculosis. The sensitivity, specificity, and positive and negative predictive values for the LCx test, compared with culture results, were 93.90, 92.31, 70.00, and 98.75%, respectively; these values rose in resolved cases to 95.53, 99.25, 97.27, and 98.75%, respectively. With respiratory specimens, for which the LCx system is licensed, the sensitivity reached 98.97%. In patients with a final clinical diagnosis of tuberculosis the sensitivity of the LCx system was 89.36% compared to 82.98% for cultures and 78.72% for microscopy. We conclude that the LCx test is user friendly, rapid, fairly sensitive, and highly specific. It can also be effectively used on extrapulmonary specimens provided possible false-negative results are taken into account. However, the use of LCx test appears to be less appropriate for the monitoring of antituberculosis therapy, as the majority of samples from treated tuberculosis patients gave consistently positive results, despite the sterilization of cultures.
Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication
Authors: Salzman N. P. , Sebring E. D.
Published: January 2020

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.
Induced Structural Defects in T-Even Bacteriophage
Authors: Cummings Donald J. , Chapman V. A. , De Long S. S. , Mondale L.
Published: January 2020

Multiple aberrant substructures of T-even bacteriophage particles occurred when amino acid analogues or antimetabolites were present during phage growth. Certain aberrant substructures were induced by specific analogues or antimetabolites. In particular, it was observed by electron microscopy that
l
-canavanine, an arginine analogue, gave rise to polyheads;
l
-azetidine-2-carboxylic acid, a proline analogue, gave rise to polytail tubes; and 1,2,4-trizaole-3-alanine, a histidine analogue, proflavine, and actinomycin D all gave rise to small heads. These aberrant substructures were similar to those reported earlier with conditional lethal mutants (amber) of T4D in a restrictive host.
Effect of Resistance to Chloramphenicol on Bacteriophage Sensitivity of Group A Streptococci
Authors: Friend Patric L. , Slade Hutton D.
Published: January 2020

Several phage hosts of group A streptococci became resistant to lysis by bacteriophage as a consequence of having acquired the ability to grow in the presence of chloramphenicol. The phage was adsorbed to the streptococcal cell, and P
32
-labeling of the phage showed that the phage genome penetrated the chloramphenicol (CM)- resistant cells as it did the parent cells. However, artificial lysis of the infected CM-resistant cells with chloroform or enzymes revealed no intracellular mature phage particles. Lysates of infected CM-resistant cells contained no phage-related antigenic materials which possessed serum-blocking power, although they were readily detected in lysates of infected parent cells. The CM-resistant cells were not lysogenized by the phage. Only cells resistant to more than 10 μg/ml of chloramphenicol were resistant to phage, and this threshold effect was taken as an indication of at least two different loci of chloramphenicol resistance on the streptococcal genome. Strains resistant to high levels of other antibiotics, such as streptomycin and erythromycin, showed no resistance to lysis by phage. Evidence indicated that the mutant cells were deficient in an essential function associated with the phage genome.
Behavior of λ Bacteriophage in a Recombination Deficient Strain of
Escherichia coli
Authors: Brooks Katherine , Clark Alvin J.
Published: January 2020

The behavior of λ phage in the Rec
−
strain JC-1569 is compared with that in the Rec
+
strain JC-1557. No difference deemed significant was noted in the adsorption rate, latent period, burst size, frequency of lysogenization, and frequency of vegetative phage recombination. The location of the prophage and its mode of insertion in the Rec
−
lysogen of wild-type λ (λ
+
) were inferred to be normal from the results of conjugational crosses. Spontaneous and ultraviolet (UV) irradiation induction of λ
+
were markedly reduced in the Rec
−
lysogen. On the other hand, thermal induction of a mutant lambda (λ
cI857
) lysogen of the Rec
−
strain was not reduced and was only slightly affected by UV irradiation. Phage subject to inhibition by λ immunity failed to multiply in UV-irradiated cells of the Rec
−
λ
+
lysogen, whereas those not inhibited by this immunity did multiply. It was concluded that the failure of UV to induce λ
+
in the Rec
−
lysogen was not due to damage to the prophage, but rather to the inability of the irradiated cells to respond by lifting immunity. Preliminary evidence indicates that a single mutation confers recombination deficiency and the inability to lift immunity after UV irradiation. Possible relationships between recombination and the lifting of immunity are enumerated.
Herpes Simplex Virus Products in Productive and Abortive Infection I. Stabilization with Formaldehyde and Preliminary Analyses by Isopycnic Centrifugation in CsCl
Authors: Spring Susan B. , Roizman Bernard
Published: January 2020

Lysates of HEp-2 cells productively infected with herpes simplex virus yielded two bands on isopycnic centrifugation in CsCl gradients, ranging from 1.2 to 1.6 g/cm
3
. One band, designated α, had a mean buoyant density of 1.27 g/cm
3
and contained herpes virions. Band β had a mean density of 1.305 g/cm
3
and contained primarily complement-fixing viral antigens and little or no viral deoxyribonucleic acid (DNA). The products banding in the α and β bands were unstable; fivefold or higher amounts were recovered by treating the cell extract with formaldehyde prior to centrifugation. Formaldehyde treatment increased the buoyant density of viral products in both the α and β bands by about 0.015 g/cm
3
. In addition, it stabilized hitherto inapparent products, forming a broad band γ with a density range of 1.37 to 1.45 g/cm
3
. The material in the γ band was heterogeneous; it contained viral DNA, cellular DNA, and viral antigen. Formalinized lysates of DK cells abortively infected with herpes simplex virus yielded a β band undifferentiated from that formed by extracts of productively infected cells. The γ band was less dense and narrower. The α band was entirely missing.
Soluble Antigens of Vaccinia-infected Mammalian Cells II. Time Course of Synthesis of Soluble Antigens and Virus Structural Proteins
Authors: Wilcox Wesley C. , Cohen Gary H.
Published: January 2020

Virus-induced soluble antigens produced in mammalian cells after infection with vaccinia virus can be divided into two classes on the basis of molecular weight. Synthesis of the low molecular weight antigens begins early in the course of infection (1 to 2 hr), and is switched-off rather abruptly 4 to 5 hr after infection in a manner similar to that reported for the early enzymes characteristic of this same system. It was demonstrated, however, that these antigens do not include virus-induced thymidine kinase, a major virus-induced enzyme, nor is it likely that the low molecular weight antigens described here share identity with any of the virus-induced enzymes. A portion of the low molecular weight antigens appear to be incorporated into the structure of newly synthesized virus, probably as internal proteins. In contrast, synthesis of the high molecular weight antigen class is initiated later in the course of infection (4 to 5 hr), just prior to the appearance of newly synthesized virus. Antiserum directed specifically against virus structural proteins forms precipitin bands with all of the high molecular weight antigens recognizable by immunoelectrophoresis. This evidence, coupled with the observation that the high molecular weight antigen fraction elicits production of specific virus-neutralizing antibody, strongly suggests that this antigen class represents virus structural subunits produced in excess.
Interaction of Encephalomyocarditis Virus with Ultraviolet-irradiated Host Cells
Authors: Balduzzi P. C.
Published: January 2020

Ultraviolet light (UV) impaired the capacity of L cells to support growth of encephalomyocarditis virus. The loss of capacity was partially restored by high multiplicity of infection (MOI). This phenomenon was not due to an increased probability of an infectious virus particle reaching a site of replication undamaged by UV, since UV-inactivated virus at high MOI induced restoration of the capacity to support multiplication of nonirradiated virus adsorbed at low MOI. Multiplicity reactivation of UV-irradiated virus did not play a role in this phenomenon since restoration of capacity took place without multiplication of the UV-irradiated restoring virus. The evidence indicates that restoration of capacity was not due to viral interactions involving genetic exchange. The ability to restore capacity was a property more radioresistant than infectivity, suggesting that the former is a function only of part of the viral genome.
Electron Microscopy of Adenovirus 12 Replication II. High-Resolution Autoradiography of Infected KB Cells Labeled with Tritiated Thymidine
Authors: Martinez-Palomo A. , Granboulan N.
Published: January 2020

Incorporation of tritiated thymidine by KB cells infected with oncogenic adenovirus 12 was studied by means of high-resolution electron microscopic autoradiography. After a 1-hr pulse with tritiated thymidine, infected and control cultures were fixed at 8, 16, 24, 30, and 36 hr. Infected cultures showed a higher percentage of labeled cells. During early stages, the frequency of silver grains in the nucleus and in the nucleolus was higher in infected material. From 24 hr on, there was an inhibition of nuclear and nucleolar deoxyribonucleic acid (DNA) synthesis. At late stages, one-third of the label was located over nuclear inclusions, type II and IV, previously shown to be composed of DNA and protein, while a large majority of the remaining grains were located over the nucleoplasm. The possibility is considered, that the early increase in nuclear and nucleolar DNA synthesis produced by adeno 12 replication could in part be due to newly synthesized cellular DNA, as has been reported by others with respect to other oncogenic DNA viruses.
A New Procedure for Quantitative Measurements of Virus Particles in Crude Preparations
Authors: Strohmaier K.
Published: January 2020

A new method is described for the quantitative measurement of virus concentration in crude preparations by density gradient centrifugation and electron microscopy. The centrifugation is carried out in a specially designed centrifuge tube which permits separation and sedimentation of virus particles at different levels according to their sedimentation velocity. The gradient of a mixture of heavy and normal water (D
2
O-H
2
O) is designed to sediment the virus particles with constant velocity so that the optimal time of centrifugation can easily be calculated. The virus particles are collected on carbon-coated nickel grids floating on mercury at the bottom of the centrifuge tube and are counted by means of electron microscopy. The efficiency of the method is demonstrated with a crude plant extract of tobacco mosaic virus.
Two Loci Controlling Genetic Cellular Resistance to Avian Leukosis-Sarcoma Viruses
Authors: Crittenden Lyman B. , Stone Howard A. , Reamer Richard H. , Okazaki William
Published: January 2020

Female chickens known to be heterozygous for resistance to subgroups A and B of the avian leukosis-sarcoma viruses were mated to males known to be homozygously resistant to both. The progeny were assayed both on the chorioallantoic membrane (CAM) and in tissue culture for resistance to representative viruses of the A, B, and tentatively defined C subgroups. Segregation ratios of resistance to A and B subgroup viruses agreed with the previously suggested hypothesis of single-autosomal-recessive genes controlling resistance to each subgroup. Mixed infection on the CAM and replicate plate infection in tissue culture with subgroup A and B viruses showed that resistance to the A and B subgroups was inherited independently. Assays with viruses tentatively classified as subgroup C indicated that they were largely composed of a mixture of subgroup A and B viruses or of particles possessing the host range specificity of both. However, virus stocks of the subgroup C category, as well as some stocks classified as subgroup B, produced small numbers of pocks or foci on individuals known to be resistant to subgroup A and B viruses. It is suggested that these Rous sarcoma virus stocks carry between 1 and 10% of a true subgroup C virus.
Growth of a Dihydrofolate Reductaseless Mutant of Bacteriophage T4
Authors: Mathews Christopher K.
Published: January 2020

A mutant of bacteriophage T4 was isolated which was unable to induce virus-specific dihydrofolate reductase in infected cells. The mutant was able to form several other early enzymes of pyrimidine metabolism. Growth of the mutant in a wild-type host,
Escherichia coli
B, was compared with that of the parent strain, T4BO
1
, and T4
td
8, a mutant which lacks the ability to induce thymidylate synthetase. Growth studies were carried out in minimal medium, which gave higher growth rates and phage yields than the supplemented media used in previous studies. The reductase mutant formed deoxyribonucleic acid and plaque-forming particles at a rate slightly higher than the synthetase mutant but 1.5-to 2-fold lower than that of the wild-type phage under all conditions studied. The addition of thymine to a culture infected by the mutant increased the growth rate significantly, suggesting that the genetic lesion leads to a partial thymidylate deficiency. Like other viral genes controlling steps in thymidylate metabolism, the dihydrofolate reductase gene appears to be useful but not completely essential for growth.
Physicochemical Characterization of Adeno-associated Satellite Virus Type 4 and Its Nucleic Acid
Authors: Parks Wade P. , Green Maurice , Piña Magdalena , Melnick Joseph L.
Published: January 2020

Based on protein, deoxyribonucleic acid (DNA), and phosphorus analyses, adeno-associated satellite virus type 4 has a DNA content of 26.5%, significantly larger than type 1 which contains 18.9% DNA. Type 4 DNA contains 58 to 62% guanine + cytosine, as calculated from thermal denaturation temperature and buoyant density measurements. Simian adenovirus SV15, which serves as a helper virus for type 4 satellite, contains 13.7% DNA with a guanine + cytosine content of 55 to 57%. The DNA extracted from type 4 satellite virus is a linear, double-stranded molecule, as shown by thermal denaturation temperature profile measurements and by electron microscopy. The contour length and sedimentation coefficient of type 4 DNA indicate a molecular weight of 3.0 × 10
6
daltons.
Semliki Forest Virus in HEp-2 Cell Cultures
Authors: Erlandson Robert A. , Babcock Virginia I. , Southam Chester M. , Bailey Roller B. , Shipkey Frederick H.
Published: January 2020

The growth and development of Semliki Forest virus (SFV), an arbovirus of serological group A, in HEp-2 cells in tissue culture was examined by various techniques at frequent intervals. Infectivity and fluorescent-antibody studies demonstrated the presence of infective virus and viral antigens within the cells at 8 hr after infection. The antigen was particulate and distributed throughout the cytoplasm. Thereafter, there was rapid progression of virus production and cell destruction. By electron microscopy, tubular structures bounded by a fine membrane were observed in cytoplasm at 12 hr. Rows of small (25 mμ) virus particles were often present on the outer surface of these membranes, and at later times they became progressively more encrusted with the small virus particles. These structures subsequently increased rapidly in number, size, and complexity, and the space between the membrane and the tubules increased, thus forming vacuoles which contained tubules and were covered with the small particles. At later times (24 hr and later) larger (42 to 50 mμ) particles were observed, usually inside of the vacuoles. These larger particles (and occasionally the smaller ones) were also seen at the cell periphery and in the extracellular space. The large SFV particles appear to form by three distinct processes: (i) from the smaller particles, (ii) by development on an intravacuolar membrane, and (iii) at the ends of the tubules. The mode of development of SFV is unique among viruses studied to date, but in some characteristics it resembles that of other group A arboviruses. Its development differs from that of most arboviruses of group B and other serological groups.
Transformation of Simian Virus 40-resistant Hamster Cells with an Adenovirus 7-Simian Virus 40 Hybrid
Authors: Diamond Leila
Published: January 2020

When the hamster cell lines BHK21 and Nil-2 were infected at a multiplicity of 100 with the adenovirus 7-simian virus 40 (SV40) hybrid (strain LLE46), SV40 T antigen was induced in 0.1 to 6% of the cells during the first 96 hr postinfection, morphological changes occurred 3 to 7 weeks later, and eventually all the cells contained SV40 T antigen, but no adeno 7 T antigen. Results were similar when primary and secondary monolayer cultures of hamster embryo (HE) cells were infected with the adeno 7-SV40 hybrid, and when
primary
HE cells were infected with SV40. However, infection of BHK21, Nil-2, and
secondary
HE cells with the same multiplicity of SV40 did
not
induce SV40 T antigen or morphological transformation. This suggests that the target cells required for infection with SV40 virions, but not those required for infection with the hybrid, are lost or altered in secondary HE cultures and in the two cell lines. In most of the virus-host cell systems in which SV40 T antigen and transformation were induced, there was a decrease in the number of T antigen-positive cells after the initial infection. This was followed by a lag period of up to 2 months before the onset of a progressive increase in the number of positive cells. The beginning of the rise in T antigen production coincided with the first morphological changes.
Transfection in
Agrobacterium tumefaciens
Authors: Milani Victor J. , Heberlein Gary T.
Published: January 2020

Intact cells of
Agrobacterium tumefaciens
were examined for ability to take up biologically active LR-4 phage deoxyribonucleic acid (DNA) from the surrounding medium. DNA incorporation as measured by subsequent plaque formation (transfection) failed to occur when the bacteria were grown in defined minimal salts media, and was restricted to a 4-hr period in the early log phase of growth in enriched media. In the latter case, maximal transfection frequencies were obtained after a 25- to 30-min incubation with 22.5 μg of phage DNA/ml. Higher DNA concentrations or longer incubation times were inhibitory. Transfection was completely inhibited by deoxyribonuclease but not by ribonuclease, trypsin, or phage-specific antisera.
Effect of Rifampin on the Growth of Bacteriophage T5
Authors: Beck Man L. David , Witonsky Philip , McCorquodale D. J.
Published: January 2020

A rifampin-resistant mutant of
Escherichia coli
F with an altered ribonucleic acid (RNA) polymerase was isolated and was shown to support the growth of phage T5 in the presence of rifampin. In contrast, wild-type, rifampin-sensitive cells of
E. coli
F did not support the growth of T5 in the presence of rifampin. We concluded, therefore, that no phage-specific RNA polymerase is essential to the development of phage T5. Rather, the host RNA polymerase, or at least that portion of the host RNA polymerase that is responsible for rifampin sensitivity, is required for the transcription of all essential regions of the T5 deoxyribonucleic acid. These conclusions are supported by in vitro measurements of the rifampin sensitivity of the RNA polymerase activities extracted from infected and uninfected cells. The rifampin sensitivity of the RNA polymerase activity extracted from uninfected cells was similar to the rifampin sensitivity of the RNA polymerase activity extracted 30 min after infection.
Virus-Associated Nucleases: Location and Properties of Deoxyribonucleases and Ribonucleases in Purified Frog Virus 3
Authors: Kang H. S. , McAuslan B. R.
Published: January 2020

At least three nuclease activities are associated with purified frog virus 3. These activities are endodeoxyribonuclease (
p
H 7.5, double-stranded [DS] and single-stranded [SS] deoxyribonucleic acid [DNA]); endodeoxyribonuclease (
p
H 5.0, DS and SS DNA); endoribonuclease (DS and SS ribonucleic acid [RNA],
p
H 7.5). These activities are not adsorbed to the surface of the virion but are within the viral capsid and require detergent disruption of virions to unmask enzyme activity. Only one activity, deoxyribonuclease (
p
H 5.0, SS and DS DNA) appears to be core-associated after detergent disruption of virions. The ribonuclease degrades poliovirus replicative-form RNA, reovirus native RNA, and poly(I) poly(C) to a product with a sedimentation coefficient of about 6
S
. Qβ 6
S
DS RNA and 4
S
transfer RNA are not degraded. The ribonuclease appears to be a late function of the virus and is elicited in a soluble form as well as a virus-associated form.
Temperature-Dependent Transformation of Cells Infected with a Mutant of Bryan Rous Sarcoma Virus
Authors: Bader John P.
Published: January 2020

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.
Defective Deoxyribonucleic Acid Replication of T4rII Bacteriophage in Lambda-Lysogenic Host Cells
Authors: Szargel Raymonde , Shalitin Channa
Published: January 2020

Sedimentation of the replicative deoxyribonucleic acid through alkaline sucrose gradients showed that rII single chains reached the half-mature size at a time when wild-type molecules formed long chains (dimers and trimers of genome size). Long rII single chains could be observed on substitution of tris(hydroxymethyl)aminomethane buffer for Na
+
K
+
phosphate in the growth medium.
Filamentous Bacterial Viruses VII. Inhibition of fd Deoxyribonucleic Acid Synthesis After a Temperature Jump into Protein-Synthesis Inhibitors
Authors: Tseng B. Y. , Marvin D. A.
Published: January 2020

Synthesis of fd deoxyribonucleic acid (DNA) was stopped by transferring infected bacteria from 32 C into chloramphenicol or serine hydroxamate at 42 C, but not by addition of these antibiotics at 32 C, and not by a temperature change in the absence of antibiotics. The inhibition of fd DNA synthesis by serine hydroxamate at 42 C was reversed by excess serine. The ability to synthesize fd DNA at 42 C in chloramphenicol was rescued by delaying the addition of chloramphenicol for a few minutes after the transfer from 32 to 42 C. The colony-forming ability of abortively infected bacteria was also rescued from “killing” by delaying the addition of chloramphenicol after a transfer from 32 to 42 C.
Demonstration of Infectious Deoxyribonucleic Acid in Transformed Cells I. Recovery of Simian Virus 40 from Yielder and Nonyielder Transformed Cells
Authors: Boyd Virginia A. L. , Butel Janet S.
Published: January 2020

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 μg/culture of 10
6
cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 μg of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.
Characterization of an Endonuclease Associated with Simian Virus 40 Virions
Authors: Kidwell William R. , Saral Rein , Martin Robert G. , Ozer Harvey L.
Published: January 2020

An endonucleolytic activity associated with purified simian virus 40 (SV40) virions has been found. The enzyme is present in virions prepared from a number of different host lines. The enzyme is present in all early and late temperature-sensitive mutants examined. Some aspects of the endonucleolytic activity have been examined with SV40 deoxyribonucleic acid as substrate.
Fate of Infecting Simian Virus 40-Deoxyribonucleic Acid in Nonpermissive Cells: Integration into Host Deoxyribonucleic Acid
Authors: Collins Carolyn J. , Sauer Gerhard
Published: January 2020

Nonpermissive 3T3 cells were infected with purified superhelical simian virus 40 (SV40) deoxyribonucleic acid I (DNA I). One hour after infection, approximately 60% of the intracellular SV40 DNA was converted to relaxed forms. One day after infection, all intracellular SV40 DNA was present as slow-sedimenting material, and no SV40 DNA I was detectable. At 2 days after infection there appeared viral DNA sequences cosedimenting with cellular DNA during alkaline velocity centrifugation. Furthermore, by both alkaline equilibrium gradient centrifugation and by DNA-ribonucleic acid hybridization analysis, covalent linkage of viral DNA sequences to cellular DNA was demonstrated. Integration of SV40 DNA into cellular DNA did not appear to require DNA synthesis, although DNA synthesis followed by mitotic division of the cells enhanced the amount of viral DNA integrated. Based on data obtained by two different methods, it was calculated that 1,100 to 1,200 SV40 DNA equivalents must be integrated per cell by 48 hr after infection.
Isolation and Preliminary Characterization of Bacteriophages for
Bdellovibrio bacteriovorus
Authors: Althauser Mary , Samsonoff William A. , Anderson Carol , Conti S. F.
Published: January 2020

Ten bacteriophages that attack and lyse saprophytic strains of
Bdellovibrio bacteriovorus
were isolated. Morphological, serological, and host-range studies revealed that there were four different bdellovibrio phages present among the isolates. One of the phages lysed a strain of
B. bacteriovorus
that requires the presence of a suitable bacterial host for growth. The phage attached to the bdellovibrio cells in the absence of the bacterial host cells; lysis occurred only in the presence of host cells. The 19 saprophytic bdellovibrio strains employed in the phage host-range studies were grouped on the basis of their susceptibility to phage lysis.
Inhibition of Sindbis Virus Release by Media of Low Ionic Strength: an Electron Microscope Study
Authors: Waite Marilynn R. F. , Brown Dennis T. , Pfefferkorn Elmer R.
Published: January 2020

Release of Sindbis virus from infected cells is inhibited by lowering the ionic strength of the medium. To determine the nature of the inhibited step, we examined, by electron microscopy, both freeze-etched and thin-sectioned preparations which had been fixed with either glutaraldehyde or formaldehyde. Inhibitory medium had two different effects on Sindbis virus release: virus budding was partially inhibited, and those virions which did mature were precipitated on the surface of the cell. Freeze-etched, inhibited cells showed very few viral buds. After shift to normal medium, the number of budding virions increased dramatically, far exceeding the quantity found in normal controls. Thus, low ionic strength medium clearly inhibited an early stage of virus maturation. The results were the same regardless of the fixative. Thin sections of glutaraldehyde-fixed, inhibited cells contained large extracellular aggregates of mature virus which were not present in similar, formaldehyde-fixed preparations. Fixation of radioactively-labeled, inhibited cultures revealed that approximately half of the virus that could be released from inhibited cells by raising the ionic strength of the medium could also be released by formaldehyde, but not by glutaraldehyde. This fraction probably represents mature virus attached to the cell surface by the ionic conditions.
Covalent Linkage Between Ribonucleic Acid Primer and Deoxyribonucleic Acid Product of the Avian Myeloblastosis Virus Deoxyribonucleic Acid Polymerase
Authors: Verma Inder M. , Meuth Nora L. , Baltimore David
Published: January 2020

Initiation of deoxyribonucleic acid (DNA) synthesis by the avian myeloblastosis virus DNA polymerase was previously suggested to involve a ribonucleic acid (RNA) primer, the initial product being a DNA molecule joined by a phosphodiester bond to the RNA primer. The existence and nature of such an RNA-DNA joint was investigated by assaying for transfer of a
32
P atom from an α-
32
P-deoxyribonucleotide to a 2′(3′)-ribonucleotide after alkaline hydrolysis of the polymerase product. Such a transfer was observed, but only from α-
32
P-deoxyadenosine triphosphate and only to 2′(3′)-adenosine monophosphate. This same transfer was observed in both the endogenous DNA polymerase reaction of purified virions and the reconstructed reaction of purified DNA polymerase plus purified 60 to 70
S
viral RNA. These results indicate a high level of specificity for the initiation process and support the idea of a low-molecular-weight initiator RNA as part of the 60 to 70
S
RNA complex.
Transcription of the Influenza Ribonucleic Acid Genome by a Virion Polymerase III. Completeness of the Transcription Process
Authors: Bishop David H. L. , Roy Polly , Bean William J. , Simpson Robert W.
Published: January 2020

The virion ribonucleic acid (RNA) polymerase of influenza strain A
0
/WS transcribed at least 81% of the viral genome in vitro. The polymerase is shown to be associated with each of the major size classes of the virion RNA-ribonucleoprotein complexes. Under optimal in vitro conditions, at least 45% of the RNA contained in a population of influenza virions was involved in a repetitive transcription process. The detectable proteins associated with enzymatically active complexes containing RNA, ribonucleoprotein, and polymerase have been identified by polyacrylamide gel electrophoresis.
Recovery of Cell-Bound Interferon
Authors: Stewart William E. , De Clercq Erik , De Somer Pierre
Published: January 2020

Interferon could be recovered from homologous cells to which it was applied but could not be recovered from heterologous cells. The amount of interferon that could be recovered from cells corresponded to the sensitivity of the cells to the antiviral activity of the interferon: mouse embryo fibroblasts, which were 5 to 10 times as sensitive as L-929 cells to interferon, bound 5 to 10 times more interferon than the latter, whereas Lpa cells, which were only one-third as sensitive as L-929 cells to interferon, bound only one-third as much as the latter. The concentration of cell-bound interferon was as much as 150 times the extracellular concentration of interferon applied to the cells. Interferon bound to cells at 4 C with the same efficiency as it did to cells at 37 C, and actinomycin D-treated cells bound interferon as well as normal cells. Even though the total amount of interferon bound to cells was as much as 30% of the amount of interferon applied to them, no loss of antiviral activity was detectable from the medium.
Effect of Bacteriophage Ghost Infection on Protein Synthesis in
Escherichia coli
Authors: Fukuma Isamu , Kaji Akira
Published: January 2020

The rate of protein synthesis by
Escherichia coli
markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.
Macromolecular Content of Inclusions Produced by a Canine Adenovirus
Authors: Shahrabadi Mahmood S. , Roy K. L. , Yamamoto T.
Published: January 2020

Early inclusions induced by a canine adenovirus in a canine cell line, appearing before the formation of infectious virus particles, were purified by differential centrifugation in sucrose followed by CsCl density gradient centrifugation. Chemical analysis of these inclusions revealed that they contained deoxyribonucleic acid (DNA), ribonucleic acid, and protein. On the basis of density gradient centrifugation, the DNA extracted from the inclusions was found to be viral DNA. Electron microscope autoradiography showed that these inclusions were the sites of DNA synthesis. In addition, association of DNA polymerase activity with the inclusions was detected by incorporation of radioactivity from
3
H-thymidine triphosphate into a DNA product. The in vitro product of the enzyme had a density equal to that of viral DNA rather than host DNA. The level of DNA polymerase activity in exponentially growing infected and uninfected whole cells was similar, but in cells in stationary phase the enzyme activity of infected cells was twice that in noninfected cells. Furthermore, nuclei isolated from infected cells showed a fourfold increase in DNA polymerase activity over the noninfected cells.
Location of Ferritin-Labeled Concanavalin A Binding to Influenza Virus and Tumor Cell Surfaces
Authors: Klein Paul A. , Adams W. R.
Published: January 2020

Concanavalin A (Con-A) was linked to ferritin with glutaraldehyde and chromatographed on Sepharose 6B to separate unconjugated Con-A and ferritin from covalently cross-linked molecules. Ehrlich ascites tumor cells were infected with WSA influenza virus, stained at intervals with the ferritin-labeled Con-A and examined by electron microscopy. The surfaces of most mature viruses were specifically stained, providing direct evidence that influenza viruses maturing in this cell type have exposed Con-A receptor sites. The ferritin cores of the staining reagent were found at an average distance of 21.3 nm from the virus membrane and 10.8 nm from the uninfected cell membrane. This finding was interpreted to mean that the population of Con-A receptor sites on influenza virus particles is located at an average distance from the virus membrane twice that of the population of Con-A receptor sites found on uninfected cells. The structural elements of viral membranes can provide a reliable means for evaluating electron microscopy staining reagents, thereby enhancing their usefulness as probes for the study of membrane relationships.
Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus VI. Polyadenylic Acid Sequences in Viral Messenger Ribonucleic Acid
Authors: Bachenheimer Steven L. , Roizman Bernard
Published: January 2020

Evidence is presented by use of radiolabeling and pancreatic and T1 ribonuclease digestion that some of the ribonucleic acid specified by herpes simplex virus contains polyadenylic acid sequences. The polyadenylic sequences are not transcribed from viral DNA.