bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplex reverse transcription-PCR for surveillance of influenza A and B viruses in England and Wales in 1995 and 1996
Authors: Ellis J S , Fleming D M , Zambon M C
Published: January 2020

Multiple-target (multiplex) reverse transcription-PCR (RT-PCR) for detection, typing, and subtyping of the hemagglutinin gene of influenza type A (H3N2 and H1N1) and type B viruses was developed and applied prospectively to virological surveillance of influenza in England in the 1995-1996 winter season. During this season both influenza A H3N2 and H1N1 viruses were circulating, although at different times. Six hundred nineteen combined nose and throat swabs taken by general practitioners in sentinel practices from individuals presenting with "influenzalike illness" were analyzed by culture, multiplex RT-PCR, and immunofluorescence. Of the 619 samples, 246 (39.7%) were positive by multiplex RT-PCR compared with 200 (32.3%) which yielded influenza viruses on culture. There was 100% correlation between multiplex RT-PCR typing and subtyping and the influenza types and subtypes obtained from culture. There was also excellent correlation between the temporal detection of influenza A H3N2 and H1N1 viruses by multiplex RT-PCR and by culture. During the peak weeks of influenza virus activity, a total of 259 specimens were received, of which 101 (38.9%) yielded influenza viruses on culture while 149 (57.5%) were positive in multiplex RT-PCR, providing an increase in detection of influenza viruses of approximately 20%. The increased detection of influenza virus occurred in all the age groups sampled. Samples which were positive by multiplex RT-PCR but negative by culture were not detected significantly earlier or later in the winter of 1995-1996 but were detected during the peak weeks of clinical influenza virus activity. Multiplex RT-PCR was successfully used in surveillance of influenza to provide accurate, sensitive diagnosis directly on clinical specimens sent through the post.
Application of the Etest to the antimicrobial susceptibility testing of Mycobacterium marinum clinical isolates
Authors: Flynn C M , Kelley C M , Barrett M S , Jones R N
Published: January 2020

Mycobacterium marinum, a well-recognized cutaneous pathogen, is usually treated by chemotherapy without available standardized in vitro susceptibility testing information. In this study, we have attempted to apply the stable-gradient method (Etest; AB Biodisk, Solna, Sweden) to susceptibility testing of M. marinum in order to assess the activities of eight antimicrobial agents against 60 recent clinical strains of M. marinum collected from 10 geographic sites within the United States. Two plated media (5% sheep blood Mueller-Hinton agar and Middlebrook 7H11 agar) were compared, and 7H11 agar was found to be superior in supporting the growth of all strains. Four reference strains of M. marinum were tested on five occasions with eight drugs (160 tests) in order to evaluate Etest reproducibility. Results were observed to be within 1 log2 dilution of the all-test median MIC for 97.5% of the Etests. Our MIC results for the 60 strains clearly demonstrate the best in vitro potency against M. marinum isolates to be as follows (rank order): trimethoprim-sulfamethoxazole (MIC at which 90% of the isolates are inhibited [MIC90], 0.25 and 4.25 microg/ml, respectively) = ethambutol > clarithromycin (MIC90, 1 microg/ml) > minocycline = doycycline (MIC90, 4 microg/ml) > amikacin (MIC90, 8 microg/ml). Rifampin was only marginally active against the M. marinum strains tested (MIC90, at the National Committee for Clinical Laboratory Standards) breakpoint of 1 microg/ml), and ciprofloxacin was not active (MIC90, 8 microg/ml). These data should enhance the empiric drug selection for contemporary M. marinum infections and also provide evidence that the Etest can be utilized to guide chemotherapy with alternative agents.
Detection of group B rotaviruses in fecal samples from diarrheic calves and adult cows and characterization of their VP7 genes
Authors: Chang K O , Parwani A V , Smith D , Saif L J
Published: January 2020

Groups A, B, and C rotaviruses have been identified in cattle. Group B rotaviruses are associated with sporadic cases of diarrhea in calves and adult cows. From diagnostic submissions to our laboratory, 90 fecal samples from cases of calf diarrhea, 81 fecal samples from cases of adult cow diarrhea (winter dysentery), and 20 fecal samples from case control normal adult cows were tested for group B rotaviruses by polyacrylamide gel electrophoresis (PAGE), and reverse transcription (RT)-PCR (targeting 279 bp of the VP7 gene). In addition, 53 fecal samples from diarrheic adult cows were tested for group B rotaviruses by immune electron microscopy (IEM). By RT-PCR, five samples from calves were group B rotavirus positive (5.6%). Fifteen samples from adult cows with diarrhea were group B rotavirus positive (18.5%), and none of the control fecal samples from normal cows were positive for group B rotaviruses. By PAGE, one calf sample (RT-PCR positive) was group B rotavirus positive (short electropherotype), but none of the adult cow samples were positive for group B rotaviruses. By IEM, 5 (9.4%) of the 53 fecal samples from diarrheic adult cows were group B positive (all were also RT-PCR positive). The VP7 genes of three strains (WD653 from an adult cow and the ATI and Mebus calf strains) were sequenced. The VP7 genes from the three bovine strains showed high (over 90%) nucleotide and deduced amino acid homologies, but lower homologies (48 to 61%) were seen between these genes and the genes from rodent (IDIR) and human (ADRV) group B rotaviruses. Although there were some differences of degree, all inoculated gnotobiotic calves (n = 6) showed abnormal feces between 1 and 3 days after inoculation with each of three strains of group B bovine rotaviruses, and group B rotaviruse, were detected in the feces for up to 2 weeks by RT-PCR but for shorter periods by PAGE or IEM.
Coinfection with Bartonella clarridgeiae and Bartonella henselae and with different Bartonella henselae strains in domestic cats
Authors: Gurfield A N , Boulouis H J , Chomel B B , Heller R , Kasten R W , Yamamoto K , Piemont Y
Published: January 2020

Bartonella clarridgeiae and several strains of Bartonella henselae, the agent of cat scratch disease, with variations in the 16S rRNA gene have been found to infect the blood of cats. An epidemiologic study of Bartonella infection in domestic French cats revealed that of 436 cats sampled, 5 cats (1.1%) were coinfected with B. henselae and B. clarridgeiae and 2 cats (0.5%) were coinfected with two strains of B. henselae with variations in the 16S rRNA gene, B. henselae type I and type II. In an indirect immunofluorescence assay, coinfected cats tested positive for both Bartonella species at titers of > or = 128. Identification of the colonies was achieved by preformed enzyme analysis, PCR-restriction fragment length polymorphism analysis of the citrate synthase gene, and 16S rRNA gene sequencing. Colony size differences in mixed culture allowed differentiation of the Bartonella species. The coinfection of cats with two Bartonella species or variants of the same species raises concern about the possibility of dual infection in humans. The development of a polyvalent vaccine targeted against the most pathogenic or invasive strains may be a means of protecting cats and man from infection.
Characterization of a neurotoxigenic Clostridium butyricum strain isolated from the food implicated in an outbreak of food-borne type E botulism
Authors: Meng X , Karasawa T , Zou K , Kuang X , Wang X , Lu C , Wang C , Yamakawa K , Nakamura S
Published: January 2020

Neurotoxigenic Clostridium butyricum was isolated from the food implicated in an outbreak of clinically diagnosed type E botulism in China. PCR assay showed that the isolate (LCL 155) contained the type E botulinum toxin gene. This appears to be the first report of neurotoxigenic C. butyricum causing food-borne botulism.
Rapid detection of mycobacteria in clinical specimens by using the automated BACTEC 9000 MB system and comparison with radiometric and solid-culture systems
Authors: Pfyffer G E , Cieslak C , Welscher H M , Kissling P , Rüsch-Gerdes S
Published: January 2020

Recovery rates of acid-fast bacilli (AFB) and the mean time to their detection from clinical specimens were determined by using the fluorescent BACTEC 9000 MB system. Data were compared to those assessed by the radiometric BACTEC 460 system and by cultivation on solid media. A total of 3,095 specimens were processed with N-acetyl-L-cysteine-NaOH by two laboratories. The contamination rates for the BACTEC 9000 MB system were 6.8% (center 1) and 9.8% (center 2). A total of 451 mycobacterial isolates were detected (Mycobacterium tuberculosis complex, n = 296; nontuberculous mycobacteria [NTM], n = 155). These isolates originated from 94 (20.8%) smear-positive and 357 (79.2%) smear-negative specimens. The BACTEC 9000 MB system was significantly better than solid media (P < 0.05) in detecting AFB, but it was less efficient than the radiometric system (P < 0.01). The BACTEC 9000 MB system plus solid media (combination A) recovered 393 (87.1%) of the isolates, while the BACTEC 460 system plus solid media (combination B) detected 430 (95.3%) of all AFB isolates. Between combination A and B there was no statistically significant difference for the detection of isolates from smear-positive specimens (P > 0.05), in contrast to the recovery of AFB from smear-negative specimens for M. tuberculosis complex, P < 0.05; for NTM, P < 0.01). The mean time to detection of M. tuberculosis complex was 12.2 days for smear-positive specimens and 18.1 days for smear-negative specimens with the BACTEC 9000 MB system; 9.3 and 15.6 days, respectively, with the BACTEC 460 system; and 21.2 and 28.4 days, respectively, with solid media. For NTM, the average detection times were 15.1, 17.3, and 31.3 days by the three methods, respectively. In conclusion, the BACTEC 9000 MB system is a rapid, less labor-intensive detection system which allows for higher levels of recovery of AFB than solid media. There is no risk of cross contamination, which is known to be the case for the BACTEC 460 system, and data management is greatly facilitated. As a whole, however, the BACTEC 9000 MB system should only be used in conjunction with solid media.
Serological cross-reactions between Bartonella and Chlamydia species: implications for diagnosis
Authors: Maurin M , Eb F , Etienne J , Raoult D
Published: January 2020

Diagnosis of Chlamydia or Bartonella infections continues to rely mainly on serology. However, serological cross-reactions between members of these genera have recently been described. Sera from eight patients originally diagnosed as having Chlamydia pneumoniae endocarditis reacted with both Chlamydia sp. and Bartonella quintana antigens (microimmunofluorescence technique). Adsorption of sera with B. quintana or C. pneumoniae antigens removed anti-C. pneumoniae antibodies, whereas adsorption with C. pneumoniae antigens did not change antibody titers to B. quintana. Western blot analysis confirmed the presence of cross-reacting antigens and showed antibody patterns in all sera to be compatible with a Bartonella infection. These patients were therefore probably suffering from Bartonella-induced rather than Chlamydia-induced endocarditis. In contrast, sera from 10 patients presumed to be suffering from C. pneumoniae pneumonia did not display anti-B. quintana antibodies, although cross-reacting antigens were revealed by Western blotting. This work highlights the possibility that cases of infective Bartonella endocarditis are erroneously diagnosed as chlamydial infections.
Detection of vancomycin-resistant enterococci in fecal samples by PCR
Authors: Satake S , Clark N , Rimland D , Nolte F S , Tenover F C
Published: January 2020

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.
Identification of clinically relevant viridans group streptococci to the species level by PCR
Authors: Garnier F , Gerbaud G , Courvalin P , Galimand M
Published: January 2020

A PCR assay that allows identification of clinically relevant viridans group streptococci (Streptococcus gordonii, S. mitis, S. mutans, S. oralis, S. salivarius, and S. sanguis) to the species level and identification of milleri group streptococci (S. anginosus, S. constellatus, and S. intermedius) to the group level was developed. This assay was based on specific amplification of internal fragments of genes encoding D-alanine:D-alanine ligases which are species specific and ubiquitous in prokaryotes possessing peptidoglycan. The specificity of this assay was tested on 9 reference strains and 91 characterized clinical isolates. This assay offers a specific and rapid alternative to phenotypic or DNA-DNA hybridization methods for identification of clinically relevant viridans group streptococci.
Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans
Authors: Pujol C , Joly S , Lockhart S R , Noel S , Tibayrenc M , Soll D R
Published: January 2020

Randomly amplified polymorphic DNA (RAPD) analysis, multilocus enzyme electrophoresis (MLEE), and Southern blot hybridization with moderately repetitive DNA probes have emerged as effective fingerprinting methods for the infectious fungus Candida albicans. The three methods have been compared for their capacities to identify identical or highly related isolates, to cluster weakly related isolates, to discriminate between unrelated isolates, and to assess microevolution within a strain. By computing similarity coefficients between 29 isolates from three cities within the continental United States, strong concordance of the results is demonstrated for RAPD analysis, MLEE, and Southern blot hybridization with the moderately repetitive probe Ca3, and weaker concordance of the results is demonstrated for these three fingerprinting methods and Southern blot hybridization with the moderately repetitive probe CARE2. All methods were also demonstrated to be able to resolve microevolution within a strain, with the Ca3 probe exhibiting the greatest resolving power. The strong correlations demonstrated between polymorphic markers assessed by the four independent fingerprinting methods and the nonrandom association between loci demonstrated by RAPD analysis and MLEE provide evidence for strong linkage disequilibrium and a clonal population structure for C. albicans. In addition, a synapomorphic allele, Pep-3A, was found to be present in all members of one of the three clusters discriminated by RAPD analysis, MLEE, and Ca3 fingerprinting, supporting the concordance of the clustering capacities of the three methods, the robustness of the clusters, and the clonal nature of the clusters.
Use of the isolator 1.5 microbial tube for culture of synovial fluid from patients with septic arthritis
Authors: Yagupsky P , Press J
Published: January 2020

Synovial fluid specimens obtained from patients with arthritis were plated onto solid media (conventional cultures) or inoculated into an Isolator 1.5 microbial tube (Isolator cultures), and the yield and time to detection of organisms were compared. Overall, 144 specimens obtained from 137 patients were processed, and 31 (21.5%) cultures obtained from 29 patients were positive by at least one method. Staphylococcus aureus was isolated from 12 patients, Streptococcus pneumoniae and Kingella kingae were isolated from 4 patients each, group G streptococci were isolated from 3 patients, Staphylococcus epidermidis and members of the family Enterobacteriaceae were isolated from 2 patients each, and Streptococcus mitis and Peptostreptococcus prevotii were isolated from 1 patient each. Overall, the causative organism was detected in 31 of 31 (100.0%) Isolator cultures and 24 of 31 (77.4%) conventional cultures (P < 0.02). Twenty-nine of 31 (93.5%) positive Isolator cultures and 20 of 24 (83.3%) conventional cultures were positive by the second day of incubation. Among the 24 cultures positive by both methods, higher numbers of CFU per milliliter were detected with the Isolator system in 13 cultures and with conventional cultures in 2 cultures (P < 0.002). Inoculation of synovial fluid into an Isolator 1.5 microbial tube improves the recovery of organisms causing septic arthritis.
Replication of Bacteriophage Ribonucleic Acid: Some Physical Properties of Single-stranded, Double-stranded, and Branched Viral Ribonucleic Acid
Authors: Franklin Richard M.
Published: January 2020

Replicative intermediate (RI) is considered to be the double-stranded ribo-nucleic acid (RNA) template for synthesis of viral RNA, with bound nascent single-stranded viral RNA. A theoretical description of RI is based on the analysis of a steady state of biopolymerization on a template which determines not only nucleotide sequence, but also chain length. The hydrodynamic properties of RI isolated from
Escherichia coli
infected with bacteriophage R17 are compared with those of RNA isolated from R17 (single-stranded RNA) and of replicative form (RF) isolated from
E. coli
infected with R17. RF is double-stranded RNA template without any single-stranded component. Whereas
S
for R17 RNA is a function of the ionic strength (Γ/2) of the solvent,
S
is almost invariant with Γ/2 for RF. By contrast for RI lies between the sedimentation constants for R17 RNA and RF and varies with Γ/2 as does R17 RNA. The weight distribution of
S
for RI demonstrates the heterogeneity of this material, and the variation in the weight distribution with ionic strength demonstrates the duality of structure in RI. Using [η], the
w
for RI is estimated to be 2.6 × 10
6
daltons, as compared with the theoretical value of 2.9 × 10
6
daltons.
Electron Microscopy of Chick Fibroblasts Infected by Defective Rous Sarcoma Virus and Its Helper
Authors: Courington Doris , Vogt Peter K.
Published: January 2020

Nonproducing Rous sarcoma cells of the chicken and their virus-producing as well as uninfected counterparts were studied with an electron microscope. The structural peculiarities of transformed cells included cytoplasmic annulate lamellae, aggregates of membrane-bound, glycogen-like granules, and empty, virus-like shells. Of 69 individual lines of nonproducing Rous sarcoma cells, 64 contained small numbers of viral particles. These particles were morphologically indistinguishable from mature avian tumor virus but lacked demonstrable infectivity. In sessile normal and leukosis virus-infected fibroblasts, microtubules and fibrils occurred in parallel arrays at the periphery of the cytoplasm. This cortical organization was absent from rounded Rous sarcoma cells. The characteristics of microtubular arrangement seemed to reflect differences in the locomotory activity of normal and transformed cells.
Selective Gene Transcription in Bacteriophage T4 by Putrescine
Authors: Shalitin Channa
Published: January 2020

Messenger ribonucleic acid (mRNA) preparations from T4-infected cells formed in the presence or absence of putrescine have been characterized and compared. Hybrid competition experiments indicate that these mRNA molecules are derived from distinct genetic loci. The results are consistent with the hypothesis that putrescine might control differential transcription of the phage genome during morphogenesis. The data are also in accord with previously observed changes in the population of mRNA formed at different times after infection.
Purification and Properties of a Thermophilic Bacteriophage Lytic Enzyme
Authors: Welker N. E.
Published: January 2020

A phage lytic enzyme was isolated from lysates of
Bacillus stearothermophilus
(NCA 1503-4R). The enzyme was purified 1,998-fold with a 27% recovery of enzyme activity. By use of polyacrylamide gel electrophoresis and sucrose gradient centrifugation the enzyme was judged free from protein contaminants. The lytic enzyme was active over a
p
H range of 6.0 to 7.0, with a maximum at 6.3, and it was stable between
p
H 7.0 and 8.0 and at 5.0 and unstable between
p
H 5.5 and 6.5. The temperature coefficient (Q
10
) was 2.27 between 35 and 45 C, 2.01 between 45 and 55 C, and 2.00 between 50 and 60 C. Lytic enzyme in 0.1
m
sodium phosphate was not inactivated after a 1-hr exposure to temperatures below 65.5 C, whereas a 1% inactivation was observed at 70.6 C. A 2-hr exposure at 60.1, 65.5, and 70.6 C resulted in an inactivation of 1.2, 9.6, and 12.0%, respectively. A sodium phosphate concentration of at least 0.1
m
was necessary for the prolonged exposure of lytic enzyme at 55 C (
p
H 6.3), whereas 0.005
m
was required for maximal lytic activity. Lytic activity was stimulated 169, 165, and 160% by 10
−4
m
Mg
++
, Ca
++
, and Mn
++
, respectively. Lytic activity was inhibited 75% by 10
−4
m
ethylenediaminetetraacetic acid (EDTA). The EDTA inhibition could be reversed by the addition of excess Mg
++
, Ca
++
, or Mn
++
. Lytic activity was not affected by NaCl, KCl, or NH
4
Cl. Lytic activity was inhibited 100, 91, 25, 61, and 56% by 10
−4
m
Hg
++
, Cu
++
, Zn
++
,
p
-chloromercuribenzoate, and
p
-hydroxymercuribenzoate, respectively. Cysteine or 2-mercaptoethanol did not stimulate lytic activity, nor were these sulfhydryl compounds required for maintenance of enzyme activity during handling or storage. Cell walls were rapidly solubilized when incubated with lytic enzyme. Lytic action was complete after 1.5 min, with a 70% reduction in optical density (OD). Cell walls without lytic enzyme showed no reduction in OD during this period. The solubilization of N-terminal amino groups paralleled the reduction in OD and reached a level of 0.3 μmole/mg of cell wall after 4 min of incubation. Cell walls with and without lytic enzyme treatment showed a 3- and a 1.3-fold increase, respectively, in N-terminal amino groups after 3 hr of incubation. There was no release of reducing power in either the untreated cell wall suspensions or those treated with lytic enzyme. Electron micrographs of treated and untreated cell walls showed that the enzyme partially degrades the cell wall with the release of small wall fragments.
Adenovirus Proteins II. N-Terminal Amino Acid Analysis
Authors: Laver William G. , Suriano J. Robert , Green Maurice
Published: January 2020

Adenovirus types 2, 4, 5, 6, 18, 21 and 27, were analyzed for N-terminal amino acids by use of
35
S-labeled phenylisothiocyanate of high specific radioactivity. Two free N-terminal amino acids, alanine and glycine, were found in these viruses in the molar ratio (alanine-glycine) of 2.5:1, with the exception of type 2 where the ratio was 3.6:1 and type 5 where the ratio was 5.5:1 Adenovirus type 2 was disrupted by acetone treatment, and two protein fractions were obtained after sucrose gradient centrifugation. One of these fractions, which was associated with the viral deoxyribonucleic acid and comprised approximately 18% of the total protein of the virus, was greatly enriched with respect to N-terminal alanine and glycine.
Induction of Cellular Deoxyribonuleic Acid Synthesis by Simian Virus 40
Authors: Kit Saul , de Torres Ramon A. , Dubbs Del R. , Salvi Maria L.
Published: January 2020

The incorporation of
3
H-thymidine (
3
H-dT) into deoxyribonucleic acid (DNA) has been studied in uninfected confluent monolayer cultures of monkey kidney and mouse kidney cells, simian virus 40 (SV40)-infected cells, and in SV40-transformed mouse kidney cells. Radioautographic measurements revealed that during the period from 28 to 51 hr after productive SV40 infection of monkey kidney cultures about 80% of the cells synthesized DNA, compared to about 16% in uninfected cultures. At 28 to 43 hr after abortive SV40 infection of mouse kidney cultures, 24 to 37% of the cells synthesized DNA, compared to about 6 to 8% in uninfected cultures. The infected monkey kidney and mouse kidney cultures, respectively, incorporated about 5 to 10 times and 3 to 5 times as much
3
H-dT into DNA as did uninfected cultures. Moreover, the net DNA synthesized by SV40-infected monkey kidney cultures, estimated by colorimetric methods, substantially exceeded that of uninfected cultures.

Nitrocellulose chromatography and band centrifugation experiments were performed to elucidate the kinds of DNA synthesized in the cultures. In uninfected monkey kidney cultures and at 2 to 12 hr after SV40 infection, almost all of the
3
H-dT labeled DNA sedimented more rapidly than SV40 DNA, and the radioactive DNA was denatured by heating for 12 min at 100 C (cellular DNA). Almost all of the labeled DNA obtained from abortively infected mouse kidney cultures and from SV40-transformed cells also had the properties of cellular DNA. However, approximately one-third to one-half of the labeled DNA obtained from monkey kidney cultures 28 to 51 hr after infection sedimented more slowly than cellular DNA and was not denatured by the heating (SV40 DNA). It is concluded that cellular DNA synthesis was induced during either the productive or abortive SV40 infections.
Growth and Intracellular Development of a New Respiratory Virus
Authors: Hamre Dorothy , Kindig David A. , Mann Judith
Published: January 2020

The multiplication of a new, ether-sensitive, ribonucleic acid virus, 229E, isolated from the human respiratory tract, has been studied in cultures of WI-38 human diploid cells. In thin sections of these cells examined with the electron microscope, particles appeared in vesicles in the cytoplasm of cells at a time corresponding to the initial increase in infectious virus. Antigen was also detected in the cytoplasm of cells by the immunofluorescent technique. Extracellular particles of similar morphology were prominent soon after. These events preceded a detectable cytopathic effect. Later, an electron-dense particle appeared within vacuoles in the cytoplasm but was never found extracellularly. Its role in virus development is not known. Complement-fixing antigen developed along with the increase in infectious virus.
Factors Influencing the Enhancement of the Infectivity of Poliovirus Ribonucleic Acid by Diethylaminoethyl-Dextran
Authors: Pagano Joseph S. , McCutchan James H. , Vaheri Antti
Published: January 2020

The enhancement by diethylaminoethyl-dextran (DEAE-D) of the infectivity of poliovirus ribonucleic acid (RNA) for cell cultures was demonstrated by infective-center as well as by plaque assays, both in nonprimate (L) and primate cell systems (MK, HeLa, LLC-MK
2
). The sensitivity of plaque assays was greatly improved by using a tris (hydroxymethyl)aminomethane-buffered synthetic medium (basal medium Eagle) and freshly confluent cell monolayers. Enhancement of nucleic acid infectivity was directly dependent on the molecular weight of the DEAE-D. Two observations bearing on the action of DEAE-D appeared important: ribonuclease activity was reduced by DEAE-D, and cells pretreated with DEAE-D remained susceptible to infection with RNA in isotonic medium. Appreciable susceptibility of the treated cells persisted for at least 2 hr; the susceptible state could be reversed at will by an application of heparin. Enhancement of nucleic acid infectivity was independent of an effect of DEAE-D on intact virus and agar inhibitors.
Induction of Specific Chromosomal Aberrations by Adenovirus Type 12 in Human Embryonic Kidney Cells
Authors: zur Hausen Harald
Published: January 2020

Nonrandom chromosomal breaks in chromosomes 1 and 17 were provoked in human embryonic kidney cells 24 hr after infection with adenovirus type 12. These chromosomal changes disappeared in persistently infected cultures. Neutralization of the virus with type-specific antiviral serum prior to infection prevented the occurrence of chromosomal aberrations. No viral deoxyribonucleic acid (DNA) synthesis, as determined by autoradiography, was seen in metaphases containing adenovirus type 12-induced chromosomal aberrations. Ultraviolet irradiation of the virus reduced chromosomal aberrations linearly. This reduction in aberrations was fourfold slower than the inactivation of viral infectivity. At 24 hr after infection of cells with purified
3
H-labeled adenovirus type 12, the isotope was found to be associated with the nuclei. The uptake of isotope was reduced ninefold when the labeled virus was neutralized with type-specific antiviral serum. This difference is considered to account for neutralization of labeled virions. In metaphases infected with labeled viruses, most of the clustered grains were seen only on one arm of the chromatid, even after 72 hr. Isochromatid labeling was found, however, in a small percentage of chromosomes, and increased with time after infection. This increase was threefold between 24 and 72 hr after infection, whereas the mean grain counts decreased twofold during the same period. This has been tentatively interpreted to mean that most of the viral DNA molecules or parts thereof are merely attached to cellular chromatin, but a small fraction of them becomes gradually integrated as time proceeds. Certain chromosomal sites appeared to be preferentially labeled when chromosome 2 was used as a model for evaluation.
Purification of Large Quantities of Influenza Virus by Density Gradient Centrifugation
Authors: Reimer C. B. , Baker R. S. , vanFrank R. M. , Newlin T. E. , Cline G. B. , Anderson N. G.
Published: January 2020

New zonal centrifuges can conveniently process as much as five orders of magnitude (10
5
) greater sample volumes than conventional swinging-bucket rotors. The continuous-sample-flow-with-banding versions may be used in series with ancillary purification procedures. Here we have studied the combined process: absorption and elution of influenza virus with barium sulfate followed by concentration and isopycnic banding of the virus in a buffered sucrose gradient. Kilogram quantities of impurity have been rapidly separated from grams of purified virus, which have been conveniently concentrated several hundred-fold by the purification process. Experimental vaccines made by these procedures are being evaluated.
Replicative Intermediates of Bacteriophage T7 Deoxyribonucleic Acid
Authors: Center Melvin S.
Published: January 2020

After infection with bacteriophage T7, parental and newly synthesized deoxyribonucleic acid (DNA) exhibit an extremely fast sedimentation rate in neutral sucrose gradients. This fast-sedimenting component (intermediate I) has a sedimentation constant of about 1,500
S
and contains T7 DNA as determined by DNA-DNA hybridization experiments. Pulse-chase experiments indicate that the fast-sedimenting material is metabolically active and serves as a precursor to the formation of T7 DNA. Intermediate I contains about 2.5 to 7% of the total
3
H-labeled protein formed between 3 and 9.5 min after T7 infection. Treatment of intermediate I with Pronase results in the release of the DNA from the complex. At early times after infection, a second intermediate (intermediate II) can be detected which contains both parental and newly synthesized DNA sedimenting slower than intermediate I but 2 to 3 times as fast as mature T7 DNA. Intermediates I and II containing parental DNA are formed after infection of the nonpermissive host with an amber mutant in gene 1, a gene whose expression is necessary for the synthesis of most T7 proteins. The two intermediates are also observed when infection with T7 wild type is carried out in the presence of chloramphenicol.
Nuclear Disruption After Infection of
Escherichia coli
with a Bacteriophage T4 Mutant Unable to Induce Endonuclease II
Authors: Snustad D. Peter , Warner Huber R. , Parson Kathleen A. , Anderson Dwight L.
Published: January 2020

Nuclear disruption after infection of
Escherichia coli
with a bacteriophage T4 mutant deficient in the ability to induce endonuclease II indicates that either (i) the endonuclease II-catalyzed reaction is not the first step in host deoxyribonucleic acid (DNA) breakdown or (ii) nuclear disruption is independent of nucleolytic cleavage of the host chromosome. M-band analysis demonstrates that the host DNA remains membrane-bound after infection with either an endonuclease II-deficient mutant or T4 phage ghosts.
Biosynthetic Properties of a Polyoma Nucleoprotein Complex: Evidence for Replication Sites
Authors: Green Melvin H.
Published: January 2020

Under normal growth conditions,
all
of the newly synthesized polyoma deoxyribonucleic acid (py DNA) that could be extracted from infected mouse cell cultures by the Triton procedure of Green, Miller, and Hendler was in the form of a 55
S
nucleoprotein complex. Inhibition of protein synthesis by cycloheximide reduced the sedimentation rate of the polyoma complex synthesized during the first hour after addition of the drug to 25 to 35
S
. Since the 55
S
and the 25 to 35
S
complexes each contain closed circular 20
S
py DNA, it is suggested that the slower complex contains less protein per DNA molecule and that there is normally a small or unstable pool of protein available for binding to newly replicated py DNA. In the presence of cycloheximide, the newly formed 25 to 35
S
complex was not derived from preexisting 55
S
complex. Thus, some py DNA which was not solubilized by the Triton method served as a template for replication. Further evidence for the existence of polyoma replication sites is provided by the demonstration that, during the inhibition of protein synthesis, a class of newly replicated py DNA can be solubilized by the sodium dodecyl sulfate procedure of Hirt, but not by the Triton method. It is postulated that continuous protein synthesis is required to release py DNA from replication sites in the form of a Triton-extractable nucleoprotein complex.
A
2
(N2) Neuraminidase of the X-7 Influenza Virus Recombinant: Determination of Molecular Size and Subunit Composition of the Active Unit
Authors: Bucher D. J. , Kilbourne E. D.
Published: January 2020

Neuraminidase activity of influenza virus was directly seen on sodium dodecyl sulfate polyacrylamide gels with the aid of the synthetic substrate, methoxyphenol neuraminic acid. Neuraminidase (NA) appeared as a high-molecular-weight fraction with a size in the range of 220,000 to 250,000 daltons. Isolation of this fraction from the X-7 strain of influenza virus, dissociation with sodium dodecyl sulfate, and reduction showed the presence of two polypeptides of 66,000 (NA
1
) and 58,000 (NA
2
) molecular weights in equimolar concentration. We postulate that the minimum active unit for the viral A
2
neuraminidase is a tetramer composed of two NA
1
and two NA
2
subunits.
Isolation and Characterization of Simian Virus 40 Ribonucleic Acid
Authors: Weinberg R. A. , Warnaar S. O. , Winocour E.
Published: January 2020

Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization in formamide was used to isolate simian virus 40-specific RNA. Early in the lytic cycle, a 19
S
viral RNA species was observed. Late in the lytic cycle, 16
S
and 19
S
viral species were found. The 16
S
and 19
S
species of viral RNA were localized in the cytoplasm. High-molecular-weight heterogeneous RNA, containing viral sequences, was isolated from the nuclear fraction of infected cells late in the lytic cycle. This RNA may contain non-viral sequences linked to viral sequences. The formamide hybridization technique can be used to isolate intact late lytic viral RNA which is at least 99% pure.
Synthesis of Viral Ribonucleic Acid During Restricted Adenovirus Infection
Authors: Fox Robert I. , Baum Stephen G.
Published: January 2020

The mechanism by which simian virus 40 converts the abortive adenovirus type 7 infection of monkey cells into an efficient lytic infection has been investigated. Analysis of ribonucleic acid (RNA) synthesis during unenhanced and enhanced infection of monkey cells has shown that adenovirus RNA synthesized in the abortive infection contains both “early” and “late” sequences. In hybridization competition experiments, early adenovirus RNA from human cells prevented the hybridization of only 20% of the adenovirus RNA transcribed in unenhanced infection. Further, the RNA from unenhanced cells was able to completely block the hybridization of RNA synthesized during enhanced infection. Finally, virus-associated RNA, which is a late RNA transcribed in lytic adenovirus infection, is also produced in the unenhanced infection. An accompanying paper describes a marked deficiency in adenoviral capsid protein synthesis in the unenhanced infection. We conclude that RNA sequences, which are sufficient to code for the synthesis and assembly of structural proteins of adenovirus, are transcribed but are not efficiently translated in the unenhanced adenovirus infection of monkey cells.
Rescue of Epstein-Barr Virus from Somatic Cell Hybrids of Burkitt Lymphoblastoid Cells
Authors: Glaser Ronald , Rapp Fred
Published: January 2020

A Burkitt lymphoblastoid cell line, P3J-HR-1, was fused and hybridized to a human sternal marrow cell line. The somatic cell hybrids were negative when examined for Epstein-Barr virus (EBV) markers. When the hybrid cells were exposed to 5-iododeoxyuridine, both EBV-specific antigens and virus particles were induced as determined by the immunofluorescence test and by electron microscopy. The data presented suggest that the EBV genome can be transferred from a lymphoblastoid cell to another cell type during cell hybridization, that the EBV genome can persist in the hybrid cells for long periods of time, and that synthesis of the virus can be induced in the heterokaryons.
Capsid Polypeptides of Mouse Elberfeld Virus I. Amino Acid Compositions and Molar Ratios in the Virion
Authors: Stoltzfus C. Martin , Rueckert Roland
Published: January 2020

The four major polypeptide chains (alpha, beta, gamma, delta) constituting the capsid protein of mouse Elberfeld (ME) virus were isolated by preparative electrophoresis on polyacrylamide gels, and the amino acid composition of each chain was determined. In addition, the molecular weights of the smallest chains of ME virus, mengovirus, and poliovirus, which had previously been determined by gel electrophoretic methods, were redetermined by gel filtration chromatography in 6
m
guanidine hydrochloride. Each was found to have a molecular weight about 7,300. Using the reevaluated molecular weights and the known amino acid compositions of the chains, the molar ratio of each chain in the ME virion was determined by quantitative analysis of the distribution of radioactivity in the electrophoretically separated chains of virus which had been specifically radiolabeled with leucine or with methionine. Equimolar proportions of all four chains were found in the virion.
Replication of the Parvovirus MVM I. Dependence of Virus Multiplication and Plaque Formation on Cell Growth
Authors: Tattersall Peter
Published: January 2020

A plaque assay has been developed for the minute virus of mice. The infectious unit is a single particle. Plaque size is determined by the extent of cell division in the infected monolayer. Infection of quiescent and serum-stimulated cells suggests that virus multiplication depends on host function(s) that are not normally expressed in resting cells.
Control of Interferon Synthesis: Effect of Diethyl-aminoethyl-Dextran on Induction by Polyinosinic-Polycytidylic Acid
Authors: Vilček Jan , Barmak Sandra L. , Havell Edward A.
Published: January 2020

Interferon production in cultures of rabbit kidney cells (RKC) stimulated with 10 to 250 μg of polyinosinic-polycytidylic acid (poly I·poly C) per ml peaked at 3 to 4 hr after the exposure of cells to inducer and rapidly declined thereafter. On the other hand, RKC stimulated with poly I·poly C (10 or 2 μg/ml) in the presence of diethylaminoethyl (DEAE)-dextran (100 or 20 μg/ml, respectively) produced a protracted interferon response, with the release of interferon continuing for over 24 hr. The kinetics of interferon production in RKC stimulated with lower concentrations of the mixture of poly I·poly C and DEAE-dextran were similar to the response produced by poly I·poly C alone (10 to 250 μg/ml). Only the responses that terminated early were paradoxically enhanced by treatment with low doses of actinomycin D or with cycloheximide. Cells stimulated with 50 μg of poly I·poly C/ml showed hyporesponsiveness to a second interferon induction with poly I·poly C when restimulated 7 hr after primary induction. This hyporesponsiveness could be overcome by restimulating with higher concentrations of the poly I·poly C-DEAE-dextran complex. The results are compatible with the hypothesis that the early termination of interferon production and hyporesponsiveness to repeated induction with poly I·poly C are due to a cellular repressor exerting negative control on interferon synthesis, and that the increased cellular uptake of poly I·poly C in the presence of DEAE-dextran may effectively neutralize the repressor. These results also suggested that the often observed different kinetics and the varied effects of inhibitors of ribonucleic acid or protein synthesis on interferon responses in various cells and in cells stimulated with different inducers (such as with viruses as compared with polynucleotides) need not imply the existence of fundamentally different mechanisms of interferon production.
Transcription by Infectious Subviral Particles of Reovirus
Authors: Shatkin A. J. , LaFiandra A. J.
Published: January 2020

Digestion of purified reovirus with chymotrypsin in the presence of 0.15 M NaCl converts virions to infectious subviral particles (SVP
i
). The SVP
i
have an active ribonucleic acid (RNA) polymerase and are similar in composition to the partially uncoated virions which have been isolated from infected L cells. SVP
i
have a buoyant density of 1.40 g/ml in CsCl and sediment at 420
S
as compared to 1.37 g/ml and 630
S
for virions. They consist of 30% less protein and include the polypeptides of the inner structural layer, λ
1
, λ
2
, and σ
3
, and a polypeptide derived by cleavage of μ
2
, a constituent of the outer shell. The genome RNA is retained within SVP
i
, but more than 60% of the “adenine-rich,” single-stranded RNA is released by the proteolytic treatment. Infection of L cells with SVP
i
or virions results in the transcription of all 10 genome segments. In cycloheximide-treated SVP
i
-infected cells, transcription occurs predominantly from one medium and two small genome segments, the same pattern of early messenger RNA (mRNA) observed in virion-infected cells. In contrast, SVP
i
incubated in vitro synthesize mRNA corresponding to all genome segments.
Isolation and Properties of
rex
−
Mutants of Bacteriophage Lambda
Authors: Gussin Gary N. , Peterson Victoria
Published: January 2020

Twenty-five
rex
−
mutants of phage lambda have been isolated. Three of the mutants, including one amber mutant, map at three distinct sites within the
rex
region of the lambda genetic map. The existence of the amber mutant provides further evidence that
rex
and
c
I are distinct genes, since it seems to be identical to wild-type lambda in its ability to establish or maintain lysogeny.
Properties of a Ribonucleoprotein Particle Isolated from Nonidet P-40-Treated Rous Sarcoma Virus
Authors: Davis Nancy L. , Rueckert Roland R.
Published: January 2020

A ribonucleoprotein particle containing about 20% ribonucleic acid (RNA), and containing little if any phospholipid or glucosamine, was recovered in high yield after treatment of Schmidt-Ruppin strain of Rous sarcoma virus and B77 virus with the nonionic detergent Nonidet P-40. This structure, which probably derives from the internal ribonucleoprotein filament described in electron microscopy studies, contained 80 to 90% of the viral 60 to 70
S
RNA and only about 10% of the protein present in intact virions. It sedimented in glycerol density gradients at approximately 130
S
and had a buoyant density in sucrose of about 1.34 g/ml. Studies with
32
P-labeled virus indicated that the ribonucleoprotein particle contained approximately 30 4
S
RNA molecules per 10
7
daltons of high-molecular-weight viral RNA. Intact virions contained about 70 4
S
RNA molecules per 10
7
daltons of high-molecular-weight RNA. Electrophoretic studies in dodecyl sulfate-containing polyacrylamide gels showed that the ribonucleoprotein particle contained only 5 of the 11 polypeptides found in the virion; of these the major component was a polypeptide weighing 14,000 daltons.
Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus
Authors: Lewandowski L. J. , Traynor B. L.
Published: January 2020

Iodination of reovirus, cytoplasmic polyhedrosis virus (CPV), and wound tumor virus (WTV), and their respective subviral forms, followed by analysis of the labeled polypeptides by using polyacrylamide gel electrophoresis, has been used to compare the protein contents of these three diplornaviruses. This approach, when combined with electron microscopy and buoyant density determinations, appears capable of localizing individual polypeptides in some of the viral and subviral forms. CPV (
p
= 1.435 g/cm
3
) seems to resemble reovirus cores (
p
= 1.440 g/cm
3
) in both ultrastructure and polypeptide composition. CPV is composed of five polypeptides with molecular weights of about 151,000, 142,000, 130,000, 67,000, and 33,000. The polyhedral matrix, which in nature encapsulates the virions, is, in turn, composed mainly of two polypeptide species with molecular weights of about 30,000 and 20,000, and several minor proteins. The proteins of WTV consist mainly of four species of polypeptide with molecular weights of about 156,000, 122,000, 63,000, and 44,000, and several minor components. These molecular weight determinations are consistent with the hypothesis that, as has been suggested for reovirus, the viral proteins of CPV and WTV seem to be coded for by monocistronic mes senger RNA molecules transcribed from distinct segments of the double-stranded RNA viral genomes.