bluuebunny/crossref_metadata_2025 huggingface dataset:
Osteomyelitis Caused by
Staphylococcus schleiferi
and Evidence of Misidentification of This
Staphylococcus
Species by an Automated Bacterial Identification System
Authors: Calvo Jorge , Hernández José L. , Fariñas Maria C. , García-Palomo Daniel , Agüero Jesús
Published: December 2019

We report a case of sternal osteomyelitis due to
Staphylococcus schleiferi
in a patient who underwent thoracic surgery. This constitutes the first documented case of osteomyelitis caused by this
Staphylococcus
species. We also relate our experience in the utilization of commercially available MicroScan panels for the identification of this microorganism.
Rapid Detection of West Nile Virus from Human Clinical Specimens, Field-Collected Mosquitoes, and Avian Samples by a TaqMan Reverse Transcriptase-PCR Assay
Authors: Lanciotti Robert S. , Kerst Amy J. , Nasci Roger S. , Godsey Marvin S. , Mitchell Carl J. , Savage Harry M. , Komar Nicholas , Panella Nicholas A. , Allen Becky C. , Volpe Kate E. , Davis Brent S. , Roehrig John T.
Published: December 2019

The authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number (≈500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.
Detection and Quantification of
Plasmodium falciparum
in Blood Samples Using Quantitative Nucleic Acid Sequence-Based Amplification
Authors: Schoone Gerard J. , Oskam Linda , Kroon Nel C. M. , Schallig Henk D. F. H. , Omar Sabah A.
Published: December 2019

A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of
Plasmodium
parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 10
8
Plasmodium falciparum
parasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.
Diagnostic Performance of the Roche AMPLICOR PCR in Detecting
Neisseria gonorrhoeae
in Genitourinary Specimens from Female Sex Workers in Cotonou, Benin
Authors: Mukenge-Tshibaka L. , Alary M. , Bernier F. , van Dyck E. , Lowndes C. M. , Guédou A. , Anagonou S. , Joly J. R.
Published: December 2019

The objective of this study was to evaluate the diagnostic performance of the Roche multiplex AMPLICOR
Chlamydia trachomatis/Neisseria gonorrhoeae
PCR test for the detection of
Neisseria gonorrhoeae
infection in female urine specimens and wet and dry endocervical swabs. Endocervical swabs and urine specimens were collected from 342 female sex workers from Cotonou, Benin, and were tested using the AMPLICOR
C. trachomatis/N. gonorrhoeae
test (Roche Diagnostic Systems, Inc., Branchburg, N.J.) with internal control detection. Endocervical swabs were also cultured on Thayer-Martin medium. A series of alternate standards that included a combination of all the tests but not the test being evaluated was used to assess the performance of the test with each type of specimen. The sensitivity, specificity, and positive and negative predictive values for the urine were 53.8, 98.9, 93.5, and 87.5%, respectively. Corresponding figures for the wet swab were 91.5, 100, 100, and 97.4%, respectively. Those for the dry swab were 96.3, 96.2, 88.5, and 98.8%, respectively. Based on this study, the AMPLICOR PCR assay showed a low sensitivity for detection of
N. gonorrhoeae
infection in urine specimens, whereas the test was found to be highly sensitive and specific with endocervical specimens.
Peritonitis Due to
Brevibacterium otitidis
in a Patient Undergoing Continuous Ambulatory Peritoneal Dialysis
Authors: Wauters Georges , Van Bosterhaut Bernard , Avesani Véronique , Cuvelier René , Charlier Jacqueline , Janssens Michèle , Delmée Michel
Published: December 2019

Brevibacterium otitidis
is a coryneform rod and, as far as is known, is isolated only from infected ears. We report the first known case of peritonitis caused by
B. otitidis
in a patient undergoing continuous ambulatory peritoneal dialysis.
Cryptic Genetic Diversity in
Dientamoeba fragilis
Authors: Johnson Jeanette A. , Clark C. Graham
Published: December 2019

Uncertainty surrounding the role of
Dientamoeba fragilis
in human disease could be due in part to the existence of pathogenic and nonpathogenic variants. Evidence for two genetically distinct forms was obtained using PCR-restriction fragment length polymorphism analysis of ribosomal genes. Future studies in humans will need to take
D. fragilis
diversity into account.
Genetic Heterogeneities and Phenotypic Characteristics of Strains of the GenusAbiotrophiaand Proposal ofAbiotrophia para-adiacenssp. nov
Authors: Kanamoto Taisei , Sato Setsuko , Inoue Masakazu
Published: December 2019

The genusAbiotrophiarepresents a heterogeneous group of fastidious cocci that show a dependence on pyridoxal hydrochloride analogs for growth. The genetic heterogeneity in the genusAbiotrophiawas examined by DNA-DNA hybridization, PCR assay of genomic DNA sequences, and restriction fragment length polymorphism and sequence homology analyses of the PCR-amplified 16S rRNA gene. Nine type or reference strains ofAbiotrophia defectiva,Abiotrophia adiacens, andAbiotrophia elegansand 36 oralAbiotrophiaisolates including the ones presumptively identified asGemella morbillorumby the rapid ID32 STREP system were divided into four groups:A. defectiva(genotype 1),A. adiacens(genotype 2),A. elegans(genotype 4), and a fourth species (genotype 3) which we propose be namedAbiotrophia para-adiacenssp. nov. A PCR assay specific for detection and identification of the novelAbiotrophiaspecies was developed.A. para-adiacensgenerally produced β-glucosidase but did not produce α- or β-galactosidase or arginine dihydrolase, did not ferment, trehalose, pullulan, or tagatose, and was serotype IV, V, or VI. Thus, it was distinguished phenotypically fromA. adiacens,A. elegans, andA. defectivaas well as, apparently, from the recently described speciesAbiotrophia balaenopteraesp. nov., which produces arginine dihydrolase and which ferments pullulan but not sucrose (P. A. Lawson et al., Int. J. Syst. Bacteriol. 49:503–506, 1999). Strain ATCC 27527, currently listed asG. morbillorum, was a member of the speciesA. para-adiacens.
A New Alkaline pH-Adjusted Medium Enhances Detection of β-Hemolytic Streptococci by Minimizing Bacterial Interference Due to
Streptococcus salivarius
Authors: Dierksen Karen P. , Ragland Nancy L. , Tagg John R.
Published: December 2019

A new selective medium (CNA-P) that reduces or eliminates the inhibitory activity of bacteriocin-producing
Streptococcus salivarius
against β-hemolytic streptococci has been developed and compared with sheep blood agar (SBA) for the sensitive detection of small numbers of β-hemolytic streptococci in clinical specimens. CNA-P has as its basis a commercial medium (Difco Columbia CNA agar) supplemented with 5% (vol/vol) sheep blood, and the CNA is further modified by addition of 100 mM PIPES buffer [piperazine-
N
,
N
′-bis(2-ethanesulfonic acid)] (pH 7.5) to maintain cultures at an alkaline pH during incubation. CNA-P was shown to inhibit the production and/or release of four different types of
S. salivarius
bacteriocins or bacteriocin-like inhibitory molecules. The efficacies of CNA-P and SBA for detection of β-hemolytic streptococci in 1,352 pharyngeal samples from 376 children were compared. The β-hemolytic streptococcal isolates recovered from the samples included 314 group A (
S. pyogenes
), 61 group G, 33 group B, and 5 group C streptococci. Of 314 samples that yielded
S. pyogenes
, 300 were positive on CNA-P (96%) and 264 (86%) were positive on SBA. A significantly greater number of
S. pyogenes
isolates from these samples were recovered only on CNA-P (50 of 314) compared with the number of isolates recovered only on SBA (14 of 314). In addition, the degree of positivity, a measure of the total numbers of
S. pyogenes
isolates on the plate, was significantly higher on CNA-P than on SBA (2.40 versus 2.07;
P
< 0.001). Interestingly, CNA-P was also found to enhance the hemolytic activity of streptolysin O, allowing detection of streptolysin S-deficient
S. pyogenes
strains which might otherwise go undetected on SBA and other isolation media.
Presence of Type III Secretion Genes in
Burkholderia pseudomallei
Correlates with Ara
−
Phenotypes
Authors: Winstanley Craig , Hart C. Anthony
Published: December 2019

Dot blot hybridization and PCR amplification of 14 Ara
+
and 8 Ara
−
Burkholderia pseudomallei
strains showed that type III secretion (TTS) genes were present in all the Ara
−
strains but absent from all but one of the Ara
+
strains. The link between TTS genes and an Ara
−
phenotype suggests a role for TTS in virulence.
Comparison of a New Neuraminidase Detection Assay with an Enzyme Immunoassay, Immunofluorescence, and Culture for Rapid Detection of Influenza A and B Viruses in Nasal Wash Specimens
Authors: Noyola Daniel E. , Clark Bruce , O'Donnell Frederick T. , Atmar Robert L. , Greer Jewel , Demmler Gail J.
Published: December 2019

The performance of a new, rapid, easy-to-perform assay based on neuraminidase enzyme activity for detection of influenza virus types A and B was compared to detection by culture, indirect immunofluorescence, and enzyme immunoassay in 479 nasal wash specimens from children with respiratory infections. Compared to isolation of influenza virus by culture, the neuraminidase assay had a sensitivity of 70.1%, specificity of 92.4%, positive predictive value of 76.3%, and negative predictive value of 89.9%. There was a higher sensitivity for the detection of influenza A virus (76.4%) than for influenza B virus (40.9%). Indirect immunofluorescence showed a sensitivity of 59.8% and specificity of 97% compared to culture isolation for detection of influenza A and B viruses. Enzyme immunoassay showed a sensitivity of 89.7% and specificity of 98.1% for the detection of influenza A alone. The quality of the nasal wash specimen had a significant effect on the detection of influenza virus by all of the assays. A strong response of the neuraminidase assay was more likely to represent a culture-confirmed influenza infection. This new rapid neuraminidase assay was useful for the detection of influenza A and B viruses in nasal wash specimens.
Comparative Evaluation of Nine Different Enzyme-Linked Immunosorbent Assays for Determination of Antibodies against
Treponema pallidum
in Patients with Primary Syphilis
Authors: Schmidt Bruno L. , Edjlalipour Marzieh , Luger Anton
Published: December 2019

Nine different enzyme-linked immunosorbent assays (ELISAs) with a sonicate or recombinant proteins of
Treponema pallidum
as antigen have been evaluated comparatively by testing 52 highly selected sera from patients with primary syphilis, all negative in the microhemagglutination test for
T. pallidum
(MHA-TP). Eight tests exhibited greater sensitivity (48.5 to 76.9%) than the commonly used Venereal Disease Research Laboratory test (44.2%). Higher sensitivity could be related to (i) the volume and dilution of the serum, (ii) the design of the assay (capture and competitive tests showed higher sensitivity than sandwich-based assays), and (iii) the ability to detected specific immunoglobulin M antibodies. The specificity of the ICE Syphilis and the Enzygnost Syphilis tests was 99.5 and 99.8%, respectively, as determined by routine testing of 2,053 unselected sera in comparison with the MHA-TP test. ELISAs tested offered high sensitivity in patients with primary syphilis; however, recommendations to use these tests as screening assays do need further data on specificity and reactivity in late stages of the disease.
A Panel of Monoclonal Antibodies Targeting the Rabies Virus Phosphoprotein Identifies a Highly Variable Epitope of Value for Sensitive Strain Discrimination
Authors: Nadin-Davis S. A. , Sheen M. , Abdel-Malik M. , Elmgren L. , Armstrong J. , Wandeler A. I.
Published: December 2019

A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross-reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination.
Phylogeny of
Porphyromonas gingivalis
by Ribosomal Intergenic Spacer Region Analysis
Authors: Rumpf Robert W. , Griffen Ann L. , Leys Eugene J.
Published: December 2019

Periodontitis has been associated with the presence of
Porphyromonas gingivalis
, and previous studies have shown phenotypic differences in the pathogenicities of strains of
P. gingivalis
. An accurate and comprehensive phylogeny of strains of
P. gingivalis
would be useful in determining if there is an evolutionary basis to pathogenicity in this species. Previous phylogenies of
P. gingivalis
strains based on random amplified polymorphic DNA (RAPD) analysis and multilocus enzyme electrophoresis (MLEE) show little agreement. While the 16S ribosomal gene is the standard for phylogenetic reconstruction among bacterial species, it is insufficiently variable for this purpose. In the present study, the phylogeny of
P. gingivalis
was constructed on the basis of the sequence of the most variable region of the ribosomal operon, the intergenic spacer region (ISR). Heteroduplex analysis of the ISR has been used to study the variability of
P. gingivalis
strains in periodontitis. In the present study, typing by heteroduplex analysis was compared to ISR sequence-based phylogeny and close agreement was observed. The two strains of
P. gingivalis
whose heteroduplex types are strongly associated with periodontitis were found to be closely related and were well separated from strains whose heteroduplex types are less strongly associated with disease, suggesting a relationship between pathogenicity and phylogeny.
Pooling Cervical Swabs and Testing by Ligase Chain Reaction Are Accurate and Cost-Saving Strategies for Diagnosis of
Chlamydia trachomatis
Authors: Kapala J. , Copes D. , Sproston A. , Patel J. , Jang D. , Petrich A. , Mahony J. , Biers K. , Chernesky M.
Published: December 2019

Specimen pooling to achieve efficiency when testing urine specimens for
Chlamydia trachomatis
nucleic acids has been suggested. We pooled endocervical swabs from 1,288 women and also tested individual swabs by ligase chain reaction (LCR). Out of 53 positive specimens, pools of 4 or 8 specimens missed two positives, providing 96.2% accuracy compared to individual test results. Dilution and positive-control spiking experiments showed that negative specimens with inhibitors of LCR in the pool reduced the signal. Conversely, two extra positives, detected only through pooling, were negative by individual testing but became positive after storage, suggesting that fresh positive specimens with labile inhibitors may be positive in a pool because of dilution of inhibitors. For this population of women with a 4% prevalence of
C. trachomatis
infection, substantial savings in cost of reagents (55 to 63%) and technologist time (50 to 63%) made pooling strategies a desirable alternative to individual testing.
False-Positive Results for
Mycobacterium celatum
with the AccuProbe
Mycobacterium tuberculosis
Complex Assay
Authors: Somoskövi Ákos , Hotaling Jacqueline E. , Fitzgerald Marie , Jonas Vivian , Stasik Denise , Parsons Linda M. , Salfinger Max
Published: December 2019

Mycobacterium celatum
type 1 was found to cross-react in the AccuProbe
Mycobacterium tuberculosis
complex assay. Subsequently, we found a statistically significant increase in the relative light units with lower temperatures, suggesting that it is necessary to perform this AccuProbe assay at between 60 and 61°C. We also recommend the inclusion of
M. celatum
type 1 as a negative control.
Prevalence and Risk Factors of Tinea Unguium and Tinea Pedis in the General Population in Spain
Authors: Perea Sofia , Ramos Maria Jose , Garau Margarita , Gonzalez Alba , Noriega Antonio R. , del Palacio Amalia
Published: December 2019

This study prospectively evaluated the prevalence and risk factors of tinea unguium and tinea pedis in the general adult population in Madrid, Spain. One thousand subjects were clinically examined, and samples of nails and scales from the interdigital spaces of the feet were taken from those patients presenting with signs or symptoms of onychomycosis and/or tinea pedis, respectively. In addition, a sample from the fourth interdigital space of both feet was collected from all individuals with a piece of sterilized wool carpet. Tinea unguium was defined as a positive direct examination with potassium hydroxide and culture of the etiological agent from subjects with clinically abnormal nails. Patients with positive dermatophyte cultures of foot specimens were considered to have tinea pedis. The prevalence of tinea unguium was 2.8% (4.0% for men and 1.7% for women), and the prevalence of tinea pedis was 2.9% (4.2% for men and 1.7% for women). The etiological agents of tinea unguium were identified asTrichopyton rubrum (82.1%), followed by Trichopyton mentagrophytes var. interdigitale (14.3%) andTrichopyton tonsurans (3.5%). Trichophyton rubrum (44.8%) and Trichophyton mentagrophytes(44.8%), followed by Epidermophyton floccosum (7%) andT. tonsurans (3.4%), were the organisms isolated from patients with tinea pedis. The percentage of subjects who suffered simultaneously from both diseases was 1.1% (1.7% for men and 0.6% for women). In a multivariate logistic regression analysis, age (relative risk [RR], 1.03) and gender (RR, 2.50) were independent risk factors for tinea unguium, while only gender (RR, 2.65) was predictive for the occurrence of tinea pedis. In both analyses, the presence of one of the two conditions was associated with a higher risk for the appearance of the other disease (RR, >25).
Molecular Variability of Pseudallescheria boydii, a Neurotropic Opportunist
Authors: Rainer J. , de Hoog G. S. , Wedde M. , Gräser Y. , Gilges S.
Published: December 2019

The sequences of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) domain data obtained by restriction fragment length polymorphism analysis with 18S rDNA and fingerprinting (M13) for clinical and environmental strains of Pseudallescheria boydii (anamorph, Scedosporium apiospermum) were compared to those for related species of Pseudallescheria,Petriella, and Scedosporium. The infraspecific variability of P. boydii was considerable. There were five different lengths in the 18S rDNAs within P. boydii due to the occurrence of introns. In several cases, strains isolated from a single pond or ditch proved to be genetically very different. Nevertheless, some lineages had a regional distribution. The variability found is unlikely to be explained by meiotic recombination alone. Pseudallescheria fusoidea, Pseudallescheria ellipsoidea, and Pseudallescheria angusta were found to be synonyms for P. boydii. Scedosporium prolificans was found amid Petriella species in the ITS tree and showed no infraspecific variability. The type strain of Rhinocladium lesnei proved to be identical to Graphium putredinis. Acladium castellanii, which is morphologically reminiscent ofS. apiospermum, was also found to be a separate species, but with an unknown affiliation.
Multilaboratory Validation of Rapid Spot Tests for Identification of Escherichia coli
Authors: York Mary K. , Baron Ellen Jo , Clarridge Jill E. , Thomson Richard B. , Weinstein Melvin P.
Published: December 2019

To validate the accuracy of rapid tests for identification ofEscherichia coli, five laboratories sequentially collected 1,064 fresh, clinically significant strains with core criteria of indole-positive, oxidase-negative, nonspreading organisms on sheep blood agar plates (BAP), having typical gram-negative rod plate morphology, defined as good growth on gram-negative rod-selective media. An algorithm using beta-hemolysis on BAP, lactose reaction on eosin-methylene blue or MacConkey agar,l-pyrrolidonyl-β-naphthylamide (PYR), and 4-methylumbelliferyl-β-d-glucuronide (MUG) was evaluated. Identifications using the algorithm were compared to those obtained using commercial kit system identifications. One thousand strains wereE. coli and 64 were not E. coli by kit identifications, which were supplemented with conventional biochemical testing of low probability profiles. Of the 1,064 isolates meeting the core criteria, 294 were beta-hemolytic and did not require further testing to be identified as E. coli. None of the 64 non-E. coli strains were hemolytic, although other indole-positive, lactose-negative species were found to be hemolytic when further strains were examined in a follow-up study. Of the remaining strains, 628 were identified as E. coli by a lactose-positive and PYR-negative reaction. For nonhemolytic, lactose-negative E. coli, PYR was not helpful, but a positive MUG reaction identified 65 of 78 isolates as E. coli. The remaining 13 E. coli strains required kit identifications. This scheme for E. coli identification misidentified three non-E. coli strains as E. coli, for an error rate of 0.3%. A total of 13 kit identifications, 657 PYR tests, and 113 MUG tests were needed to identify 1,000 E. coli strains with the algorithm. The use of this rapid system saves laboratory resources, provides timely identifications, and yields rare misidentifications.
Whole-Blood Hemagglutination Inhibition Test for Venereal Disease Research Laboratory (VDRL) Antibodies
Authors: Meyer M. P. , Baughn R. E.
Published: December 2019

Nontreponemal antibody tests such as the Venereal Disease Research Laboratory (VDRL) test are carried out on serum and widely used as screening tests for syphilis. The aim of the present study was to develop a screening test for syphilis making use of whole blood and VDRL liposomes. Antibody to human red blood cells was conjugated to VDRL liposomes and reacted with a diluted sample of patient whole blood. A total of 951 samples were tested by the new test and the VDRL tube test. All 49 VDRL samples positive by the VDRL test showed inhibition of hemagglutination in the whole-blood test (sensitivity, 100%). Of 902 samples with negative results by the VDRL test, 901 caused hemagglutination when tested with the liposomes (specificity, 99.9%). The hemagglutination inhibition method tests for syphilis in a simple one-step procedure in which whole blood is added to a tube containing liposomes. The new test has potential for point-of-care testing in developing countries.
Pseudo-Outbreak of Imipenem-Resistant Acinetobacter baumannii Resulting from False Susceptibility Testing by a Rapid Automated System
Authors: Tsakris Athanassios , Pantazi Anastassia , Pournaras Spyros , Maniatis Antonios , Polyzou Aleka , Sofianou Danai
Published: December 2019

Introduction of the Vitek GNS-506 susceptibility testing cards in the Hippokration General Hospital, Thessaloniki, Greece, resulted in an apparently high prevalence of imipenem-resistant Acinetobacter baumannii. When 35 of these isolates were further tested by disk diffusion, broth microdilution, and agar dilution assays, 32 were imipenem sensitive by all tests and three were sensitive or intermediate, depending on the method. The pseudoresistant acinetobacters did not form a genetically homogeneous group. It is suggested that the detection of imipenem-resistant A. baumannii isolates by this system should be confirmed by an additional susceptibility test.
Tracking Members of the Simian Immunodeficiency Virus deltaB670 Quasispecies Population In Vivo at Single-Cell Resolution
Authors: Reinhart Todd A. , Rogan Michael J. , Amedee Angela Martin , Murphey-Corb Michael , Rausch Dianne M. , Eiden Lee E. , Haase Ashley T.
Published: December 2019

Genetically distinct lentiviruses constitute a quasispecies population that can evolve in response to selective forces. To move beyond characterization of the population as a whole to the behavior of individual members, we devised an in situ hybridization approach that uses genotype-specific probes. We used probes that detect simian immunodeficiency viruses (SIV) that differ in sequence in the V1 region of the surface envelope glycoprotein (
env
) gene to investigate the replication and cellular tropisms of four viral variants in the tissues of infected rhesus macaques. We found that the V1 genotypic variants replicated in spatially defined patterns and to different extents at each anatomic site. The two variants that replicated most extensively in animals with AIDS were detected in both macrophages and T lymphocytes in tissues. By extension of this approach, it will be possible to investigate the role of individual lentiviruses in a quasispecies in pathogenesis and to evaluate the effects of antiviral or immunotherapeutic treatment on select members of a quasispecies.
Inactivation of the RNase Activity of Glycoprotein E
rns
of Classical Swine Fever Virus Results in a Cytopathogenic Virus
Authors: Hulst M. M. , Panoto F. E. , Hoekman A. , van Gennip H. G. P. , Moormann R. J. M.
Published: December 2019

Envelope glycoprotein E
rns
of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E
rns
are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E
rns
was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E
rns
, the mutated proteins had lost their RNase activity. The mutated proteins reacted with E
rns
-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E
rns
. This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E
rns
(FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E
rns
gene and by neutralizing polyclonal antibodies directed against E
rns
, indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E
rns
plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.
Stimulation of Cyclin-Dependent Kinase Activity and G1- to S-Phase Transition in Human Lymphocytes by the Human T-Cell Leukemia/Lymphotropic Virus Type 1 Tax Protein
Authors: Schmitt Iris , Rosin Oliver , Rohwer Peter , Gossen Manfred , Grassmann Ralph
Published: December 2019

The human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) induces a malignant lymphocytic disease. The HTLV-1 transactivator protein, Tax, is believed to be crucial for the development of the disease since it is transforming in vitro and induces tumors in transgenic animals. Although the transcriptional modulation of viral and cellular gene expression by Tax has been analyzed thoroughly, it has remained unclear how the Tax functions act on the cell cycle of primary T cells. To investigate the mechanism of Tax-mediated T-cell stimulation, we transduced primary human cord blood T cells with a conditional, tetracycline repressor-basedtaxexpression system. Permanent Tax expression results in an abnormal proliferation of T cells which closely resemble HTLV-1-infected lymphocytes. Suppression of Tax synthesis stopped lymphocyte growth and caused cell cycle arrest in the G1phase. Upon reinduction oftaxexpression, the arrested cells entered the S phase. This showed that Tax has mitogenic activity, which is required for stimulating the G1- to S-phase transition of immortalized lymphocytes. In mammalian cells, the G1-phase progression is controlled by the serial activation of several cyclin-dependent kinases (Cdks), starting with Cdk4 and Cdk6. In the presence of Tax, both Cdk4 and Cdk6 were activated. The suppression of Tax synthesis, however, resulted in a significant reduction of the Cdk4 and Cdk6 activities but did not influence the expression of Cdk4, Cdk6, or cognate D-type cyclin proteins. These data suggest that Tax induces Cdk4 and Cdk6 activity in primary human T lymphocytes; this Cdk activation is likely to account for the mitogenic Tax effect and for the abnormal T-cell proliferation of HTLV-1-infected lymphocytes.
Local Periocular Vaccination Protects against Eye Disease More Effectively Than Systemic Vaccination following Primary Ocular Herpes Simplex Virus Infection in Rabbits
Authors: Nesburn Anthony B. , Slanina Susan , Burke Rae Lyn , Ghiasi Homayon , Bahri S. , Wechsler Steven L.
Published: December 2019

Vaccination of experimental animals can provide efficient protection against ocular herpes simplex virus type 1 (HSV-1) challenge. Although it is suspected that local immune responses are important in protection against ocular HSV-1 infection, no definitive studies have been done to determine if local ocular vaccination would produce more efficacious protection against HSV-1 ocular challenge than systemic vaccination. To address this question, we vaccinated groups of rabbits either systemically or periocularly with recombinant HSV-2 glycoproteins B (gB2) and D (gD2) in MF59 emulsion or with live KOS (a nonneurovirulent strain of HSV-1). Three weeks after the final vaccination, all eyes were challenged with McKrae (a virulent, eye disease-producing strain of HSV-1). Systemic vaccination with either HSV-1 KOS or gB2/gD2 in MF59 did not provide significant protection against any of the four eye disease parameters measured (conjunctivitis, iritis, epithelial keratitis, and corneal clouding). In contrast, periocular vaccination with gB2/gD2 in MF59 provided significant protection against conjunctivitis and iritis, while ocular vaccination with live HSV-1 KOS provided significant protection against all four parameters. Thus, local ocular vaccination provided better protection than systemic vaccination against eye disease following ocular HSV-1 infection. Since local vaccination should produce a stronger local immune response than systemic vaccination, these results suggest that the local ocular immune response is very important in protecting against eye disease due to primary HSV-1 infection. Thus, for clinical protection against primary HSV-1-induced corneal disease, a local ocular vaccine may prove more effective than systemic vaccination.
The Epstein-Barr Virus Rta Protein Activates Lytic Cycle Genes and Can Disrupt Latency in B Lymphocytes
Authors: Ragoczy Tobias , Heston Lee , Miller George
Published: December 2019

The transition of Epstein-Barr virus (EBV) from latency into the lytic cycle is associated with the expression of two immediate-early viral genes, BZLF1 and BRLF1. Overexpression of ZEBRA, the product of BZLF1, is sufficient to disrupt latency in B lymphocytes and epithelial cells by stimulating expression of lytic cycle genes, including BRLF1. The BRLF1 product Rta functions as a transcriptional activator in both B lymphocytes and epithelial cells. However, Rta has recently been reported to disrupt latency in an epithelial specific manner (S. Zalani, E. Holley-Guthrie, and S. Kenney, Proc. Natl. Acad. Sci. USA 93:9194–9199, 1996). Here we demonstrate that expression of Rta is also sufficient for disruption of latency in a permissive B-cell line. In HH514-16 cells, transfection of Rta leads to synthesis of ZEBRA, viral DNA replication, and late gene expression. However, Rta by itself is less potent than ZEBRA in the ability to activate most early and late lytic cycle genes. In light of previous work implicating ZEBRA in the activation of Rta, we suggest a cooperative model for EBV entry into the lytic cycle. Expression of either BZLF1 or BRLF1 triggers expression of the other immediate-early factor, and together these activators act individually or in synergy on downstream targets to activate the viral lytic cycle.
Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide
Authors: Nüesch Jürg P. F. , Corbau Romuald , Tattersall Peter , Rommelaere Jean
Published: December 2019

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities;transregulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1Orevealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1Oshowed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1Oby rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1Owas incubated with protein kinase C.
Varicella-Zoster Virus (VZV) ORF32 Encodes a Phosphoprotein That Is Posttranslationally Modified by the VZV ORF47 Protein Kinase
Authors: Reddy Sanjay M. , Cox Edward , Iofin Ilya , Soong Weily , Cohen Jeffrey I.
Published: December 2019

Varicella-zoster virus (VZV) encodes five gene products that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 32 (ORF32), is predicted to encode a protein of 16 kDa. VZV ORF32 protein was shown to be phosphorylated and located in the cytosol of virus-infected cells. Antibody to ORF32 protein immunoprecipitated 16- and 18-kDa phosphoproteins from VZV-infected cells. Since VZV encodes two protein kinases that might phosphorylate ORF32 protein, immunoprecipitations were performed with cells infected with VZV mutants unable to express either of the viral protein kinases. Cells infected with VZV unable to express the ORF66 protein kinase contained both the 16- and 18-kDa ORF32 phosphoproteins; however, cells infected with the VZV ORF47 protein kinase mutant showed only the 16-kDa ORF32 phosphoprotein. Treatment of [
35
S]methionine-labeled proteins with calf intestine alkaline phosphatase resulted in a decrease in size of the ORF32 proteins from 16 and 18 kDa to 15 and 17 kDa, respectively. VZV unable to express ORF32 protein replicated in human melanoma cells to titers similar to those seen with parental virus; however, VZV unable to express ORF32 was impaired for replication in U20S osteosarcoma cells. Thus, VZV ORF32 protein is posttranslationally modified by the ORF47 protein kinase. Since the VZV ORF47 protein kinase has recently been shown to be critical for replication in human fetal skin and lymphocytes, its ability to modify the ORF32 protein suggests that the latter protein may have a role for VZV replication in human tissues.
Herpes Simplex Virus Type 1 Glycoprotein gC Mediates Immune Evasion In Vivo
Authors: Lubinski John M. , Wang Liyang , Soulika Athena M. , Burger Reinhard , Wetsel Rick A. , Colten Harvey , Cohen Gary H. , Eisenberg Roselyn J. , Lambris John D. , Friedman Harvey M.
Published: December 2019

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.
Cell Surface Proteoglycans Are Necessary for A27L Protein-Mediated Cell Fusion: Identification of the N-Terminal Region of A27L Protein as the Glycosaminoglycan-Binding Domain
Authors: Hsiao Jye-Chian , Chung Che-Sheng , Chang Wen
Published: December 2019

We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y.-S. Chang, and W. Chang, J. Virol. 72:1577–1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.
Neuronal Death Induced by Brain-Derived Human Immunodeficiency Virus Type 1 Envelope Genes Differs between Demented and Nondemented AIDS Patients
Authors: Power C. , McArthur J. C. , Nath A. , Wehrly K. , Mayne M. , Nishio J. , Langelier T. , Johnson R. T. , Chesebro B.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) infection of the brain results in viral replication primarily in macrophages and microglia. Despite frequent detection of viral genome and proteins in the brains of AIDS patients with and without HIV dementia, only 20% of AIDS patients become demented. To investigate the role of viral envelope gene variation in the occurrence of dementia, we examined regions of variability in the viral envelope gene isolated from brains of AIDS patients. Brain-derived HIV-1 V1-V2 envelope sequences from seven demented and six nondemented AIDS patients displayed significant sequence differences between clinical groups, and by phylogenetic analysis, sequences from the demented group showed clustering. Infectious recombinant viruses containing brain-derived V3 sequences from both clinical groups were macrophagetropic, and viruses containing brain-derived V1, V2, and V3 sequences from both clinical groups spread efficiently in macrophages. In an indirect in vitro neurotoxicity assay using supernatant fluid from HIV-1-infected macrophages, recombinant viruses from demented patients induced greater neuronal death than viruses from nondemented patients. Thus, the HIV-1 envelope diversity observed in these patient groups appeared to influence the release of neurotoxic molecules from macrophages and might account in part for the variability in occurrence of dementia in AIDS patients.
The Human T-Cell Leukemia Virus Type 1 Oncoprotein Tax Inhibits the Transcriptional Activity of c-Myb through Competition for the CREB Binding Protein
Authors: Colgin Mark A. , Nyborg Jennifer K.
Published: December 2019

Tax, the transforming protein of human T-cell leukemia virus type 1 (HTLV-1), is required for strong activation of HTLV-1 transcription. This activation is mediated through interaction with the KIX domain of the cellular coactivator CREB binding protein (CBP). In this study we examined the possibility that the Tax-KIX interaction may mediate effects on cellular gene transcription in vivo, as a growing number of cellular transcription factors have been shown to utilize CBP as a coactivator. We tested the ability of Tax to deregulate the activity of the cellular transcription factor, c-Myb, since both Tax and c-Myb interact with the KIX domain of CBP. Our results show that in vivo, Tax antagonizes the transcriptional activity of c-Myb and, reciprocally, c-Myb antagonizes the transcriptional activity of Tax. Furthermore, c-Myb competes for KIX binding to Tax in vitro, indicating that these two transcription factors bind CBP in a mutually exclusive manner. This novel mechanism of transcriptional interference by Tax may promote globally deregulated cellular gene expression in the HTLV-1-infected cell, possibly leading to leukemogenesis.
Functionality and Cell Anchorage Dependence of the African Swine Fever Virus GeneA179L, a Viralbcl-2Homolog, in Insect Cells
Authors: Brun Alejandro , Rodríguez Fernando , Escribano José M. , Alonso Covadonga
Published: December 2019

The African swine fever virus geneA179Lhas been shown to be a functional member of theced9/bcl-2family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed theA179Lgene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of theA179Lgene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-d-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested thatA179Lmight regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.
A Pathogenic Threshold of Virus Load Defined in Simian Immunodeficiency Virus- or Simian-Human Immunodeficiency Virus-Infected Macaques
Authors: Ten Haaft Peter , Verstrepen Babs , Überla Klaus , Rosenwirth Brigitte , Heeney Jonathan
Published: December 2019

To determine if a specific pathogenic threshold of plasma viral RNA could be defined irrespective of virus strain, RNA levels in the plasma of more than 50 infected rhesus macaques (
Macaca mulatta
) were measured. Animals were inoculated intravenously with either simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) strains of known pathogenic potential (SIV
8980
, SIV
smm-3
, SIV
mac32H/J5
, SIV
mac32H/1XC
, reverse transcriptase-SHIV, SHIV
89.6p
) or with attenuated strains (SHIV
W6.1D
, SHIV
sf13
, SHIV
han-2
, SIV
macΔnef
, SHIV
sf33
). In animals inoculated with nonpathogenic strains, shortly after the primary peak of viremia viral RNA levels declined and remained below 10
4
RNA equivalents/ml of plasma between 6 and 12 weeks postinoculation. Animals infected with documented pathogenic strains maintained viral RNA levels higher than 10
5
RNA equivalents/ml of plasma. In animals infected with strains with low virulence, a decline in plasma RNA levels was observed, but with notable individual variation. Our results demonstrate that the disease-causing potential was predicted and determined by a threshold plasma virus load which remained greater than 10
5
RNA equivalents/ml of plasma 6 to 12 weeks after inoculation. A threshold virus load value which remained below 10
4
RNA equivalents/ml of plasma was indicative of a nonpathogenic course of infection.