bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of Local Variations in Antibiotic Consumption and Geographical Differences of Erythromycin and Penicillin Resistance in
Streptococcus pneumoniae
Authors: García-Rey César , Aguilar Lorenzo , Baquero Fernando , Casal Julio , Dal-Ré Rafael
Published: July 2002

A geographical analysis of how commonly prescribed oral antibiotics are quantitatively and qualitatively responsible for the different local rates of erythromycin and penicillin resistance in
Streptococcus pneumoniae
in Spain is presented. From 1998 to 1999 a multicenter surveillance study yielded 1,684 consecutive
S. pneumoniae
isolates from community-acquired respiratory infections. Data on antibiotic sales in the retail market for the same period were gathered, and the corresponding defined doses per 1,000 inhabitants per day were calculated. Macrolides and β-lactams were considered separately. Macrolides were subdivided into thrice-, twice-, and once-a-day macrolides, and β-lactams were split into aminopenicillins and cephalosporins. Univariate Spearman nonparametric coefficients (
R
) were calculated, and variables proving to be significantly associated (
P
< 0.1) were entered into several multiple lineal regression models. Ample variation in both resistance rates and antibiotic consumption was seen. Multivariate analyses showed that integrated consumption of both macrolides and β-lactams accounted well for erythromycin (
R
2
= 0.722;
P
= 0.002) and penicillin (
R
2
= 0.706;
P
= 0.002) resistance. Macrolides were more important drivers for local differences in both erythromycin and penicillin resistance than β-lactams were. Consumption of once-a-day macrolides was key for local erythromycin resistance variations. Cephalosporins were slightly more important penicillin resistance drivers than aminopenicillins were.
Identification by 16S rRNA Gene Sequencing of
Lactobacillus salivarius
Bacteremic Cholecystitis
Authors: Woo Patrick C. Y. , Fung Ami M. Y. , Lau Susanna K. P. , Yuen Kwok-Yung
Published: July 2002

An anaerobic, nonsporulating, gram-positive bacterium was isolated from blood and bile pus cultures of a 70-year-old man with bacteremic acute cholecystitis. The API 20A system showed that it was 70%
Actinomyces naeslundii
and 30%
Bifidobacterium
species, whereas the Vitek ANI system and the ATB ID32A Expression system showed that it was “unidentified.” The 16S rRNA gene of the strain was amplified and sequenced. There were 3 base differences between the nucleotide sequence of the isolate and that of
Lactobacillus salivarius
subsp.
salivarius
or
L. salivarius
subsp.
salicinius
, indicating that the isolate was a strain of
L. salivarius
. The patient responded to cholecystectomy and a 2-week course of antibiotic treatment. Identification of the organism in the present study was important because the duration of antibiotic therapy would have been entirely different depending on the organism. If the bacterium had been identified as
Actinomyces
, penicillin for 6 months would have been the regimen of choice. However, it was
Lactobacillus
, and a 2-week course of antibiotic was sufficient.
Modeling of 5′ Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for
Salmonella enterica
Authors: Knutsson Rickard , Löfström Charlotta , Grage Halfdan , Hoorfar Jeffrey , Rådström Peter
Published: July 2002

The performance of a 5′ nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched
Salmonella enterica
cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (
C

T

) and the fluorescence intensity by a normalized reporter value (Δ
R

n

). The
C

T

response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from
S. enterica
serovar Enteritidis was studied in double-distilled H
2
O (ddH
2
O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on Ampli
Taq
Gold or r
Tth
. A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH
2
O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the r
Tth
mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 × 10
−13
g/microwell for the r
Tth
mixture and 2 × 10
−12
g/microwell for the Ampli
Taq
Gold mixture. To verify the improved amplification capacity of the r
Tth
mixture, BPW was inoculated with 1 CFU of
S. enterica
serovar Enteritidis per ml and the mixture was incubated at 30°C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the r
Tth
mixture resulted in an earlier PCR detection during enrichment than use of the Ampli
Taq
Gold mixture. For accurate detection (
C

T

≤ 30) of
S. enterica
serovar Enteritidis inoculated in BPW, the r
Tth
mixture required 8.4 h of enrichment, while the Ampli
Taq
Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5′ nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures.
Development and Validation of a PCR-Based Enzyme-Linked Immunosorbent Assay with Urine for Use in Clinical Research Settings To Detect
Trichomonas vaginalis
in Women
Authors: Kaydos S. Cornelia , Swygard Heidi , Wise Shelly L. , Sena Arlene C. , Leone Peter A. , Miller William C. , Cohen Myron S. , Hobbs Marcia M.
Published: July 2002

Trichomonas vaginalis
infection is highly prevalent worldwide and is associated with poor birth outcomes and enhanced human immunodeficiency virus transmission. Traditional detection methods rely on microscopic examination of vaginal specimens (wet mount) and culture, which can be insensitive and time-consuming. More than 3,000 women attending two sexually transmitted disease clinics were enrolled in this cross-sectional study to evaluate urine-based PCR for detection of
T. vaginalis
using a combined reference standard of wet mount and culture from vaginal swab. The prevalence of trichomoniasis in the population was 16.7% (502 of 3,009 women) using the reference standard. PCR with urine combined with agarose gel-based detection was 66.9% sensitive and 98.3% specific compared to the reference standard. Detection of PCR products using an unlabeled enzyme-linked immunosorbent assay (ELISA) improved the sensitivity to 86.4%, but specificity fell to 86.1%. Using a digoxigenin-labeled ELISA for detection of amplified
T. vaginalis
DNA from urine, the sensitivity and specificity of the PCR improved to 90.8 and 93.4%, respectively, compared to wet mount or culture from vaginal swabs. For clinical research settings in which vaginal specimens are not available and culture conditions are not feasible, urine-based PCR-ELISA may be useful for the detection of trichomoniasis in women.
Experimental Infection of Domestic Cats withBartonella koehleraeand Comparison of Protein and DNA Profiles with Those of OtherBartonellaSpecies Infecting Felines
Authors: Yamamoto Kazuhiro , Chomel Bruno B. , Kasten Rickie W. , Hew Carrie M. , Weber David K. , Lee Wilson I. , Droz Sara , Koehler Jane E.
Published: July 2002

Bartonella koehlerae, a recently described felineBartonellaspecies, was isolated from two naturally infected cats in northern California. We experimentally infected domestic cats withB. koehleraeto establish the microbiological and immunological characteristics of this infection in cats and to compare it to infections with those caused byB. henselaeandB. clarridgeiae. Four cats were inoculated intradermally withB. koehlerae(8.6 × 107to 3.84 × 108CFU/ml). None of the cats presented any obvious clinical signs, but all cats developed bacteremia, which peaked at 3.36 × 104to 1.44 × 106CFU/ml of blood between day 14 and day 36 postinoculation.B. koehlerae-inoculated cats had a bacteremia duration (mean, 74 days) shorter than did cats inoculated withB. clarridgeiae(mean, 324 days) (P= 0.03). None of the four cats inoculated withB. koehleraehad bacteremia relapse. As shown by enzyme-linked immunosorbent assay (ELISA) usingB. koehleraeouter membrane protein (OMP) antigens, the four cats developed a species-specific antibody response, and ELISA testing using other felineBartonellaOMP antigens showed statistically lower optical density values. All four cats developed similar antibody reactivity patterns toB. koehleraeOMP antigens as seen by Western blotting, each with at least 20 seroreactive protein bands. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein profile differences were observed for both whole-cell lysate and OMPs fromB. koehlerae, compared withB. henselaeandB. clarridgeiae. B. koehleraewas more closely related toB. henselaethan toB. clarridgeiaeby protein profile, and this relatedness was also confirmed by analysis of the genomic DNA profiles by pulsed-field gel electrophoresis.
Community-Acquired Bacteremic
Acinetobacter
Pneumonia in Tropical Australia Is Caused by Diverse Strains of
Acinetobacter baumannii
, with Carriage in the Throat in At-Risk Groups
Authors: Anstey Nicholas M. , Currie Bart J. , Hassell Marilyn , Palmer Didier , Dwyer Brian , Seifert Harald
Published: July 2002

Acinetobacter
isolates from eight subjects with community-acquired
Acinetobacter
pneumonia (CAAP), a major cause of fatal community-acquired pneumonia in tropical Australia, were phenotypically and genotypically confirmed by pulsed-field gel electrophoresis analysis to be broadly diverse
Acinetobacter baumannii
strains. Wet-season throat carriage of
A. baumannii
was found in 10% of community residents with excess levels of alcohol consumption, the major at-risk group for CAAP.
Immune Responses to the
Mycobacterium tuberculosis
-Specific Antigen ESAT-6 Signal Subclinical Infection among Contacts of Tuberculosis Patients
Authors: Doherty T. Mark , Demissie Abebech , Olobo Joseph , Wolday Dawit , Britton Sven , Eguale Tewodros , Ravn Pernille , Andersen Peter
Published: July 2002

Diagnosis of latent
Mycobacterium tuberculosis
infection is considered essential for tuberculosis control but is hampered by the lack of specific reagents. We report that strong recognition of tuberculosis complex-specific antigen ESAT-6 by healthy household contacts of tuberculosis patients correlates with the subsequent development of active tuberculosis during a 2-year follow-up period.
PCR Detection and Serological Evidence of Granulocytic Ehrlichial Infection in Roe Deer (
Capreolus capreolus
) and Chamois (
Rupicapra rupicapra
)
Authors: Liz Jorge S. , Sumner John W. , Pfister Kurt , Brossard Michel
Published: July 2002

The role of wild mammals, such as roe deer (
Capreolus capreolus
) and chamois (
Rupicapra rupicapra
), in the epidemiology of granulocytic ehrlichiae in Switzerland was investigated. We tested blood samples for
Ehrlichia phagocytophila
genogroup 16S rRNA gene sequences by PCR and for immunoglobulin G antibodies against granulocytic ehrlichiae by indirect fluorescent-antibody assay (IFA). Overall means of 60.9% of 133 roe deer serum samples and 28.2% of 39 chamois serum samples were seroreactive by IFA. PCR results were positive for 18.4% of 103 roe deer serum samples as well. None of the 24 chamois blood samples tested were positive by PCR. Partial 16S rRNA gene and
groESL
heat shock operon sequences of three roe deer samples tested showed strong degrees of homology (≥99.7 and ≥98.6%, respectively) with the sequences of granulocytic ehrlichiae isolated from humans. These results confirm that chamois, and particularly roe deer, are commonly infected with granulocytic ehrlichiae and provide evidence that these wild mammals are potential reservoirs for granulocytic ehrlichiae in Switzerland.
Reactivity of Convalescent-Phase Hemolytic-Uremic Syndrome Patient Sera with the Megaplasmid-Encoded TagA Protein of Shiga Toxigenic
Escherichia coli
O157
Authors: Paton Adrienne W. , Paton James C.
Published: July 2002

A cosmid library of Shiga toxigenic
Escherichia coli
(STEC) O157:H7 strain EDL933 DNA was screened for clones capable of reacting with convalescent-phase serum from a patient with hemolytic-uremic syndrome (HUS), in an attempt to identify candidate virulence genes. One of the immunoreactive clones contained a portion of the large plasmid pO157, and the immunoreactive gene product was identified as TagA. The function of this 898-amino-acid protein is unknown, but it exhibits 42% amino acid sequence identity and 63% similarity to a 312-amino-acid region of a ToxR-regulated lipoprotein of
Vibrio cholerae
. Antibodies to
E. coli
O157 TagA were detected in sera from other HUS patients with O157 STEC infection but not in those from patients whose illnesses were caused by other STEC types or in healthy controls. These data demonstrate that TagA is expressed in vivo and provide circumstantial evidence for a role in the pathogenesis of the disease. The
tagA
gene is present only in STEC strains belonging to serogroup O157, and so antibodies to TagA are a potentially useful serological marker for infections due to such strains.
Identification of Two Phylogenetically Related Organisms from Feces by PCR for Detection ofSalmonellaspp
Authors: Gentry-Weeks Claudia , Hutcheson H. Joel , Kim Lisa Marie , Bolte Denise , Traub-Dargatz Josie , Morley Paul , Powers Barbara , Jessen Michael
Published: July 2002

Two previously reported PCR methods were evaluated to determine whether they are as sensitive and specific as conventional culture methods in detectingSalmonellaspp. from feces. Bovine and equine feces were enriched overnight in brain heart infusion broth and assayed using PCR methods and primer sets described by other investigators. A total of 774 fecal specimens were tested using a primer set (invE-Aprimer set) that amplifies a region spanning the invasin E and A genes ofSalmonella entericaserovar Typhimurium. A subset of these fecal specimens (306 of the 774 total) were tested using primers (hisJprimer set) that amplify a portion of the histidine transport J gene. The PCR required 24 h to obtain results, whereas it took 5 to 7 days to identifySalmonellaspp. by culture. PCR detection ofSalmonellaspp. using thehisJprimers and theinvE-Aprimers had a sensitivity of 93.3 and 80%, respectively, and a specificity of 85.6 and 98.6%, respectively, compared with bacterial culture. Amplification of 42 culture-negative fecal specimens (of 306 total specimens) generated a DNA fragment that corresponded to the molecular weight of the amplifiedhisJgene. ThehisJ-generated amplicons from six culture-negative and six culture-positive specimens were sequenced and analyzed using DNA sequence alignment and phylogenetic analysis software. A neighbor-joining dendrogram of the DNA sequences of both sets ofhisJamplicons revealed two distinct groups—one group of amplicons from culture-positive specimens identical to thehisJgene ofS. entericaserovar Typhimurium and a second group of amplicons from culture-negative specimens that were more closely related tohisJofS. entericaserovar Typhimurium than to otherhisJsequences present in nucleotide databases.
Performance of Four Chromogenic Urine Culture Media after One or Two Days of Incubation Compared with Reference Media
Authors: Aspevall Olle , Osterman Björn , Dittmer Rakel , Stén Lena , Lindbäck Emma , Forsum Urban
Published: July 2002

Four chromogenic urine culture media were compared to culture on blood agar, MacConkey agar, and CLED (cysteine-, lactose-, and electrolyte-deficient) agar for detection of uropathogens in 1,200 urine specimens. After 2 nights of incubation, 96% of all isolates were recovered on blood agar, 96% were recovered on CLED agar, 92% were recovered on CPS ID2, 96% were recovered on CHROMagar Orientation from BBL, 95% were recovered on CHROMagar Orientation from The CHROMagar Company, and 95% were recovered on Chromogenic UTI Medium.
Probable Mother-to-Infant Transmission of
Pneumocystis carinii
f. sp.
hominis
Infection
Authors: Miller Robert F. , Ambrose Helen E. , Novelli Vas , Wakefield Ann E.
Published: July 2002

A mother and her 4.5-week-old infant had
Pneumocystis carinii
pneumonia contemporaneously. Genotyping of
P. carinii
f. sp.
hominis
DNA at three independent loci showed the same genotype in samples from mother and infant. These data suggest transmission of
P. carinii
organisms from the mother to her infant.
Detection of Ethambutol-Resistant
Mycobacterium tuberculosis
Strains by Multiplex Allele-Specific PCR Assay Targeting
embB306
Mutations
Authors: Mokrousov Igor , Narvskaya Olga , Limeschenko Elena , Otten Tatiana , Vyshnevskiy Boris
Published: July 2002

We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the
embB
gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical
Mycobacterium tuberculosis
isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the
embB
306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis.
Calculation of the Stability of the IS
6110
Banding Pattern in Patients with Persistent
Mycobacterium tuberculosis
Disease
Authors: Warren R. M. , van der Spuy G. D. , Richardson M. , Beyers N. , Borgdorff M. W. , Behr M. A. , van Helden P. D.
Published: July 2002

The interpretation of molecular epidemiologic data of
Mycobacterium tuberculosis
infection is dependent on the understanding of the stability and evolutionary characteristics of the DNA fingerprinting marker used to classify clinical isolates. This study investigated the stability of the IS
6110
banding pattern in serial tuberculosis isolates collected from patients resident in an area with a high incidence of tuberculosis. Evolutionary changes were observed in 4% of the strains, and a half-life (
t
1/2
) of 8.74 years was calculated, assuming a constant rate of change over time. This rate may be composed of a high rate of change seen during the early disease phase (
t
1/2
= 0.57 years) and a low rate of change seen in the late disease phase (
t
1/2
= 10.69 years). The early rate probably reflects change occurring during active growth prior to therapy, while the low late rate may reflect change occurring during or after treatment. We demonstrate that the calculation of these rates is strongly influenced by the time interval between onset of disease and sputum sampling. These calculations are further complicated by partial replacement of the original strain population, resulting in the sporadic appearance of clonal variants in sputum specimens. Therefore, the true extent of genetic diversity may be underestimated within each host, thereby influencing molecular epidemiological data used to establish transmission chains.
Random Amplified Polymorphic DNA and Amplified Fragment Length Polymorphism Analyses ofPasteurella multocidaIsolates from Fatal Fowl Cholera Infections
Authors: Huber Brad S. , Allred Darin V. , Carmen John C. , Frame David D. , Whiting David G. , Cryan Jason R. , Olson Terry R. , Jackson Paul J. , Hill Karen , Laker Miriam T. , Robison Richard A.
Published: July 2002

Fowl cholera, a disease caused byPasteurella multocida, continues to be a major problem for the poultry industry. The sources of pathogenic organisms responsible for most sporadic epidemics remain unconfirmed, although attenuated vaccines that retain a low level of virulence have occasionally been implicated in outbreaks of the disease. One of the vaccines most commonly used to prevent fowl cholera is the M-9 strain. In the present study, 61 clinical isolates from turkeys that died of fowl cholera from 1997 to 1999 on 36 Utah farms were analyzed and compared to the M-9 vaccine strain. Genetic analyses of the isolates were done by random amplified polymorphic DNA (RAPD) analysis and amplified fragment length polymorphism (AFLP) fingerprinting. The results of these genetic analyses were correlated with the vaccination status of the flock, isolate serotype, and geographic location. Although both genetic techniques effectively identified similar subtle genomic differences, RAPD analysis provided only 77% of the detail provided by AFLP analysis. While a relationship between genetic profile and serotype was evident, no significant relationship indicating geographic influence was found (P= 0.351). Interestingly, organisms isolated from vaccinated flocks were significantly closer genetically to the M-9 vaccine strain than isolates from unvaccinated birds were (P= 0.020). Statistical analyses revealed that this relationship could not have been determined by serotyping alone (P= 0.320), demonstrating the value of AFLP and RAPD analyses in the characterization of disease-causing strains.
Integrated Hepatitis B Virus DNA Preserves the Binding Sequence of Transcription Factor Yin and Yang 1 at the Virus-Cell Junction
Authors: Nakanishi-Matsui Mayumi , Hayashi Yasuyuki , Kitamura Yoshiyuki , Koike Katsuro
Published: July 2002

Accumulated findings have indicated that hepatitis B virus (HBV) DNA integrates into the cellular DNA of HBV-infected chronic hepatitis tissues. The integrated sequence (IS) of HBV DNA at the virus-cell junction is conserved in a 25-bp region which is adjacent to direct repeat 1. A cellular protein which we purified from the nuclear extract of HepG2 cells binds to the IS and was designated IS binding protein 3 (ISBP3). The amino acid sequence of ISBP3 was determined and found to be identical to that of transcription initiation factor Yin and Yang 1 (YY1). An antibody against C-terminal amino acids of YY1 recognized ISBP3 in a Western blot analysis and an electrophoretic mobility shift assay. Furthermore, ISBP3 also interacted with Y3, which corresponds to the YY1 binding sequence, to enhance intramolecular recombination of polyomavirus DNA. Although YY1 is known as a transcription factor, the IS did not exhibit any effect on the transcription of precore and pregenome RNAs. The possible involvement of YY1 in the intramolecular recombination of linear replicative HBV DNA has been examined (Y. Hayashi et al., unpublished data). Data suggest that YY1 is involved in the joining reaction between HBV DNA and cellular DNA to form the virus-cell junction.
Primary Murine Small Intestinal Epithelial Cells, Maintained in Long-Term Culture, Are Susceptible to Rotavirus Infection
Authors: Macartney Kristine K. , Baumgart Daniel C. , Carding Simon R. , Brubaker Jeffery O. , Offit Paul A.
Published: July 2002

We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-IC
cl2
. Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-IC
cl2
cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology.
Variability and Immunogenicity of Caprine Arthritis-Encephalitis Virus Surface Glycoprotein
Authors: Valas S. , Benoit C. , Baudry C. , Perrin G. , Mamoun R. Z.
Published: July 2002

The complete surface glycoprotein (SU) nucleotide sequences of three French isolates of caprine arthritis-encephalitis virus (CAEV) were determined and compared with those of previously described isolates: three American isolates and one French isolate. Phylogenetic analyses revealed the existence of four distinct and roughly equidistant evolutionary CAEV subtypes. Four conserved and five variable domains were identified in the SU. The fine specificities of antibodies produced against these domains during natural infection were examined using a pepscan analysis. Nine immunogenic segments were delineated throughout the conserved and variable domains of SU, two of them corresponding to conserved immunodominant epitopes. Antigenic determinants which may be involved in the immunopathogenic process induced by CAEV were identified. These results also provide sensitive and specific antigen peptides for the serological detection and differentiation of CAEV and visna/maedi virus infections.
Antigenic Drift in the Influenza A Virus (H3N2) Nucleoprotein and Escape from Recognition by Cytotoxic T Lymphocytes
Authors: Voeten J. T. M. , Bestebroer T. M. , Nieuwkoop N. J. , Fouchier R. A. M. , Osterhaus A. D. M. E. , Rimmelzwaan G. F.
Published: July 2002

Viruses exploit different strategies to escape immune surveillance, including the introduction of mutations in cytotoxic T-lymphocyte (CTL) epitopes. The sequence of these epitopes is critical for their binding to major histocompatibility complex (MHC) class I molecules and recognition by specific CTLs, both of which interactions may be lost by mutation. Sequence analysis of the nucleoprotein gene of influenza A viruses (H3N2) isolated in The Netherlands from 1989 to 1999 revealed two independent amino acid mutations at the anchor residue of the HLA-B27-specific CTL epitope SRYWAIRTR (383 to 391). A R384K mutation was found in influenza A viruses isolated during the influenza season 1989–1990 but not in subsequent seasons. In the influenza season 1993–1994, a novel mutation in the same CTL epitope at the same position was introduced. This R384G mutation proved to be conserved in all influenza A viruses isolated from 1993 onwards. Both mutations R384K and R384G abrogated MHC class I presentation and allowed escape from recognition by specific CTLs.
Histone Acetylation and Reactivation of Epstein-Barr Virus from Latency
Authors: Jenkins Peter J. , Binné Ulrich K. , Farrell Paul J.
Published: July 2002

Induction of the viral BZLF1 gene has previously been shown to be one of the first steps in the reactivation of Epstein-Barr virus (EBV). Using an EBV oriP episomal vector system, we have reconstituted the regulation of the promoter for BZLF1 on stably transfected episomes, mapped promoter elements required for that regulation, and investigated mechanisms that may control the switch between latency and the lytic cycle. Changes in histone acetylation at the promoter for the BZLF1 gene appear to be a key part of the reactivation mechanism of this herpesvirus.
Entry versus Blockade of Brain Infection following Oral or Intraperitoneal Scrapie Administration: Role of Prion Protein Expression in Peripheral Nerves and Spleen
Authors: Race Richard , Oldstone Michael , Chesebro Bruce
Published: July 2002

Naturally occurring transmissible spongiform encephalopathy (TSE) diseases such as bovine spongiform encephalopathy in cattle are probably transmitted by oral or other peripheral routes of infection. While prion protein (PrP) is required for susceptibility, the mechanism of spread of infection to the brain is not clear. Two prominent possibilities include hematogenous spread by leukocytes and neural spread by axonal transport. In the present experiments, following oral or intraperitoneal infection of transgenic mice with hamster scrapie strain 263K, hamster PrP expression in peripheral nerves was sufficient for successful infection of the brain, and cells of the spleen were not required either as a site of amplification or as transporters of infectivity. The role of tissue-specific PrP expression of foreign PrP in interference with scrapie infection was also studied in these transgenic mice. Peripheral expression of heterologous PrP completely protected the majority of mice from clinical disease after oral or intraperitoneal scrapie infection. Such extensive protection has not been seen in earlier studies on interference, and these results suggested that gene therapy with mutant PrP may be effective in preventing TSE diseases.
In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
Authors: Chang Ming , Marquardt Anthony P. , Wood Brent L. , Williams Ocean , Cotler Scott J. , Taylor Shari L. , Carithers Robert L. , Gretch David R.
Published: July 2002

Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver injury resulting from chronic hepatitis C virus (HCV) infection is not well understood. To examine the relationship between HCV replication in liver tissue and hepatocellular injury, a strand-specific in situ hybridization procedure was developed. The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expressing HCV RNAs. For the current study, both genomic (sense) and replicative-intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 liver tissue specimens from infected patients versus 0 of 11 liver tissue specimens from noninfected controls. The distribution pattern for HCV replicative-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and was located primarily in the cytoplasm of hepatocytes, with some signal in fibroblasts and/or macrophages in the surrounding fibroconnective tissue. However, HCV replicative-intermediate RNA showed a more focal pattern of distribution and was exclusively localized in the cytoplasm of hepatocytes. There was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury. However, a highly significant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflammatory activity (
P
, < 0.0001). Furthermore, the ratio of cells staining positive for HCV replicative-intermediate versus genomic RNA correlated with the histological severity of liver injury (
P
, 0.0065), supporting the hypothesis that active replication of HCV in liver tissue may be a significant determinant of hepatocellular injury.
Biochemical Characterization of Rotavirus Receptors in MA104 Cells
Authors: Guerrero Carlos A. , Zárate Selene , Corkidi Gabriel , López Susana , Arias Carlos F.
Published: July 2002

We have tested the effect of metabolic inhibitors, membrane cholesterol depletion, and detergent extraction of cell surface molecules on the susceptibility of MA104 cells to infection by rotaviruses. Treatment of cells with tunicamycin, an inhibitor of protein N glycosylation, blocked the infectivity of the SA-dependent rotavirus RRV and its SA-independent variant nar3 by about 50%, while the inhibition of O glycosylation had no effect. The inhibitor of glycolipid biosynthesis
d
,
l
-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human rotavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with β-cyclodextrin reduced the infectivity of the three viruses by more than 90%. The involvement of N-glycoproteins, glycolipids, and cholesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells incubated with the nonionic detergent octyl-β-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to which they became refractory to infection, suggesting that OG extracts the potential virus receptor(s) from the cell surface. Accordingly, when preincubated with the viruses, the OG extract inhibited the virus infectivity by more than 95%. This inhibition was abolished when the extract was treated with either proteases or heat but not when it was treated with neuraminidase, indicating the protein nature of the inhibitor. Two protein fractions of around 57 and 75 kDa were isolated from the extract, and these fractions were shown to have rotavirus-blocking activity. Also, antibodies to these fractions efficiently inhibited the infectivity of the viruses in untreated as well as in neuraminidase-treated cells. Five individual protein bands of 30, 45, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were finally isolated from the OG extract. These proteins are good candidates to function as rotavirus receptors.
Disrupting Surfaces of Nef Required for Downregulation of CD4 and for Enhancement of Virion Infectivity Attenuates Simian Immunodeficiency Virus Replication In Vivo
Authors: Iafrate A. John , Carl Silke , Bronson Scott , Stahl-Hennig Christiane , Swigut Tomek , Skowronski Jacek , Kirchhoff Frank
Published: July 2002

The multifunctional simian and human immunodeficiency virus (SIV and HIV) Nef proteins are important for virulence. We studied the importance of selected Nef functions using an SIV Nef with mutations in two regions that are required for CD4 downregulation. This Nef mutant is defective for downregulating CD4 and, in addition, for enhancing SIV infectivity and induction of SIV replication from infected quiescent peripheral blood mononuclear cells, but not for other known functions, including downregulation of class I major histocompatibility complex (MHC) cell surface expression. Replication of SIV containing this Nef variant in rhesus monkeys was attenuated early during infection. Subsequent increases in viral load coincided with selection of reversions and second-site compensatory changes in Nef. Our results indicate that the surfaces of Nef that mediate CD4 downregulation and the enhancement of virion infectivity are critical for SIV replication in vivo. Furthermore, these findings indicate that class I MHC downregulation by Nef is not sufficient for SIV virulence early in infection.
Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo
Authors: Twizere Jean-Claude , Kerkhofs Pierre , Burny Arsène , Portetelle Daniel , Kettmann Richard , Willems Luc
Published: July 2002

Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of thetaxgene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.
Epstein-Barr Virus LMP2A Transforms Epithelial Cells, Inhibits Cell Differentiation, and Activates Akt
Authors: Scholle Frank , Bendt Katharine M. , Raab-Traub Nancy
Published: July 2002

The Epstein-Barr virus LMP2A protein was expressed in a human keratinocyte cell line, HaCaT, and effects on epithelial cell growth were detected in organotypic raft cultures and in vivo in nude mice. Raft cultures derived from LMP2A-expressing cells were hyperproliferative, and epithelial differentiation was inhibited. The LMP2A-expressing HaCaT cells were able to grow anchorage independently and formed colonies in soft agar. HaCaT cells expressing LMP2A were highly tumorigenic and formed aggressive tumors in nude mice. The LMP2A tumors were poorly differentiated and highly proliferative, in contrast to occasional tumors that arose from parental HaCaT cells and vector control cells, which grew slowly and remained highly differentiated. Animals injected with LMP2A-expressing cells developed frequent metastases, which predominantly involved lymphoid organs. Involucrin, a marker of epithelial differentiation, and E-cadherin, involved in the maintenance of intercellular contact, were downregulated in LMP2A tumors. Whereas activation of the mitogen-activated protein kinase pathway was not observed, phosphatidylinositol-3-kinase (PI3-kinase)-dependent activation of the serine-threonine kinase Akt was detected in LMP2A-expressing cells and LMP2A tumors. Inhibition of this pathway blocked growth in soft agar. These data indicate that LMP2A greatly affects cell growth and differentiation pathways in epithelial cells, in part through activation of the PI3-kinase–Akt pathway.
Phosphorylation Status of the Parvovirus Minute Virus of Mice Particle: Mapping and Biological Relevance of the Major Phosphorylation Sites
Authors: Maroto Beatriz , Ramı́rez Juan C. , Almendral José M.
Published: July 2002

The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the
32
P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.
Reconstitution of Marek's Disease Virus Serotype 1 (MDV-1) from DNA Cloned as a Bacterial Artificial Chromosome and Characterization of a Glycoprotein B-Negative MDV-1 Mutant
Authors: Schumacher Daniel , Tischer B. Karsten , Fuchs Walter , Osterrieder Nikolaus
Published: July 2002

The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in
Escherichia coli
as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U
S
2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform
Escherichia coli
strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.
Murine Cytomegalovirus Containing a Mutation at Open Reading Frame M37 Is Severely Attenuated in Growth and Virulence In Vivo
Authors: Lee Manfred , Xiao Jianqiao , Haghjoo Erik , Zhan Xiaoyan , Abenes Gerry , Tuong Tong , Dunn Walter , Liu Fenyong
Published: July 2002

A pool of murine cytomegalovirus (MCMV) mutants was generated by using a Tn
3
-based transposon mutagenesis procedure. One of the mutants, RvM37, which contained the transposon sequence at open reading frame M37, was characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M37 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M37 region, the viral mutant was severely attenuated in growth in both BALB/c and SCID mice after intraperitoneal infection. Specifically, titers of the Smith strain and rescued virus in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice at 21 days postinfection were about 5 × 10
5
, 2 × 10
5
, 5 × 10
4
, 5 × 10
3
, and 1 × 10
4
PFU/ml of organ homogenate, respectively; in contrast, titers of RvM37 in these organs were less than 10
2
PFU/ml of organ homogenate. Moreover, the virulence of the mutant virus appeared to be significantly attenuated because none of the SCID mice infected with RvM37 had died by 120 days postinfection, while all animals infected with the wild-type and rescued viruses had died by 26 days postinfection. Our results suggest that M37 probably encodes a virulence factor and is required for MCMV virulence in SCID mice and for optimal viral growth in vivo.
A Null Mutation in the UL36 Gene of Herpes Simplex Virus Type 1 Results in Accumulation of Unenveloped DNA-Filled Capsids in the Cytoplasm of Infected Cells
Authors: Desai Prashant J.
Published: July 2002

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KΔUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KΔUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KΔUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KΔUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KΔUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.