bluuebunny/crossref_metadata_2025 huggingface dataset:
Whole-Genome Analysis of Genes Regulated by theBacillus subtilisCompetence Transcription Factor ComK
Authors: Ogura Mitsuo , Yamaguchi Hirotake , Kobayashi Kazuo , Ogasawara Naotake , Fujita Yasutaro , Tanaka Teruo
Published: July 2002

TheBacillus subtiliscompetence transcription factor ComK is required for establishment of competence for genetic transformation. In an attempt to study the ComK factor further, we explored the genes regulated by ComK using the DNA microarray technique. In addition to the genes known to be dependent on ComK for expression, we found many genes or operons whose ComK dependence was not known previously. Among these genes, we confirmed the ComK dependence of 16 genes by usinglacZfusions, and three genes were partially dependent on ComK. Transformation efficiency was significantly reduced in ansmfdisruption mutant, although disruption of the other ComK-dependent genes did not result in significant decreases in transformation efficiency. Nucleotide sequences similar to that of the ComK box were found for most of the newly discovered genes regulated by ComK.
GranulicatellaandAbiotrophiaSpecies from Human Clinical Specimens
Authors: Christensen Jens Jørgen , Facklam Richard R.
Published: July 2002

One hundred one isolates of nutritionally variant streptococci from 97 patients were phenotypically characterized and compared with the type strains ofGranulicatella adiacens(formerlyAbiotrophia adiacens) (ATCC 49175T)Abiotrophia defectiva(ATCC 49176T), andGranulicatella elegans(formerlyAbiotrophia elegans) (DSM 11693T). Of the isolates, 55 and 43 resembledG.adiacensandA.defectiva, respectively, while 3 strains resembledG.elegans. Phenotypic characteristics useful in differentiating between species within the generaGranulicatellaandAbiotrophia(G.adiacens,G.elegans,Granulicatella balaenopterae, andA.defectiva) were production of α- and β-galactosidase; production of β-glucuronidase; hippurate hydrolysis; arginine dihydrolase activity; and acid production from trehalose, sucrose, pullulan, and tagatose. From the reports submitted with the specimens, the clinical diagnosis was endocarditis in 58% of patients and septicemia or bacteremia in 26% of patients.
Evaluation of ColorPAC
Giardia/Cryptosporidium
Rapid Assay and ProSpecT
Giardia/Cryptosporidium
Microplate Assay for Detection of
Giardia
and
Cryptosporidium
in Fecal Specimens
Authors: Katanik M. T. , Schneider S. K. , Rosenblatt J. E. , Hall G. S. , Procop G. W.
Published: July 2002

Detection of
Giardia
and
Cryptosporidium
in clinical stool specimens using the ColorPAC and ProSpecT enzyme immunoassays revealed 98.7% agreement for
Giardia
detection and 98.1% agreement for
Cryptosporidium
detection. Sensitivities were uniformly 100%. The specificities of the ColorPAC immunoassay for
Giardia
and
Cryptosporidium
detection were 100 and 99.5%, respectively, and those for the ProSpecT assay were 98.4 and 98.6%, respectively. The false-positive reactions with the ProSpecT assay occurred with specimens that were grossly bloody.
Relatedness of
Streptococcus suis
Isolates of Various Serotypes and Clinical Backgrounds as Evaluated by Macrorestriction Analysis and Expression of Potential Virulence Traits
Authors: Allgaier Achim , Goethe Ralph , Wisselink Henk J. , Smith Hilde E. , Valentin-Weigand Peter
Published: July 2002

We evaluated the genetic diversity of
Streptococcus suis
isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes
Sma
I and
Apa
I allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the
S. suis
population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains.
Detection and Heterogeneity of Herpesviruses Causing Pacheco's Disease in Parrots
Authors: Tomaszewski Elizabeth , Wilson Van G. , Wigle William L. , Phalen David N.
Published: July 2002

Pacheco's disease (PD) is a common, often fatal, disease of parrots. We cloned a virus isolate from a parrot that had characteristic lesions of PD. Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1. Five primer sets were developed from these sequences. The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD. The primer sets amplified DNA from all but one sample. Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses. A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%). A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses. PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses.
Molecular Epidemiology of Outbreak of Respiratory Syncytial Virus within Bone Marrow Transplantation Unit
Authors: Taylor Graham S. , Vipond I. Barry , Caul E. Owen
Published: July 2002

During the winter of 1995–1996, eight of nine bone marrow transplantation (BMT) unit patients were infected with the same strain of respiratory syncytial virus (RSV). This RSV strain was not detected in 20 hospitalized patients from the community, suggesting that the BMT unit infections did not occur by independent incidents of transmission from the community.
Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
by Enzyme Immunoassay, Culture, and Three Nucleic Acid Amplification Tests
Authors: Van Dyck E. , Ieven M. , Pattyn S. , Van Damme L. , Laga M.
Published: July 2002

The purpose of this study was to evaluate and compare three commercially available nucleic acid amplification tests (NAATs) for the detection of
Neisseria gonorrhoeae
and
Chlamydia trachomatis
. Roche PCR and Becton Dickinson strand displacement amplification (SDA) were performed on 733 endocervical swab specimens from commercial sex workers. Abbott ligase chain reaction (LCR) was performed on a subset of 396 samples. Endocervical specimens from all women were also tested by culture for
N. gonorrhoeae
and by Syva MicroTrak enzyme immunoassay (EIA) for
C. trachomatis
. A positive
N. gonorrhoeae
result was defined as a positive result by culture or by two NAATs, and a positive
C. trachomatis
result was defined as a positive result by two tests. According to these definitions, the sensitivities and specificities for the subsample of 396 specimens of
N. gonorrhoeae
culture, PCR, SDA, and LCR were 69.8, 95.2, 88.9, and 88.9% and 100, 99.4, 100, and 99.1%, respectively; the sensitivities and specificities of
C. trachomatis
EIA, PCR, SDA, and LCR were 42.0, 98.0, 94.0, and 90.0% and 100, 98.0, 100, and 98.6%, respectively. The performance characteristics of
N. gonorrhoeae
culture, PCR, and SDA and
C. trachomatis
EIA, PCR, and SDA for all 733 specimens were defined without inclusion of LCR results and by discrepant analysis after resolution of discordant
N. gonorrhoeae
PCR results and of discordant
C. trachomatis
EIA and PCR results by LCR testing. The sensitivities of
N. gonorrhoeae
culture, PCR, and SDA before and after LCR resolution were 67.8, 95.7, and 93.9% and 65, 95.8, and 90.0%, respectively. The sensitivities of
C. trachomatis
EIA, PCR, and SDA decreased from 39.4, 100, and 100% to 38.7, 98.7, and 94.7%, respectively. All three NAATs proved to be superior to
N. gonorrhoeae
culture and to
C. trachomatis
EIA. The accuracies of the different NAATs were quite similar. SDA was the only amplification assay with 100% specificity for detection of both
N. gonorrhoeae
and
C. trachomatis
in endocervical specimens.
The Common Ovine Shiga Toxin 2-Containing
Escherichia coli
Serotypes and Human Isolates of the Same Serotypes Possess a Stx2d Toxin Type
Authors: Ramachandran Vidiya , Hornitzky Michael A. , Bettelheim Karl A. , Walker Mark J. , Djordjevic Steven P.
Published: July 2002

Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the
stx
2
subtype of 168 Shiga toxin-producing
Escherichia coli
(STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major
stx
2
subtype in the ovine isolates was shown to be
stx
2d-Ount
(119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H
−
/H8/H40, O91:H
−
, O123:H
−
, O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H
−
possessed a
stx
2d-O111/OX3a
subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a
stx
2d
(
stx
2d-Ount/O111/OX3a
) subtype. These studies suggest that a specific
stx
2
subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of
E. coli
during outbreaks of STEC-associated diseases in humans.
Spread of Drug-Resistant Pulmonary Tuberculosis in Estonia
Authors: Krüüner Annika , Hoffner Sven E. , Sillastu Heinart , Danilovits Manfred , Levina Klavdia , Svenson Stefan B. , Ghebremichael Solomon , Koivula Tuija , Källenius Gunilla
Published: July 2002

Restriction fragment length polymorphism (RFLP) analysis of 209
Mycobacterium tuberculosis
clinical isolates obtained from newly detected pulmonary tuberculosis patients (151 male and 58 female; mean age, 41 years) in Estonia during 1994 showed that 61 isolates (29%) belonged to a genetically closely related group of isolates, family A, with a predominant IS
6110
banding pattern. These strains shared the majority of their IS
6110
DNA-containing restriction fragments, representing a predominant banding pattern (similarity, >65%). This family A comprised 12 clusters of identical isolates, and the largest cluster comprised 10 strains. The majority (87.5%) of all multidrug-resistant (MDR) isolates, 67.2% of all isolates with any drug resistance, but only 12% of the fully susceptible isolates of
M. tuberculosis
belonged to family A. These strains were confirmed by spoligotyping as members of the Beijing genotype family. The spread of Beijing genotype MDR
M. tuberculosis
strains was also frequently seen in 1997 to 1999. The members of this homogenous group of drug-resistant
M. tuberculosis
strains have contributed substantially to the continual emergence of drug-resistant tuberculosis all over Estonia.
Genotypes and Clinical Phenotypes of Hepatitis B Virus in Patients with Chronic Hepatitis B Virus Infection
Authors: Kao Jia-Horng , Chen Pei-Jer , Lai Ming-Yang , Chen Ding-Shinn
Published: July 2002

Genotype C of hepatitis B virus (HBV) has been shown to be associated with a poor clinical outcome, compared to genotype B. To explore the clinical phenotypes, with special reference to the seroconversion of hepatitis B e antigen (HBeAg) and frequency of acute exacerbation between patients infected with HBV genotypes B and C, a cohort of 272 Taiwanese patients with chronic HBV infection was analyzed. According to the status of HBeAg at enrollment and frequency of acute exacerbation during the follow-up period, five groups of patients with distinct clinical phenotypes were categorized. Of the 272 HBV carriers, 185 (68%) were infected with HBV genotype B and the remaining 87 (32%) were infected with genotype C. Among them, 150 (55%) were positive for HBeAg and patients with genotype C infection tended to have a higher positive rate of HBeAg than those with genotype B infection (63 versus 51%). Genotype B was more prevalent than genotype C in different groups of HBV carriers. However, the prevalence of genotype C in patients with multiple episodes of acute exacerbation who failed to have HBeAg seroconversion was significantly higher than in all 272 patients (50 versus 32%,
P
= 0.025), in those with HBeAg seroconversion after only one episode of acute exacerbation (50 versus 12%,
P
= 0.01), or in those negative for HBeAg at enrollment and without acute exacerbations (50 versus 23%,
P
= 0.002). In conclusion, patients with genotype C infection have a more aggressive clinical phenotype than do those with genotype B infection, which contributes to the former group's progressive liver disease and poor clinical outcomes.
Molecular Analysis of Group A
Streptococcus
Type
emm18
Isolates Temporally Associated with Acute Rheumatic Fever Outbreaks in Salt Lake City, Utah
Authors: Smoot James C. , Korgenski E. Kent , Daly Judy A. , Veasy L. George , Musser James M.
Published: July 2002

Acute rheumatic fever (ARF) and subsequent rheumatic heart disease are rare but serious sequelae of group A
Streptococcus
(GAS) infections in most western countries. Salt Lake City (SLC), Utah, and the surrounding intermountain region experienced a resurgence of ARF in 1985 which has persisted. The largest numbers of cases were encountered in 1985-1986 and in 1997-1998. Organisms with a mucoid colony phenotype when grown on blood agar plates were temporally associated with the higher incidence of ARF. To develop an understanding of the molecular population genetic structure of GAS strains associated with ARF in the SLC region, 964 mucoid and nonmucoid pharyngeal isolates recovered in SLC from 1984 to 1999 were studied by sequencing the
emm
gene. Isolates with an
emm18
allele were further characterized by sequencing the
spa
,
covR
, and
covS
genes. Peak periods of ARF were associated with GAS isolates possessing an
emm18
allele encoding the protein found in serotype M18 isolates. Among the serotype M18 isolates, the difference in the number of C repeats produced three size variants. Variation was limited in
spa
, a gene that encodes a streptococcal protective antigen, and
covR
and
covS
, genes that encode a two-component regulatory system that, when inactivated, results in a mucoid phenotype and enhanced virulence in mouse infection models. Pulsed-field gel electrophoresis showed a single restriction profile for serotype M18 organisms isolated during both peak periods of ARF. In SLC, the incidence of ARF coresurged with the occurrence of GAS serotype M18 isolates that have very restricted genetic variation.
Detection of Porcine Circovirus Types 1 and 2 in Serum and Tissue Samples of Pigs with and without Postweaning Multisystemic Wasting Syndrome
Authors: Calsamiglia Maria , Segalés Joaquim , Quintana Josefina , Rosell Carles , Domingo Mariano
Published: July 2002

Presence of porcine circovirus type 1 (PCV1) and PCV2 was studied in sera and superficial inguinal lymph nodes from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs by using in situ hybridization and PCR. PCV1 and PCV2 were found in less than 3% and more than 50% of the samples, respectively. The most sensitive technique and site was PCR in superficial inguinal lymph nodes, but in situ hybridization correlated better with presence of characteristic lesions.
Antimicrobial Susceptibility Testing of
Bacillus anthracis
: Comparison of Results Obtained by Using the National Committee for Clinical Laboratory Standards Broth Microdilution Reference and Etest Agar Gradient Diffusion Methods
Authors: Mohammed M. Jasmine , Marston Chung K. , Popovic Tanja , Weyant Robbin S. , Tenover Fred C.
Published: July 2002

We determined the patterns of antimicrobial susceptibility of 65 isolates of
Bacillus anthracis
(50 historical and 15 recent U.S. clinical isolates) to nine antimicrobial agents using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution reference method. The results for the 50 historical
B. anthracis
isolates obtained by the broth microdilution method were compared to those generated by the Etest agar gradient diffusion method. One isolate of
B. anthracis
was β-lactamase positive and resistant to penicillin (MIC, 128 μg/ml); a second isolate, which was β-lactamase negative, was borderline penicillin resistant, with the penicillin MICs for the isolate varying from 0.12 to 0.25 μg/ml; and the remainder of the isolates were β-lactamase negative and penicillin susceptible (MICs, ≤0.12 μg/ml). Approximately 78% of the isolates showed reduced susceptibility to ceftriaxone (MICs, ≥16 μg/ml). All
B. anthracis
isolates were susceptible to chloramphenicol (MICs, ≤8 μg/ml), ciprofloxacin (MICs, ≤ 1 μg/ml), clindamycin (MICs, ≤0.5 μg/ml), rifampin (MICs, ≤0.5 μg/ml), tetracycline (MICs, ≤0.06 μg/ml), and vancomycin (MICs, ≤2 μg/ml) by use of NCCLS breakpoints for staphylococci. All 15 recent
B. anthracis
isolates from the United States were susceptible to penicillin, doxycycline, and ciprofloxacin. By use of the susceptibility breakpoint for staphylococci of ≤0.5 μg/ml, 97% of the
B. anthracis
isolates tested would have been categorized as intermediate to erythromycin. No statistically significant difference was found between the results of broth microdilution testing and the results of the Etest method for any of the antimicrobial agents tested; however, the results for penicillin obtained by the Etest were 1 to 9 dilutions lower than those obtained by the broth microdilution method. The differences in the penicillin MICs by the Etest method and the difficulties of reading the Etest results through the glass of a biological safety cabinet may limit the utility of this alternate susceptibility testing method for
B. anthracis
isolates.
High-Sensitivity PCR Detection of Parvovirus B19 in Plasma
Authors: Daly P. , Corcoran A. , Mahon B. P. , Doyle S.
Published: July 2002

Parvovirus B19 (B19) is a human pathogen transmitted to susceptible individuals via respiratory secretions and contaminated blood or blood products. B19 levels in pooled plasma of less than 10
4
genome equivalents/ml may not be infectious, while those greater than 10
7
/ml are capable of transmitting infection. A World Health Organization (WHO) B19 DNA international standard has been recently introduced. The purpose of the present work was to develop a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) calibrated against the WHO B19 DNA international standard which could easily and reliably detect B19 DNA levels in plasma above 10
4
IU/ml (6.5 × 10
3
genome equivalents/ml). A B19 PCR-ELISA system was developed which uses a dinitrophenylated oligonucleotide probe to detect immobilized biotinylated amplicons following single-round PCR amplification. The level of B19 DNA (in international units per milliliter) in individual and pooled plasma specimens was evaluated. Proteinase K treatment of plasma was found to be sufficient to quantitatively release B19 DNA. The B19 PCR-ELISA had a sensitivity of detection of 1.6 × 10
3
IU/ml B19 DNA and a dynamic range extending from 8 to 1,000 IU of B19 DNA (equivalent to 1.6 × 10
3
to 2 × 10
5
IU of B19 DNA/ml). Furthermore, the antibody profile of pooled plasma products was determined in terms of B19 immunoglobulin G (IgG) (in international units per milliliter). The B19 IgG level was found to be 64.7 ± 17.5 IU/ml (mean ± standard deviation). The B19 PCR-ELISA, which is calibrated against the B19 DNA international standard, may have an application for the rapid screening of plasma minipools for B19 DNA, thereby leading to an improvement in blood product safety.
Enterococcus gallinarum
Endocarditis Occurring on Native Heart Valves
Authors: Dargere Sylvie , Vergnaud Michel , Verdon Renaud , Saloux Eric , Le Page Olivier , Leclercq Roland , Bazin Claude
Published: July 2002

We report the first case of
Enterococcus gallinarum
endocarditis developing on normal native heart valves. Using phenotypic and molecular methods, a precise identification of this naturally vancomycin-resistant species allowed an optimal antibiotic therapy and the patient's recovery.
Differential Narrow Focusing of Immunodominant Human Immunodeficiency Virus Gag-Specific Cytotoxic T-Lymphocyte Responses in Infected African and Caucasoid Adults and Children
Authors: Goulder Philip J. R. , Brander C. , Annamalai K. , Mngqundaniso N. , Govender U. , Tang Y. , He S. , Hartman K. E. , O'Callaghan C. A. , Ogg G. S. , Altfeld M. A. , Rosenberg E. S. , Cao H. , Kalams S. A. , Hammond M. , Bunce M. , Pelton S. I. , Burchett S. A. , McIntosh K. , Coovadia H. M. , Walker B. D.
Published: July 2002

Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17Gagand p24Gagsequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17Gagpeptide residues 16 to 30 (38 versus 12%;P< 0.01), while p24Gagpeptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%;P< 0.005). Within this 20-mer p24Gag, an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.
Kinetic Analysis of Human Immunodeficiency Virus Type 1 Assembly Reveals the Presence of Sequential Intermediates
Authors: Tritel Marc , Resh Marilyn D.
Published: July 2002

The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag protein precursor, but the molecular details of these processes remain poorly defined. In this study, we have combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in the lentivirus assembly process. We show that newly synthesized HIV-1 Gag rapidly forms cytoplasmic protein complexes that are resistant to detergent treatment, sensitive to protease digestion, and degraded intracellularly. A subpopulation of newly synthesized Gag binds membranes within 5 to 10 min and over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on Optiprep density gradients. These complexes likely represent assembly intermediates because they are not observed with assembly-defective Gag mutants and can be chased into extracellular viruslike particles. At steady state, nearly all of the Gag is present as membrane-bound complexes in various stages of assembly. The identification of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via an ordered process. Assembly intermediates should serve as attractive targets for the design of antiviral agents that interfere with the process of particle production.
A Cytoplasmic RNA Vector Derived from Nontransmissible Sendai Virus with Efficient Gene Transfer and Expression
Authors: Li Hai-Ou , Zhu Ya-Feng , Asakawa Makoto , Kuma Hidekazu , Hirata Takahiro , Ueda Yasuji , Lee Yun-Sik , Fukumura Masayuki , Iida Akihiro , Kato Atsushi , Nagai Yoshiyuki , Hasegawa Mamoru
Published: July 2002

We have recovered a virion from defective cDNA of
Sendai virus
(SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the
Paramyxoviridae
. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 × 10
8
to 1.0 × 10
8
cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.
Assembly of Retrovirus Capsid-Nucleocapsid Proteins in the Presence of Membranes or RNA
Authors: Zuber Guy , McDermott Jason , Karanjia Sonya , Zhao Weiyi , Schmid Michael F. , Barklis Eric
Published: July 2002

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.
Infectious Hematopoietic Necrosis Virus Matrix Protein Inhibits Host-Directed Gene Expression and Induces Morphological Changes of Apoptosis in Cell Cultures
Authors: Chiou Pinwen P. , Kim Carol H. , Ormonde Patricia , Leong Jo-Ann C.
Published: July 2002

Infectious hematopoietic necrosis virus (IHNV) infection in tissue culture cells has previously been shown to result in the shutdown of host protein synthesis, cell rounding, and cell death. We report here an investigation of the cytopathogenicity of the viral phosphoprotein (P or M1), matrix (M or M2), and nonvirion (NV) proteins in cultured fish cells. The expression of M alone potently inhibited reporter gene expression from a viral and an interferon (IFN)-inducible promoter, whereas P and NV did not produce a similar effect. Northern blot analysis further revealed a reduction in the steady-state level of reporter mRNA when the M gene was cotransfected into cells; conversely, M mRNA was not drastically reduced in the same cells. By immunofluorescence confocal microscopy, fragmented nuclei were found in some cells expressing M protein but not in cells expressing P, NV, or β-galactosidase protein. Electron microscopy revealed the morphological changes associated with apoptosis in the M-transfected cells. Furthermore, IHNV infection was shown to produce DNA “laddering” in cultured cells. Taken together, these data suggested at least two functions for M protein in an IHNV infection: down regulation of host transcription and the induction of programmed cell death. In the course of these experiments, we also discovered that NV expression was associated with cell rounding, the first biological effect on cells to be attributed to the NV gene.
Vaccine Protection against Simian Immunodeficiency Virus by Recombinant Strains of Herpes Simplex Virus
Authors: Murphy Cynthia G. , Lucas William T. , Means Robert E. , Czajak Susan , Hale Corrina L. , Lifson Jeffrey D. , Kaur Amitinder , Johnson R. Paul , Knipe David M. , Desrosiers Ronald C.
Published: July 2002

An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.
Sequence Variability of Borna Disease Virus: Resistance to Superinfection May Contribute to High Genome Stability in Persistently Infected Cells
Authors: Formella Stephan , Jehle Christian , Sauder Christian , Staeheli Peter , Schwemmle Martin
Published: July 2002

The RNA genome of Borna disease virus (BDV) shows extraordinary stability in persistently infected cell cultures. We performed bottleneck experiments in which virus populations from single infected cells were allowed to spread through cultures of uninfected cells and in which RNase protection assays were used to identify virus variants with mutations in a 535-nucleotide fragment of the M-G open reading frames. In one of the cell cultures, the major virus species (designated 2/1) was a variant with two point mutations in the G open reading frame. When fresh cells were infected with a low dose of a virus stock prepared from 2/1-containing cells, only a minority of the resulting persistently infected cultures contained detectable levels of the variant, whereas the others all seemed to contain wild-type virus. The BDV variant 2/1 remained stable in the various persistently infected cell cultures, indicating that the cells were resistant to superinfection by wild-type virus. Indeed, cells persistently infected with prototype BDV He/80 were also found to resist superinfection with strain V and vice versa. Our screen for mutations in the viral M and G genes of different rat-derived BDV virus stocks revealed that only one of four stocks believed to contain He/80 harbored virus with the original sequence. Two stocks mainly contained a novel virus variant with about 3% sequence divergence, whereas the fourth stock contained a mixture of both viruses. When the mixture was inoculated into the brains of newborn mice, the novel variant was preferentially amplified. These results provide evidence that the BDV genome is mutating more frequently than estimated from its invariant appearance in persistently infected cell cultures and that resistance to superinfection might strongly select against novel variants.
Expression of Mouse Mammary Tumor Virus Superantigen Accelerates Tumorigenicity of Myeloma Cells
Authors: Umemura Masayuki , Wajjwalku Worawidh , Upragarin Narin , Liu Tie , Nishimura Hitoshi , Matsuguchi Tetsuya , Nishiyama Yukihiro , Wilson Gary M. , Yoshikai Yasunobu
Published: July 2002

To investigate whether superantigen (SAG) from endogenous mouse mammary tumor virus functions as an immunogenic or a tumorigenic factor in tumor development, the BALB/c myeloma cell line FO was transfected with the SAG gene from the 3′Mtv-50long terminal repeat (LTR) open reading frame (ORF), the product of which was specific for Vβ6. All five transfectants expressingMtv-50LTR ORF mRNA showed stimulatory activity for Vβ6 T-cell hybridomas in vitro; this activity was inhibited by the addition of anti-Mtv-7monoclonal antibody (MAb) or anti-major histocompatibility complex class III-AdandI-EdMAb. All transfectants with the SAG gene grew more rapidly than did mock transfectants in BALB/c mice after subcutaneous inoculation, whereas all clones, including mock transfectants, grew equally well in athymic nude mice. A significant fraction of Vβ6 T cells selectively expressed activation markers, including CD44high, CD62Llow, and CD69high, and produced large amounts of interleukin 5 (IL-5) and IL-6 in BALB/c mice inoculated with transfectants. These results suggested that the expression of viral SAG enhances the tumorigenicity of a myeloma cell line through the stimulation of SAG-reactive T cells.
Identification of Carbohydrate-Binding Domains in the Attachment Proteins of Type 1 and Type 3 Reoviruses
Authors: Chappell James D. , Duong Joy L. , Wright Benjamin W. , Dermody Terence S.
Published: July 2002

The reovirus attachment protein, ς1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The ς1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of ς1 that binds cell surface carbohydrate. Chimeric and truncated ς1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-ς1 antibodies, and oligomerization indicates that the chimeric and truncated ς1 proteins are properly folded. To assess carbohydrate binding, recombinant ς1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated ς1 proteins, the sialic acid-binding domain of type 3 ς1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted β-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of ς1 protein purified from virions. In contrast, the homologous region of T1L ς1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 ς1 tail. Furthermore, our findings indicate that T1L and T3D ς1 proteins contain different arrangements of receptor-binding domains.
Cellular and Species Resistance to Murine Amphotropic, Gibbon Ape, and Feline Subgroup C Leukemia Viruses Is Strongly Influenced by Receptor Expression Levels and by Receptor Masking Mechanisms
Authors: Tailor Chetankumar S. , Nouri Ali , Kabat David
Published: July 2002

Chinese hamster ovary (CHO) cells are resistant to infections by gibbon ape leukemia virus (GALV) and amphotropic murine leukemia virus (A-MLV) unless they are pretreated with tunicamycin, an inhibitor of N-linked glycosylation. These viruses use the related sodium-phosphate symporters Pit1 and Pit2, respectively, as receptors in nonhamster cells, and evidence has suggested that the corresponding transporters of CHO cells may be masked by tunicamycin-sensitive secreted inhibitors. Although the E36 line of Chinese hamster cells was reported to secrete the putative Pit2 inhibitor and to be sensitive to the inhibitory CHO factors, E36 cells are highly susceptible to both GALV and A-MLV in the absence of tunicamycin. Moreover, expression of E36 Pit2 in CHO cells conferred tunicamycin-independent susceptibilities to both viruses. Based on the latter results, it was suggested that E36 Pit2 must functionally differ from the endogenous Pit2 of CHO cells. To test these ideas, we analyzed the receptor properties of CHO Pit1 and Pit2 in CHO cells. Surprisingly, and counterintuitively, transfection of a CHO Pit2 expression vector into CHO cells conferred strong susceptibility to both GALV and A-MLV, and similar overexpression of CHO Pit1 conferred susceptibility to GALV. Thus, CHO Pit2 is a promiscuous functional receptor for both viruses, and CHO Pit1 is a functional receptor for GALV. Similarly, we found that the natural resistance of
Mus dunni
tail fibroblasts to subgroup C feline leukemia viruses (FeLV-C) was eliminated simply by overexpression of the endogenous FeLV-C receptor homologue. These results demonstrate a novel and simple method to unmask latent retroviral receptor activities that occur in some cells. Specifically, resistances to retroviruses that are caused by subthreshold levels of receptor expression or by stoichiometrically limited masking or interference mechanisms can be efficiently overcome simply by overexpressing the endogenous receptors in the same cells.
Functional Importance of the Coiled-Coil of the Ebola Virus Glycoprotein
Authors: Watanabe Shinji , Takada Ayato , Watanabe Tokiko , Ito Hiroshi , Kida Hiroshi , Kawaoka Yoshihiro
Published: July 2002

Ebola virus contains a single glycoprotein (GP) that is responsible for receptor binding and membrane fusion and is proteolytically cleaved into disulfide-linked GP1 and GP2 subunits. The GP2 subunit possesses a coiled-coil motif, which plays an important role in the oligomerization and fusion activity of other viral GPs. To determine the functional significance of the coiled-coil motif of GP2, we examined the effects of peptides corresponding to the coiled-coil motif of GP2 on the infectivity of a mutant vesicular stomatitis virus (lacking the receptor-binding/fusion protein) pseudotyped with the Ebola virus GP. A peptide corresponding to the C-terminal helix reduced the infectivity of the pseudotyped virus. We next introduced alanine substitutions into hydrophobic residues in the coiled-coil motif to identify residues important for GP function. None of the substitutions affected GP oligomerization, but some mutations, two in the N-terminal helix and all in the C-terminal helix, reduced the ability of GP to confer infectivity to the mutant vesicular stomatitis virus without affecting the transport of GP to the cell surface, its incorporation into virions, and the production of virus particles. These results indicate that the coiled-coil motif of GP2 plays an important role in facilitating the entry of Ebola virus into host cells and that peptides corresponding to this region could act as efficient antiviral agents.
Characterization of Three
nef
-Defective Human Immunodeficiency Virus Type 1 Strains Associated with Long-Term Nonprogression
Authors: Rhodes David I. , Ashton Lesley , Solomon Ajantha , Carr Andrew , Cooper David , Kaldor John , Deacon Nicholas
Published: July 2002

Long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection provide an opportunity to investigate both viral and host factors that influence the rate of disease progression. We have identified three HIV-1-infected individuals in Australia who have been infected for over 11 years with viruses that contain deletions in the
nef
and
nef
-long terminal repeat (
nef
/LTR) overlap regions. These viruses differ from each other and from other
nef
-defective strains of HIV-1 previously identified in Australia. One individual, LTS 3, is infected with a virus containing a
nef
gene with a deletion of 29 bp from the
nef
/LTR overlap region, resulting in a truncated Nef open reading frame. In addition to the Nef defect, only viruses containing truncated Vif open reading frames of 37 or 69 amino acids could be detected in peripheral blood mononuclear cells isolated from this patient. LTS 3 had a viral load of less than 20 copies of RNA/ml of plasma. The other two long-term survivors, LTS 9 and LTS 11, had loads of less than 200 copies of RNA/ml of plasma and are infected with viruses with larger deletions in both the
nef
alone and
nef
/LTR overlap regions. These viruses contain wild-type
vif
,
vpu
, and
vpr
accessory genes. All three strains of virus had envelope sequences characteristic of macrophagetropic viruses. These findings further indicate the reduced pathogenic potential of
nef
-defective viruses.
Expression of an Altered Ribonucleotide Reductase Activity Associated with the Replication of Murine Cytomegalovirus in Quiescent Fibroblasts
Authors: Lembo David , Gribaudo Giorgio , Hofer Anders , Riera Ludovica , Cornaglia Maura , Mondo Alessandra , Angeretti Alessandra , Gariglio Marisa , Thelander Lars , Landolfo Santo
Published: July 2002

Ribonucleotide reductase (RNR) is an essential enzyme for the de novo synthesis of both cellular and viral DNA and catalyzes the conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates. The enzyme consists of two nonidentical subunits, termed R1 and R2, whose expression is very low in resting cells and maximal in S-phase cells. Here we show that murine cytomegalovirus (MCMV) replication depends on ribonucleotide reduction since it is prevented by the RNR inhibitor hydroxyurea. MCMV infection of quiescent fibroblasts markedly induces both mRNA and protein corresponding to the cellular R2 subunit, whereas expression of the cellular R1 subunit does not appear to be up-regulated. The increase in R2 gene expression is due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse R2 promoter is induced following virus infection. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in R2 promoter activity. It was found that the viral gene M45, encoding a putative homologue of the R1 subunit, is expressed 24 and 48 h after infection. Meanwhile, we observed an expansion of the deoxyribonucleoside triphosphate pool between 24 and 48 h after infection; however, neither CDP reduction nor viral replication was inhibited by treatment with 10 mM thymidine. These findings indicate the induction of an RNR activity with an altered allosteric regulation compared to the mouse RNR following MCMV infection and suggest that the virus R1 homologue may complex with the induced cellular R2 protein to reconstitute a new RNR activity.
Marking and Gene Expression by a Lentivirus Vector in Transplanted Human and Nonhuman Primate CD34+Cells
Authors: An Dong Sung , Wersto Robert P. , Agricola Brian A. , Metzger Mark E. , Lu Stephanie , Amado Rafael G. , Chen Irvin S. Y. , Donahue Robert E.
Published: July 2002

Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34+cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.
Susceptibility of Sheep for Scrapie as Assessed by In Vitro Conversion of Nine Naturally Occurring Variants of PrP
Authors: Bossers Alex , de Vries Ruth , Smits Mari A.
Published: July 2002

Polymorphisms in the prion protein (PrP) gene are associated with phenotypic expression differences of transmissible spongiform encephalopathies in animals and humans. In sheep, at least 10 different mutually exclusive polymorphisms are present in PrP. In this study, we determined the efficiency of the in vitro formation of protease-resistant PrP of nine sheep PrP allelic variants in order to gauge the relative susceptibility of sheep for scrapie. No detectable spontaneous protease-resistant PrP formation occurred under the cell-free conditions used. All nine host-encoded cellular PrP (PrP
C
) variants had distinct conversion efficiencies induced by PrP
Sc
isolated from sheep with three different homozygous PrP genotypes. In general, PrP allelic variants with polymorphisms at either codon 136 (Ala to Val) or codon 141 (Leu to Phe) and phylogenetic wild-type sheep PrP
C
converted with highest efficiency to protease-resistant forms, which indicates a linkage with a high susceptibility of sheep for scrapie. PrP
C
variants with polymorphisms at codons 171 (Gln to Arg), 154 (Arg to His), and to a minor extent 112 (Met to Thr) converted with low efficiency to protease-resistant isoforms. This finding indicates a linkage of these alleles with a reduced susceptibility or resistance for scrapie. In addition, PrP
Sc
with the codon 171 (Gln-to-His) polymorphism is the first variant reported to induce higher conversion efficiencies with heterologous rather than homologous PrP variants. The results of this study strengthen our views on polymorphism barriers and have further implications for scrapie control programs by breeding strategies.