bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects ofEscherichia coliPhysiology on Growth of Phage T7 In Vivo and In Silico
Authors: You Lingchong , Suthers Patrick F. , Yin John
Published: July 2002

Phage development depends not only upon phage functions but also on the physiological state of the host, characterized by levels and activities of host cellular functions. We establishedEscherichia coliat different physiological states by continuous culture under different dilution rates and then measured its production of phage T7 during a single cycle of infection. We found that the intracellular eclipse time decreased and the rise rate increased as the growth rate of the host increased. To develop mechanistic insight, we extended a computer simulation for the growth of phage T7 to account for the physiology of its host. Literature data were used to establish mathematical correlations between host resources and the host growth rate; host resources included the amount of genomic DNA, pool sizes and elongation rates of RNA polymerases and ribosomes, pool sizes of amino acids and nucleoside triphosphates, and the cell volume. The in silico (simulated) dependence of the phage intracellular rise rate on the host growth rate gave quantitatively good agreement with our in vivo results, increasing fivefold for a 2.4-fold increase in host doublings per hour, and the simulated dependence of eclipse time on growth rate agreed qualitatively, deviating by a fixed delay. When the simulation was used to numerically uncouple host resources from the host growth rate, phage growth was found to be most sensitive to the host translation machinery, specifically, the level and elongation rate of the ribosomes. Finally, the simulation was used to follow how bottlenecks to phage growth shift in response to variations in host or phage functions.
Controlled Comparison of BacT/ALERT FAN Aerobic Medium and BACTEC Fungal Blood Culture Medium for Detection of Fungemia
Authors: McDonald L. Clifford , Weinstein Melvin P. , Fune Jose , Mirrett Stanley , Reimer Larry G. , Reller L. Barth
Published: July 2002

Yeasts are an increasingly common cause of nosocomial bloodstream infections. Methods for their detection are many; controlled comparisons are few. The vented FAN aerobic blood culture medium has been shown to be superior to the standard BacT/ALERT aerobic medium for the detection of fungemia as well as bacteremia. The BACTEC selective fungal medium (FM) (BD Biosciences, Sparks, Md.) allowed detection of more episodes of fungemia than did a resin-containing medium with equal volumes of blood cultured. Therefore, we compared vented FAN to FM for the ability to recover fungi from the blood of patients who were at increased risk of having fungemia. From 5,109 cultures processed for which both FAN and FM bottles were adequately filled, fungi were recovered from 87 cultures. Of these, 47 were detected with both bottles, 12 were detected with FAN only, and 28 were detected with FM only (
P
< 0.05). FAN was the first bottle positive for 36 of the 47 cultures for which both bottles yielded the same fungus, whereas the FM bottle was the first bottle positive for 11 cultures (
P
< 0.001). A total of 54 episodes of fungemia were identified, with 40 detected by both media, 4 detected only by FAN, and 10 detected only by FM (
P
value, not significant). We conclude that the vented FAN aerobic bottle is comparable to the FM bottle for detection of episodes of yeast infection but has the added benefit of detecting bacteria.
Identification of Enteropathogenic
Escherichia coli
in Simian Immunodeficiency Virus-Infected Infant and Adult Rhesus Macaques
Authors: Mansfield Keith G. , Lin Kuei-Chin , Newman Joseph , Schauer David , MacKey John , Lackner Andrew A. , Carville Angela
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) was recognized as a common opportunistic pathogen of simian immunodeficiency virus-infected rhesus macaques (
Macaca mulatta
) with AIDS. Retrospective analysis revealed that 27 of 96 (28.1%) animals with AIDS had features of EPEC infection, and EPEC was the most frequent pathogen of the gastrointestinal tract identified morphologically. In 7.3% of animals dying with AIDS, EPEC represented the sole opportunistic agent of the gastrointestinal tract at death. In 20.8% of cases, it was seen in combination with one or more gastrointestinal pathogens, including
Cryptosporidium parvum, Enterocytozoon bieneusi, Mycobacterium avium, Entamoeba histolytica, Balantidium coli, Strongyloides stercoralis
, cytomegalovirus, and adenovirus. Clinically, infection was associated with persistent diarrhea and wasting and was more frequent in animals that died at under 1 year of age (
P
< 0.001, Fisher exact test). The organism was associated with the characteristic attaching and effacing lesion in colonic tissue sections and produced a focal adherence pattern on a HEp-2 assay but was negative for Shiga toxin production as assessed by PCR and a HeLa cell cytotoxicity assay. A 2.6-kb fragment encompassing the intimin gene was amplified and sequenced and revealed 99.2% identity to sequences obtained from human isolates (GenBank
AF116899
) corresponding to the epsilon intimin subtype. Further investigations with rhesus macaques may offer opportunities to study the impact of EPEC on AIDS pathogenesis and gastrointestinal dysfunction.
Molecular Characterization of Invasive and Noninvasive
Campylobacter jejuni
and
Campylobacter coli
Isolates
Authors: Carvalho Alexandro C. T. , Ruiz-Palacios Guillermo M. , Ramos-Cervantes Pilar , Cervantes Luz-Elena , Jiang Xi , Pickering Larry K.
Published: July 2002

Campylobacter jejuni
is one of the most common causes of bacterial diarrhea worldwide and is the primary bacterial cause of food-borne illness. Adherence to and invasion of epithelial cells are the most important pathogenic mechanisms of
Campylobacter
diarrhea. Molecular characterization of invasive and noninvasive
Campylobacter
isolates from children with diarrhea and symptom-free children was performed by random amplified polymorphic DNA techniques (RAPD). A distinct RAPD profile with a DNA band of 1.6 kb was observed significantly more frequently among invasive (63%) than among noninvasive (16%)
Campylobacter
isolates (
P
= 0.000005). The 1.6-kb band was named the invasion-associated marker (IAM). Using specifically designed primers, a fragment of 518 bp of the
iam
locus was amplified in 85% of invasive and 20% of noninvasive strains (
P
= 0.0000000). Molecular typing with a PCR-restriction fragment length polymorphism assay which amplified the entire
iam
locus showed a
Hin
dIII restriction fragment polymorphism pattern associated mainly with invasive strains. Although cluster analysis of the RAPD fingerprinting showed genetic diversity among strains, two main clusters were identified. Cluster I comprised significantly more pathogenic and invasive isolates, while cluster II grouped the majority of nonpathogenic, noninvasive isolates. These data indicate that most of the invasive
Campylobacter
strains could be differentiated from noninvasive isolates by RAPD analysis and PCR using specific primers that amplify a fragment of the
iam
locus.
Group A Streptococcal Genotypes from Pediatric Throat Isolates in Rome, Italy
Authors: Dicuonzo Giordano , Gherardi Giovanni , Lorino Giulia , Angeletti Silvia , De Cesaris Marina , Fiscarelli Ersilia , Bessen Debra E. , Beall Bernard
Published: July 2002

In a study assessing genetic diversity, 114 group A streptococcus (GAS) isolates were recovered from pediatric pharyngitis patients in Rome, Italy. These isolates comprised 22 different M protein gene (
emm
) sequence types, 14 of which were associated with a distinct serum opacity factor/fibronectin binding protein gene (
sof
) sequence type. Isolates with the same
emm
gene sequence type generally shared a highly conserved chromosomal macrorestriction profile. In three instances, isolates with dissimilar macrorestriction profiles had identical
emm
types; in each of these cases multilocus sequence typing revealed that isolates with the same
emm
type were clones having the same allelic profiles. Ninety-eight percent of the pharyngeal isolates had
emm
types previously found to be highly associated with
mga
locus gene patterns commonly found in pharyngeal GAS isolates.
Virulence Properties and Serotypes of Shiga Toxin-Producing
Escherichia coli
from Healthy Australian Slaughter-Age Sheep
Authors: Djordjevic Steven P. , Hornitzky Michael A. , Bailey Graham , Gill Paul , Vanselow Barbara , Walker Keith , Bettelheim Karl A.
Published: July 2002

A group of 1,623 ovine fecal samples recovered from 65 geographically distinct mutton sheep and prime lamb properties across New South Wales, Australia, were screened for the presence of Shiga toxin-producing
Escherichia coli
(STEC) virulence factors (
stx
1
,
stx
2
,
eaeA
, and
ehxA
). A subset was cultured for STEC isolates containing associated virulence factors (
eaeA
and/or
ehxA
), which were isolated from 17 of 20 (85%) and 19 of 20 (95%) tested prime lamb and mutton sheep properties, respectively. STEC isolates containing
stx
1
,
stx
2
, and
ehxA
were most commonly isolated (19 of 40 flocks; 47.5%), and this profile was observed for 10 different serotypes. Among 90 STEC isolates studied, the most common serotypes were O91:H
−
(22 isolates [24.4%]), O5:H
−
(16 isolates [17.8%]), O128:H2 (11 isolates [12.2%]), O123:H
−
(8 isolates [8.9%]), and O85:H49 (5 isolates [5.6%]). Two isolates (2.2%) were typed as O157:H
−
. A total of 78 of 90 STEC isolates (86.7%) expressed Shiga toxin in Vero cell culture and 75 of 84
ehxA
-positive isolates (89.3%) expressed enterohemolysin on washed sheep blood agar.
eaeA
was observed in 11 of 90 (12.2%) ovine STEC isolates, including serotypes O5:H
−
, O84:H
−
, O85:H49, O123:H
−
O136:H40, and O157:H
−
. Although only 2 of 90 isolates were typed as O157:H
−
, the predominant serotypes recovered during this study have been recovered from human patients with clinical disease, albeit rarely.
Human Immunodeficiency Virus Type 1 Drug Resistance Testing: a Comparison of Three Sequence-Based Methods
Authors: Erali Maria , Page Sam , Reimer Larry G. , Hillyard David R.
Published: July 2002

The use of genotypic assays for determining drug resistance in human immunodeficiency virus (HIV) type 1 (HIV-1)-infected patients is increasing. These tests lack standardization and validation. The aim of this study was to evaluate several tests used for the determination of HIV-1 drug resistance. Two genotypic tests, the Visible Genetics TruGene HIV-1 Genotyping Kit and the Applied Biosystems HIV Genotyping System, were compared using 22 clinical samples. Genotyping results were also obtained from an independent reference laboratory. The Visible Genetics and Applied Biosystems genotyping tests identified similar mutations when differences in the drug databases and reference strains were taken into account, and 19 of 21 samples were equivalent. The concordance between the two assays was 99% (249 of 252 mutation sites). Mutations identified by the reference laboratory varied the most among those identified by the three genotypic tests, possibly because of differences in the databases. The concordance of the reference laboratory results with the results of the other two assays was 80% (201 of 252). Samples with 500 to 750 HIV RNA copies/ml could be sequenced by the Visible Genetics and Applied Biosystems assays using 1 ml of input. The Visible Genetics and Applied Biosystems assays both generated an accurate sequence. However, the throughput of the Visible Genetics assay is more limited and may require additional instruments. The two assays differ technically but are similar in overall complexity. Data analysis in the two assays is straightforward, but only the reports provided by Visible Genetics contain information relating mutations to drug resistance. HIV drug resistance genotyping by sequencing is a complex technology which presents a challenge for analysis, interpretation, and reporting.
Use of Real-Time Quantitative PCR To Detect
Chlamydophila felis
Infection
Authors: Helps Chris , Reeves Nicky , Tasker Séverine , Harbour Dave
Published: July 2002

A real-time PCR assay was developed to detect and quantify
Chlamydophila felis
infection of cats. The assay uses a molecular beacon to specifically identify the major outer membrane protein gene, is highly reproducible, and is able to detect fewer than 10 genomic copies.
Characterization of Multidrug-Resistant
Salmonella enterica
Serovar Typhimurium Isolated in Japan
Authors: Izumiya Hidemasa , Terajima Jun , Matsushita Shigeru , Tamura Kazumichi , Watanabe Haruo
Published: July 2002

A total of 221 isolates of multidrug-resistant
Salmonella enterica
serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.
Epidemiology of Candidemia: 3-Year Results from the Emerging Infections and the Epidemiology of Iowa Organisms Study
Authors: Diekema D. J. , Messer S. A. , Brueggemann A. B. , Coffman S. L. , Doern G. V. , Herwaldt L. A. , Pfaller M. A.
Published: July 2002

Bloodstream infections due to
Candida
species cause significant morbidity and mortality. Surveillance for candidemia is necessary to detect trends in species distribution and antifungal resistance. We performed prospective surveillance for candidemia at 16 hospitals in the State of Iowa from 1 July 1998 through 30 June 2001. Using U.S. Census Bureau and Iowa Hospital Association data to estimate a population denominator, we calculated the annual incidence of candidemia in Iowa to be 6.0 per 100,000 of population.
Candida albicans
was the most common species detected, but 43% of candidemias were due to species other than
C. albicans
. Overall, only 3% of
Candida
species were resistant to fluconazole. However,
Candida glabrata
was the most commonly isolated species other than
C. albicans
and demonstrated some resistance to azoles (fluconazole MIC at which 90% of the isolates tested are inhibited, 32 μg/ml; 10% resistant, 10% susceptible dose dependent).
C. glabrata
was more commonly isolated from older patients (
P
= 0.02) and caused over 25% of candidemias among persons 65 years of age or older. The investigational triazoles posaconazole, ravuconazole, and voriconazole had excellent in vitro activity overall against
Candida
species.
C. albicans
is the most important cause of candidemia and remains highly susceptible to available antifungal agents. However,
C. glabrata
has emerged as an important and potentially antifungal resistant cause of candidemia, particularly among the elderly.
Introduction of New Clones of Penicillin-Nonsusceptible
Streptococcus pneumoniae
in Hong Kong
Authors: Ip Margaret , Lyon Donald J. , Yung Raymond W. H. , Tsang Lily , Cheng Augustine F. B.
Published: July 2002

Analysis of penicillin-nonsusceptible
Streptococcus pneumoniae
(PNSP) isolates in Hong Kong by use of a combination of antibiogram typing, serotyping, multilocus sequence typing, and pulsed-field gel electrophoresis indicated that the dissemination of PNSP was the result of the spread of international clones: variants of the Spain
23F
-1 or Spain
6B
-2 clones were the predominant PNSP isolates from 1994 to 1997 and remained so, but Taiwan
19F
-14 and Taiwan serotype 6B clones were disseminated in Hong Kong in 1999 and 2000. Concomitant changes in antibiotic susceptibility profiles, with the rate of susceptibility to chloramphenicol rising from 10% in the period from 1994 to 1997 to 31% (
P
< 0.001) in 1999 and 2000, were noted to accompany the shift of clones.
Rapid Identification ofPenicillium marneffeiby PCR-Based Detection of Specific Sequences on the rRNA Gene
Authors: Vanittanakom Nongnuch , Vanittanakom Pramote , Hay Roderick J.
Published: July 2002

An emerging pathogenic dimorphic fungus,Penicillium marneffei, is one of the major causes of morbidity in patients with human immunodeficiency virus infection in Southeast Asia. A PCR-hybridization assay has been developed to identify this pathogen. This study describes the use of single and nested PCR methods for the rapid identification ofP. marneffei.Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA genes ofP. marneffei.The outer primers (RRF1 and RRH1) were fungus specific. The inner primers (Pm1 and Pm2) were specific forP. marneffeiand were used in nested or single PCR. The specific fragment of approximately 400-bp was amplified from both mold and yeast forms of 13P. marneffeihuman isolates, 12 bamboo rat isolates, and 1 soil isolate, but not from other fungi, bacteria, and human DNA. The amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The sensitivities of the single PCR and nested PCR were 1.0 pg/μl and 1.8 fg/μl, respectively. The assay is useful for rapid identification ofP. marneffeicultures. Very young culture ofP. marneffei(2-day-old filamentous colony, 2 mm in diameter) could be performed by this assay. The species was identified within 7 h (single PCR) or 10 h (nested PCR), compared to 4 to 7 days for confirmation of dimorphism. The application of these PCR methods for early diagnosis of the disease needs to be studied further.
Tumor-Specific, Replication-Competent Adenovirus Vectors Overexpressing the Adenovirus Death Protein
Authors: Doronin Konstantin , Toth Karoly , Kuppuswamy Mohan , Ward Peter , Tollefson Ann E. , Wold William S. M.
Published: July 2002

We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting theadpgene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutantdl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G0to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus,dl1101/1107, or wild-type Ad. They are also more efficient thandl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1−E3−replication-defective vector expressing β-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10−4PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected withdl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.
Human Papillomavirus Type 16 E6 Induces Self-Ubiquitination of the E6AP Ubiquitin-Protein Ligase
Authors: Kao Wynn H. , Beaudenon Sylvie L. , Talis Andrea L. , Huibregtse Jon M. , Howley Peter M.
Published: July 2002

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.
Effects of Amino Acid Substitutions at Position 115 on the Fidelity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Boyer Paul L. , Hughes Stephen H.
Published: July 2002

We compared the fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and two RT mutants, Y115F and Y115V. Although neither mutation had a large effect on the overall fidelity of the enzyme, both mutations altered the spectrum of mutations and the precise nature of the mutational hot spots. The effects of Y115V were greater than those of Y115F. When we compared the behavior of the wild-type enzyme with published data, we found that, in contrast to what has been published, misalignment/slippage could account for only a small fraction of the mutations we observed. We also found that a preponderance of the mutations (both transitions and transversions) resulted in the insertion of an A. Because we were measuring DNA-dependent DNA synthesis (plus-strand synthesis), this bias could contribute to the A-rich nature of the HIV-1 genome.
Wild-Type and YMDD Mutant Murine Leukemia Virus Reverse Transcriptases Are Resistant to 2′,3′-Dideoxy-3′-Thiacytidine
Authors: Halvas Elias K. , Svarovskaia Evguenia S. , Freed Eric O. , Pathak Vinay K.
Published: July 2002

The antiretroviral nucleoside analog 2′,3′-dideoxy-3′-thiacytidine (3TC) is a potent inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). A methionine-to-valine or methionine-to-isoleucine substitution at residue 184 in the HIV-1 YMDD motif, which is located at the RT active site, leads to a high level of resistance to 3TC. We sought to determine whether 3TC can inhibit the replication of wild-type murine leukemia virus (MLV), which contains V223 at the YVDD active site motif of the MLV RT, and of the V223M, V223I, V223A, and V223S mutant RTs. Surprisingly, the wild type and all four of the V223 mutants of MLV RT were highly resistant to 3TC. These results indicate that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC. Therefore, structural differences among similar RTs might result in widely divergent sensitivities to antiretroviral nucleoside analogs.
Lymphomas and High-Level Expression of Murine Leukemia Viruses in CFW Mice
Authors: Taddesse-Heath Lekidelu , Chattopadhyay Sisir K. , Dillehay Dirck L. , Lander Marilyn R. , Nagashfar Zohreh , Morse Herbert C. , Hartley Janet W.
Published: July 2002

Historically, Swiss Webster mice of the CFW subline, both inbred and random-bred stocks, have been considered to have a low spontaneous occurrence of hematopoietic system tumors, and previous reports of infectious expression of murine leukemia viruses (MuLVs) have been rare and unremarkable. In marked contrast, in the present study of CFW mice from one source observed by two laboratories over a 2-year period, nearly 60% developed tumors, 85% of which were lymphomas, the majority of B-cell origin. All tumors tested expressed ecotropic MuLVs, and most expressed mink cell focus-inducing (MCF) MuLVs. Among normal mice of weanling to advanced age, over one-half were positive for ecotropic virus in tail or lymphoid tissues, and MCF virus was frequently present in lymphoid tissue, less often in tail. Patterns of ecotropic proviral integration indicated that natural infection occurred by both genetic and exogenous routes. Lymphomas were induced in NIH Swiss mice infected as neonates with tissue culture-propagated MuLVs isolated from normal and tumor tissue of CFW mice.
Structural Determinants of Murine Leukemia Virus Reverse Transcriptase That Affect the Frequency of Template Switching
Authors: Svarovskaia Evguenia S. , Delviks Krista A. , Hwang Carey K. , Pathak Vinay K.
Published: July 2002

Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA or between copackaged viral RNAs to generate mutations and recombination. To identify structural elements of murine leukemia virus RT important for template switching, we developed an in vivo assay in which RT template switching within direct repeats functionally reconstituted the green fluorescent protein gene. We quantified the effect of mutations in the YXDD motif, the deoxynucleoside triphosphate binding site, the thumb domain, and the RNase H domain of RT and hydroxyurea treatment on the frequencies of template switching. Hydroxyurea treatment and some mutations in RT increased the frequency of RT template switching up to fivefold, while all of the mutations tested in the RNase H domain decreased the frequency of template switching by twofold. Based on these results, we propose a dynamic copy choice model in which both the rate of DNA polymerization and the rate of RNA degradation influence the frequency of RT template switching.
Murine Model of Interstitial Cytomegalovirus Pneumonia in Syngeneic Bone Marrow Transplantation: Persistence of Protective Pulmonary CD8-T-Cell Infiltrates after Clearance of Acute Infection
Authors: Podlech Jürgen , Holtappels Rafaela , Pahl-Seibert Marcus-Folker , Steffens Hans-Peter , Reddehase Matthias J.
Published: July 2002

Interstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62Lloand CD62Lhisubsets of CD44+memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral “stand-by” memory-effector T cells persist in the lungs to prevent virus recurrence from latency.
Intranasal Administration of 2/6-Rotavirus-Like Particles with MutantEscherichia coliHeat-Labile Toxin (LT-R192G) Induces Antibody-Secreting Cell Responses but Not Protective Immunity in Gnotobiotic Pigs
Authors: Yuan Lijuan , Geyer Annelise , Hodgins Douglas C. , Fan Zhiqian , Qian Yuan , Chang Kyeong-Ok , Crawford Sue E. , Parreño Viviana , Ward Lucy A. , Estes Mary K. , Conner Margaret E. , Saif Linda J.
Published: July 2002

We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutantEscherichia coliheat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.
Template/Primer Requirements and Single Nucleotide Incorporation by Hepatitis C Virus Nonstructural Protein 5B Polymerase
Authors: Zhong Weidong , Ferrari Eric , Lesburg Charles A. , Maag David , Ghosh Saikat Kumar B. , Cameron Craig E. , Lau Johnson Y. N. , Hong Zhi
Published: July 2002

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3′ terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3′ terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3′ terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique β-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.
Phosphatidylserine-Mediated Phagocytosis of Influenza A Virus-Infected Cells by Mouse Peritoneal Macrophages
Authors: Shiratsuchi Akiko , Kaido Masako , Takizawa Takenori , Nakanishi Yoshinobu
Published: July 2002

Influenza virus induces apoptosis in cultured cell lines as well as in animal tissues. HeLa cells were infected with influenza virus A/Udon/72 (H3N2) under conditions resulting in almost 100% infection. Such cells underwent typical caspase-dependent apoptosis and were efficiently phagocytosed by macrophages prepared from peritoneal fluids of thioglycolate-treated mice. The membrane phospholipid phosphatidylserine appeared on the surfaces of virus-infected cells at around the time efficient phagocytosis became detectable. In fact, the phagocytosis was almost completely inhibited in the presence of liposomes containing phosphatidylserine, which did not influence the antibody-dependent uptake of zymosan particles by the same macrophages. These results indicate that macrophages phagocytose influenza virus-infected HeLa cells in a manner mediated by phosphatidylserine that appears on the surfaces of infected cells during the process of apoptosis.
Recognition of
N
-Glycolylneuraminic Acid Linked to Galactose by the α2,3 Linkage Is Associated with Intestinal Replication of Influenza A Virus in Ducks
Authors: Ito Toshihiro , Suzuki Yasuo , Suzuki Takashi , Takada Ayato , Horimoto Taisuke , Wells Krisna , Kida Hiroshi , Otsuki Koichi , Kiso Makoto , Ishida Hideharu , Kawaoka Yoshihiro
Published: July 2002

The hemagglutinin (HA) of H3 human influenza viruses does not support viral replication in duck intestine despite its avian origin. A Leu-to-Gln mutation at position 226 and a Ser-to-Gly mutation at position 228 in the HA of human A/Udorn/307/72 (H3N2) permit a reassortant virus [human Udorn HA, with all other genes from A/mallard/New York/6750/78 (H2N2)] to replicate in ducks. To understand the molecular basis of this change in host range restriction, we investigated the receptor specificity of duck influenza viruses as well as of human-duck virus reassortants. The results indicate that the recognition of a glycoconjugate moiety possessing
N
-glycolneuramic acid (NeuGc) linked to galactose by the α2,3 linkage (NeuGcα2,3Gal) is associated with viral replication in duck intestine. Immunofluorescence assays with NeuGcα2,3Gal-specific antiserum detected this moiety primarily on the crypt epithelial cells of duck colon. Such recognition, together with biochemical evidence of NeuGc in crypt cells, correlated exactly with the ability of the virus to replicate in duck colon. These results suggest that recognition of the NeuGcα2,3-Gal moiety plays an important role in the enterotropism of avian influenza viruses.
Site-Specific Integration of an Adeno-Associated Virus Vector Plasmid Mediated by Regulated Expression of Rep Based on Cre-
loxP
Recombination
Authors: Satoh Wataru , Hirai Yukihiko , Tamayose Kenji , Shimada Takashi
Published: July 2002

Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the
rep
gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-
loxP
recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the
rep
coding sequence; in this configuration,
rep
expression is silent. However, Cre circularizes the
rep
expression unit, directly joining the p5 promoter to the 5′ end of the
rep
78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.
Analysis of Individual Human Trigeminal Ganglia for Latent Herpes Simplex Virus Type 1 and Varicella-Zoster Virus Nucleic Acids Using Real-Time PCR
Authors: Cohrs Randall J. , Randall Jessica , Smith John , Gilden Donald H. , Dabrowski Christine , van der Keyl Harjeet , Tal-Singer Ruth
Published: July 2002

Herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) establish latent infections in the peripheral nervous system following primary infection. During latency both virus genomes exhibit limited transcription, with the HSV-1 LATs and at least four VZV transcripts consistently detected in latently infected human ganglia. In this study we used real-time PCR quantitation to determine the viral DNA copy number in individual trigeminal ganglia (TG) from 17 subjects. The number of HSV-1 genomes was not significantly different between the left and right TG from the same individual and varied per subject from 42.9 to 677.9 copies per 100 ng of DNA. The number of VZV genomes was also not significantly different between left and right TG from the same individual and varied per subject from 37.0 to 3,560.5 copies per 100 ng of DNA. HSV-1 LAT transcripts were consistently detected in ganglia containing latent HSV-1 and varied in relative expression by >500-fold. Of the three VZV transcripts analyzed, only transcripts mapping to gene 63 were consistently detected in latently infected ganglia and varied in relative expression by >2,000-fold. Thus, it appears that, similar to LAT transcription in HSV-1 latently infected ganglia, VZV gene 63 transcription is a hallmark of VZV latency.
Association of Structural Changes in the V2 and V3 Loops of the gp120 Envelope Glycoprotein with Acquisition of Neutralization Resistance in a Simian-Human Immunodeficiency Virus Passaged In Vivo
Authors: Ye Yongjun , Si Zhi Hai , Moore John P. , Sodroski Joseph
Published: July 2002

The in vivo passage of a neutralization-sensitive, laboratory-adapted simian-human immunodeficiency virus (SHIV-HXBc2) generated a pathogenic, neutralization-resistant virus, SHIV-HXBc2P 3.2. SHIV-HXBc2P 3.2 differs from SHIV-HXBc2 only in 13 amino acid residues of the viral envelope glycoproteins. Here we used antibody competition analysis to examine the structural changes that occurred in the SHIV-HXBc2P 3.2 gp120 exterior envelope glycoprotein. The relationships among the antibody epitopes on the conserved gp120 core of SHIV-HXBc2 and SHIV-HXBc2P 3.2 were similar. The third variable (V3) loop was more closely associated with the fourth conserved (C4) region and CD4-induced epitopes on the gp120 core in the HXBc2P 3.2 gp120 glycoprotein compared with the HXBc2 gp120 glycoprotein. Rearrangements of the second variable (V2) loop with respect to the CD4 binding site and associated epitopes were evident in comparisons of the two gp120 glycoproteins. Thus, the in vivo evolution of a neutralization-resistant virus involves conformational adjustments of the V2 and V3 variable loops with respect to the conserved receptor-binding regions of the gp120 core.
Cloning the Antibody Response in Humans with Chronic Inflammatory Disease: Immunopanning of Subacute Sclerosing Panencephalitis (SSPE) Brain Sections with Antibody Phage Libraries Prepared from SSPE Brain Enriches for Antibody Recognizing Measles Virus Antigens In Situ
Authors: Owens Gregory P. , Williamson R. Anthony , Burgoon Mark P. , Ghausi Omar , Burton Dennis R. , Gilden Donald H.
Published: July 2002

In central nervous system (CNS) infectious and inflammatory diseases of known cause, oligoclonal bands represent antibody directed against the causative agent. To determine whether disease-relevant antibodies can be cloned from diseased brain, we prepared an antibody phage display library from the brain of a human with subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus, and selected the library against SSPE brain sections. Antibodies that were retrieved reacted strongly with measles virus cell extracts by enzyme-linked immunosorbent assay and were specific for the measles virus nucleocapsid protein. These antibodies immunostained cells in different SSPE brains but not in control brain. Our data provide the first demonstration that diseased brain can be used to select in situ for antibodies directed against the causative agent of disease and point to the potential usefulness of this approach in identifying relevant antibodies in chronic CNS or systemic inflammatory diseases of unknown cause.
Hepatitis C Virus Core Protein Interacts with 14-3-3 Protein and Activates the Kinase Raf-1
Authors: Aoki Hiroshi , Hayashi Junpei , Moriyama Mitsuhiko , Arakawa Yasuyuki , Hino Okio
Published: July 2002

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core–14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.
The IRF-3 Transcription Factor Mediates Sendai Virus-Induced Apoptosis
Authors: Heylbroeck Christophe , Balachandran Siddharth , Servant Marc J. , DeLuca Carmela , Barber Glen N. , Lin Rongtuan , Hiscott John
Published: July 2002

Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed interferon regulatory factor 3 (IRF-3) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-3 dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-3 transcriptionally up regulates alpha/beta interferon genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF-3(5D)] and dominant negative (IRF-3 ΔN) forms of IRF-3 that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-3, we observed that IRF-3 is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-3 is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-3(5D) alone is sufficient to induce apoptosis in human embryonic kidney 293 and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-3 expression augments paramyxovirus-induced apoptosis, while expression of IRF-3 ΔN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and 3 in IRF-3-induced apoptosis. These results suggest that IRF-3, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection.
Clinical Isolates of Measles Virus Use CD46 as a Cellular Receptor
Authors: Manchester Marianne , Eto Danelle S. , Valsamakis Alexandra , Liton Paloma B. , Fernandez-Muñoz Rafael , Rota Paul A. , Bellini William J. , Forthal Donald N. , Oldstone Michael B. A.
Published: July 2002

Laboratory strains of measles viruses (MV), such as Edmonston and Halle, use the complement regulatory protein CD46 as a cell surface receptor. The receptor usage of clinical isolates of MV, however, remains unclear. Receptor usage by primary patient isolates of MV was compared to isolates that had been passaged on a variety of tissue culture cell lines. All of the isolates could infect cells in a CD46-dependent manner, but their tropism was restricted according to cell type (e.g., lymphocytes versus fibroblasts). The results indicate that patient isolates that have not been adapted to tissue culture cell lines use CD46 as a receptor. In addition, passaging primary MV patient isolates in B95-8 cells selected variants that had alternate receptor usage compared to the original isolate. Thus, changes in receptor usage by MV are dependent upon the cell type used for isolation. Furthermore, our results confirm the relevance of the CD46 receptor to natural measles infection.
Isolation and Characterization of an Equine Foamy Virus
Authors: Tobaly-Tapiero Joelle , Bittoun Patricia , Neves Manuel , Guillemin Marie-Claude , Lecellier Charles-Henri , Puvion-Dutilleul Francine , Gicquel Bernard , Zientara Stephan , Giron Marie-Louise , de Thé Hugues , Saïb Ali
Published: July 2002

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.