bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Organization ofPasteurella multocida capLoci and Development of a Multiplex Capsular PCR Typing System
Authors: Townsend Kirsty M. , Boyce John D. , Chung Jing Y. , Frost Alan J. , Adler Ben
Published: July 2002

Current serotyping methods classifyPasteurella multocidainto five capsular serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serotypes 1 to 16). In the present study, we have developed a multiplex PCR assay as a rapid alternative to the conventional capsular serotyping system. The serogroup-specific primers used in this assay were designed following identification, sequence determination, and analysis of the capsular biosynthetic loci of each capsular serogroup. The entire capsular biosynthetic loci ofP. multocidaA:1 (X-73) and B:2 (M1404) have been cloned and sequenced previously (J. Y. Chung, Y. M. Zhang, and B. Adler, FEMS Microbiol. Lett. 166:289–296, 1998; J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121–134, 2000). Nucleotide sequence analysis of the biosynthetic region (region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the capsular polysaccharide composition. The multiplex capsular PCR assay was highly specific, and its results, with the exception of those for some serogroup F strains, correlated well with conventional serotyping results. Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The multiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification ofP. multocidacapsular types.
Biochemical Identification and Characterization of DNA Groups within the
Proteus vulgaris
Complex
Authors: Janda J. Michael , Abbott Sharon L. , Khashe Shideh , Probert Will
Published: July 2002

We placed 43 isolates belonging to the
Proteus vulgaris
complex into proposed DNA groups (genomovars) using five previously recommended tests (tests for esculin hydrolysis, production of acid from salicin,
l
-rhamnose fermentation, and elaboration of DNase and lipase). On the basis of the results of these five tests, 49% of the isolates fell into DNA groups 5 and 6, 37% fell into DNA group 2, and the remaining 14% fell into DNA groups 3 and 4. Sequencing of the 16S rRNA genes of 11 members of DNA groups 5 and 6 indicated that 10 of these isolates (91%) could be unambiguously assigned to one of these two genomospecies. Overall expression of selected enzymatic and virulence-associated characteristics did not differ significantly among DNA groups.
Comparison of Sequence Analysis and the INNO-LiPA HBV DR Line Probe Assay for Detection of Lamivudine-Resistant Hepatitis B Virus Strains in Patients under Various Clinical Conditions
Authors: Aberle Stephan W. , Kletzmayr Josef , Watschinger Bruno , Schmied Brigitte , Vetter Norbert , Puchhammer-Stöckl Elisabeth
Published: July 2002

A line probe assay (INNO-LiPA HBV DR) detecting drug-resistant hepatitis B virus (HBV) strains was evaluated. Results concordant with sequence analysis were obtained with 48 of 56 serum samples from HBV-infected patients undergoing lamivudine therapy. In eight cases, additional minor subpopulations could be identified by the line probe assay.
Helicobacter winghamensis
sp. nov., a Novel
Helicobacter
sp. Isolated from Patients with Gastroenteritis
Authors: Melito P. L. , Munro C. , Chipman P. R. , Woodward D. L. , Booth T. F. , Rodgers F. G.
Published: July 2002

From 1997 to 1999 seven isolates of
Campylobacter
-like organisms from five patients that were exhibiting symptoms of gastroenteritis, including fever, stomach malaise, and diarrhea, were investigated. The organisms were isolated from stool samples and found to exhibit a diverse colony morphology; hence multiple isolates were submitted from one of the patients. All isolates were found to be identical. The organisms were catalase, urease, alkaline phosphatase, and nitrate negative but oxidase and indoxyl acetate positive. They grew at 37°C but not at 42°C, and three of the isolates from two different patients were sensitive to nalidixic acid and cephalothin. Full 16S rRNA sequence analysis not only grouped these organisms within the
Helicobacter
genus but also differentiated them from previously identified
Helicobacter
species. The closest relative by phylogenetic analysis was
Helicobacter
sp. flexispira taxon 1. Electron microscopy showed that these isolates had one or two bipolar flagella; however, the periplasmic fibers, a characteristic of the known
Helicobacter
sp. flexispira taxa, were not observed. The present isolates also lacked a flagellar sheath, a trait shared with four other
Helicobacter
spp.,
H. canadensis
,
H. mesocricetorum
,
H. pullorum
, and
H. rodentium
. On the basis of the unique phenotypic properties of these isolates and 16S rRNA sequence analysis, we propose the classification of a new
Helicobacter
species,
Helicobacter winghamensis
sp. nov.
Fourth-Generation Enzyme-Linked Immunosorbent Assay for the Simultaneous Detection of Human Immunodeficiency Virus Antigen and Antibody
Authors: Saville Rebecca D. , Constantine Niel T. , Cleghorn Farley R. , Jack Noreen , Bartholomew Courtenay , Edwards Jeffrey , Gomez Perry , Blattner William A.
Published: July 2002

The VIDAS HIV DUO Ultra, a fourth-generation immunoassay under development for the simultaneous detection of human immunodeficiency virus type 1 (HIV-1) p24 antigen and antibodies to HIV-1 and HIV-2, was evaluated. The enzyme-linked fluorescence immunoassay, performed on the automated VIDAS instrument, is claimed to detect early and established HIV infection. The assay was challenged with a total of 2,847 samples that included 74 members of 10 seroconversion panels, 9 p24 antigen-only-reactive members of a panel of group M clades, 503 consecutively collected samples from individuals seeking care in the University of Maryland Medical System, 1,010 samples from U.S. blood donors, 1,141 samples from patients in a high-incidence population in Trinidad, 83 samples from a clinic for sexually transmitted diseases in the Bahamas, 10 confirmed HIV-1 group O samples, and 16 confirmed HIV-2 samples from the Cote d'Ivoire. Reference tests were U.S. Food and Drug Administration-licensed HIV antibody screening, p24 antigen tests, HIV confirmatory assays, and the Roche Diagnostics Amplicor HIV-1 Monitor. The VIDAS HIV DUO Ultra demonstrated 100% sensitivity and 99.5% specificity overall, with a 99.7% specificity in low-risk individuals. The analytical sensitivity, as assessed by seroconversion panels and p24 antigen in samples, was equivalent to the sensitivity of the reference assays used to characterize these panels. The VIDAS HIV DUO Ultra is accurate, offers potential advantages over conventional HIV testing for time and cost savings, has walk-away capability, and correctly identifies both early and established HIV infections.
Cryptococcus neoformans
var.
neoformans
(Serotype D) Strains Are More Susceptible to Heat than
C. neoformans
var.
grubii
(Serotype A) Strains
Authors: Martinez Luis. R , Garcia-Rivera Javier , Casadevall Arturo
Published: July 2002

Cryptococcus neoformans
var.
neoformans
(serotype D) and
C. neoformans
var.
grubii
(serotype A) differ in geographic prevalence and dermatotropism, with
C. neoformans
var.
neoformans
strains being more prevalent among isolates from temperate countries as well as from skin infections. Analysis of 19 strains from each serotype revealed wide variation in thermal susceptibility, with
C. neoformans
var.
neoformans
strains being more susceptible, on average, to heat killing. The results suggest a consistent explanation for the geographic differences between serotype A and D strains and for the dermatotropism of serotype D strains.
Molecular Characterization of Methicillin-Resistant
Staphylococcus epidermidis
Clones: Evidence of Geographic Dissemination
Authors: Miragaia Maria , Couto Isabel , Pereira Sandro F. F. , Kristinsson Karl G. , Westh Henrik , Jarløv Jens O. , Carriço João , Almeida Jonas , Santos-Sanches Ilda , de Lencastre Hermínia
Published: July 2002

Denmark and Iceland are countries where the frequency of methicillin-resistant
Staphylococcus aureus
is very low due to strict infection control and restrictive antibiotic use policies. In contrast, methicillin-resistant
S. epidermidis
(MRSE) continues to be isolated as a nosocomial pathogen. The molecular typing by pulsed-field gel electrophoresis (PFGE) of 136 MRSE isolates from five hospitals in Denmark and 94 MRSE isolates from one hospital in Iceland collected in 1997 and 1998 defined 40 different patterns. Closely related PFGE types were found in isolates recovered in Iceland, Denmark, Mexico, Uruguay, Greece, and Cape Verde, evidencing for the first time the geographic clonal dissemination of MRSE strains. The large majority (87.4%) of the MRSE isolates studied were multiresistant.
Detection ofEhrlichia canisin Canine Carrier Blood and in Individual Experimentally Infected Ticks with ap30-Based PCR Assay
Authors: Stich Roger W. , Rikihisa Yasuko , Ewing S. A. , Needham Glen R. , Grover Debra L. , Jittapalapong Sathaporn
Published: July 2002

Detection of vector-borne pathogens is necessary for investigation of their association with vertebrate and invertebrate hosts. The ability to detectEhrlichiaspp. within individual experimentally infected ticks would be valuable for studies to evaluate the relative competence of different vector species and transmission scenarios. The purpose of this study was to develop a sensitive PCR assay based on oligonucleotide sequences from the uniqueEhrlichia canisgene,p30, to facilitate studies that require monitoring this pathogen in canine and tick hosts during experimental transmission. Homologous sequences forEhrlichia chaffeensis p28were compared to sequences of primers derived from a sequence conserved amongE. canisisolates. Criteria for primer selection included annealing scores, identity of the primers to homologousE. chaffeensissequences, and the availability of similarly optimal primers that were nested within the target template sequence. Thep30-based assay was at least 100-fold more sensitive than a previously reported nested 16S ribosomal DNA (rDNA)-based assay and did not amplify the 200-bp target amplicon fromE. chaffeensis,the human granulocytic ehrlichiosis agent, orEhrlichia murisDNA. The assay was used to detectE. canisin canine carrier blood and in experimentally infectedRhipicephalus sanguineusticks. Optimized procedures for preparing tissues from these hosts for PCR assay are described. Our results indicated that thisp30-based PCR assay will be useful for experimental investigations, that it has potential as a routine test, and that this approach to PCR assay design may be applicable to other pathogens that occur at low levels in affected hosts.
Results of Human Papillomavirus DNA Testing with the Hybrid Capture 2 Assay Are Reproducible
Authors: Castle Philip E. , Lorincz Attila T. , Mielzynska-Lohnas Iwona , Scott David R. , Glass Andrew G. , Sherman Mark E. , Schussler John E. , Schiffman Mark
Published: July 2002

Reproducibility of the Hybrid Capture 2 Test (HC 2) for human papillomavirus (HPV) DNA detection was evaluated by assaying frozen cervical specimens in 1997 and again in 2001 from 1,775 women with normal cervical cytology. Using a cutoff point of 1.0 pg of HPV DNA/ml between a negative and a positive test result, the result of the kappa test for agreement was 0.72 (a kappa value of >0.60 is considered good agreement). Using cutoff points of 1.0 and 10.0 pg/ml between negative and low positive and between low positive and high positive, respectively, the kappa was 0.68 and the linear-weighted kappa was 0.76. The results of this study indicate that HC 2 testing is reproducible even among cytologically normal women with low test values.
Catheter-Related Candidemia Caused byCandida lipolyticain a Patient Receiving Allogeneic Bone Marrow Transplantation
Authors: D'Antonio Domenico , Romano Ferdinando , Pontieri Eugenio , Fioritoni Giuseppe , Caracciolo Claudia , Bianchini Stefano , Olioso Paola , Staniscia Tommaso , Sferra Roberta , Boccia Stefania , Vetuschi Antonella , Federico Giovanni , Gaudio Eugenio , Carruba Giuseppe
Published: July 2002

Candida lipolyticawas recovered from the blood and the central venous catheter in a patient receiving allogeneic bone marrow transplantation. TwoC.lipolyticastrains from different geographical areas and the ATCC 9773 strain ofC.lipolyticawere used as controls.C.lipolyticawas identified by standard methods. MICs indicated antifungal susceptibilities to amphotericin B, fluconazole, and itraconazole for all strains. In vitro testing and scanning electron microscopy showed thatC.lipolyticawas capable of producing large amounts of viscid slime material in glucose-containing solution, likely responsible for the ability of the yeast to adhere to catheter surfaces. Restriction fragment length polymorphisms revealed an identical profile for all clinical isolates, unrelated to those observed for the control strains. This finding suggested the absence of microevolutionary changes in the population of the infecting strain, despite the length of the sepsis and the potential selective pressure of amphotericin B, which had been administered to the patient for about 20 days. The genomic differences that emerged between the isolates and the control strains were indicative of a certain degree of genetic diversity betweenC.lipolyticaisolates from different geographical areas.
Evaluation of a Triplex PCR Assay To DiscriminateStaphylococcus aureusfrom Coagulase-Negative Staphylococci and Determine Methicillin Resistance from Blood Cultures
Authors: Maes N. , Magdalena J. , Rottiers S. , De Gheldre Y. , Struelens M. J.
Published: July 2002

A triplex PCR targeting the 16S rRNA,mecA, andnucgenes was developed for identification of staphylococci and detection of methicillin resistance. After validation of the assay with a collection of strains of staphylococci and enterococci (n= 169), the assay was evaluated with cultures of blood with gram-positive cocci from 40 patients. Accurate results were obtained for 59 (98%) of 61 cultures within 6 h of growth detection.
Detection of Replication-Competent Human Immunodeficiency Virus Type 1 (HIV-1) in Cultures from Patients with Levels of HIV-1 RNA in Plasma Suppressed to Less Than 500 or 50 Copies Per Milliliter
Authors: Demeter Lisa M. , Bosch Ronald J. , Coombs Robert W. , Fiscus Susan , Bremer James , Johnson Victoria A. , Erice Alejo , Jackson J. Brooks , Spector Stephen A. , Squires Kathleen M. , Fischl Margaret A. , Hughes Michael D. , Hammer Scott M.
Published: July 2002

We determined the frequency with which human immunodeficiency virus (HIV) peripheral blood mononuclear cell cultures convert from positive to negative in subjects enrolled in a substudy of AIDS Clinical Trials Group (ACTG) 320, which compared the efficacy of treatment with a combination of indinavir, zidovudine, and lamivudine (indinavir arm) to that of a combination of zidovudine and lamivudine (dual-nucleoside arm). All subjects included for study had positive baseline HIV cultures. Cultures were performed in real time with 10
7
fresh patient peripheral blood mononuclear cells, using the ACTG consensus method. We found lower rates of positive HIV cultures in the indinavir treatment arm than in the dual-nucleoside treatment arm (64 versus 96% at week 24,
P
< 0.001). Within the indinavir arm of the study, we found that positive cultures were less likely to occur in samples with a plasma HIV type 1 (HIV-1) RNA level of <500 copies/ml than in those with a level of ≥500 copies/ml (44 versus 90%,
P
< 0.001). In addition, HIV cultures from samples with HIV-1 RNA levels of ≥500 copies/ml turned positive 8.5 days earlier, on average, than those from samples with levels of <500 copies/ml (
P
< 0.001). However, 38% of samples with plasma RNA levels of <50 copies/ml still were positive for HIV by culture. Thus, the rates of HIV isolation by standard culture procedures decrease as the plasma viral load decreases below 1,000 copies/ml; however, HIV isolates were still obtained from a substantial proportion of subjects with RNA levels of <50 copies/ml. The delay in the time required for HIV cultures to turn positive should be considered when attempting to obtain an HIV isolate from patients with suppression of plasma viral load.
Development and Evaluation of a PCR Method for Detection of the
Clostridium difficile
Toxin B Gene in Stool Specimens
Authors: Guilbault C. , Labbé A.-C. , Poirier L. , Busque L. , Béliveau C. , Laverdière M.
Published: July 2002

A PCR assay detecting
Clostridium difficile
toxin B gene in stool specimens was compared to the cytotoxicity assay as the reference standard for the diagnosis of
C. difficile
antibiotic-associated diarrhea (CDAD). Overall, 118 stool samples were tested. All of the specimens that were negative by the cytotoxicity assay (59 out of 118) were also negative by the PCR method (specificity of 100%). Of the 59 cytotoxin-positive samples, 54 were PCR positive (sensitivity of 91.5%). This PCR method is promising for rapid diagnosis of CDAD.
Failure of Commercial Ligase Chain Reaction To Detect
Mycobacterium tuberculosis
DNA in Sputum Samples from a Patient with Smear-Positive Pulmonary Tuberculosis Due to a Deletion of the Target Region
Authors: Gilpin Christopher M. , Dawson David J. , O'Kane Gabrielle , Armstrong John G. , Coulter Christopher
Published: July 2002

We report on a strain of
Mycobacterium tuberculosis
with a deletion in the protein antigen B gene overlapping the probe binding sites for the Abbott Diagnostics LCx
M. tuberculosis
(LCx-MTB) probe assay. A false-negative result with the LCx-MTB assay delayed a laboratory diagnosis of tuberculosis.
Cytoplasmic Trafficking of the Canine Parvovirus Capsid and Its Role in Infection and Nuclear Transport
Authors: Vihinen-Ranta Maija , Yuan Wen , Parrish Colin R.
Published: July 2002

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.
V2 Loop Glycosylation of the Human Immunodeficiency Virus Type 1 SF162 Envelope Facilitates Interaction of This Protein with CD4 and CCR5 Receptors and Protects the Virus from Neutralization by Anti-V3 Loop and Anti-CD4 Binding Site Antibodies
Authors: Ly Amy , Stamatatos Leonidas
Published: July 2002

We examined the role of asparagine-linked glycosylation of the V2 loop of the human immunodeficiency virus (HIV) SF162 envelope on viral replication potential and neutralization susceptibility. We report that the asparagines located at the amino- and carboxy-terminal sites (at positions 154 and 195, respectively), as well as within the V2 loop of the SF162 envelope (at position 186), are glycosylated during in vitro replication of this virus in human peripheral blood mononuclear cells. Our studies indicate that glycosylation of the V2 loop, in particular at its base, facilitates the interaction of the HIV envelope with the CD4 and CCR5 receptor molecules present on the surface of target cells and affects viral replication kinetics in a cell type-dependent manner. In cells expressing high numbers of receptor molecules on their surfaces, the SF162-derived V2 loop-deglycosylated mutant viruses replicate as efficiently as the parental SF162 virus, while in cells expressing small numbers of receptor molecules, the mutant viruses replicate with markedly reduced efficiency. In addition to expanding the viral tropism, V2 loop glycosylation at the three sites examined prevents neutralization by anti-CD4 binding site antibodies. In contrast, glycosylation at the amino- and carboxy-terminal sites of the V2 loop but not within the loop itself offers protection against anti-V3 loop antibodies. Thus, the epitopes masked by the sugar molecules present on the three glycosylation sites examined are not identical but overlap.
Recombinant Yellow Fever Viruses Are Effective Therapeutic Vaccines for Treatment of Murine Experimental Solid Tumors and Pulmonary Metastases
Authors: McAllister Andrés , Arbetman Alejandra E. , Mandl Stefanie , Peña-Rossi Claudia , Andino Raul
Published: July 2002

We have genetically engineered an attenuated yellow fever (YF) virus to carry and express foreign antigenic sequences and evaluated the potential of this type of recombinant virus to serve as a safe and effective tumor vaccine. Live-attenuated YF vaccine is one of the most effective viral vaccines available today. Important advantages include its ability to induce long-lasting immunity, its safety, its affordability, and its documented efficacy. In this study, recombinant live-attenuated (strain 17D) YF viruses were constructed to express a cytotoxic T-lymphocyte epitope derived from chicken ovalbumin (SIINFEKL). These recombinant viruses replicated comparably to the 17D vaccine strain in cell culture and stably expressed the ovalbumin antigen, and infected cells presented the antigen in the context of major histocompatibility complex class I. Inoculation of mice with recombinant YF virus elicited SIINFEKL-specific CD8+lymphocytes and induced protective immunity against challenge with lethal doses of malignant melanoma cells expressing ovalbumin. Furthermore, active immunotherapy with recombinant YF viruses induced regression of established solid tumors and pulmonary metastases. Thus, recombinant YF viruses are attractive viral vaccine vector candidates for the development of therapeutic anticancer vaccines.
The Enhancer I Core Region Contributes to the Replication Level of Hepatitis B Virus In Vivo and In Vitro
Authors: Bock C.-Thomas , Malek Nisar P. , Tillmann Hans L. , Manns Michael P. , Trautwein Christian
Published: July 2002

Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Long-term interaction of the immune system with the virus results in the selection of escape mutants and viral persistence. In this work we characterize mutations in the enhancer I region isolated prior to liver transplantation from the HBV genomes of 10 patients with chronic HBV infection. The HBV-genomes were sequenced, and the enhancer I region was cloned into luciferase reporter constructs to determine the transcriptional activity. Functional studies were performed by transfecting HBV replication-competent plasmids into hepatoma cells. Analyses of the replication fitness of the mutant strains were conducted by biochemical analysis. In all HBV genomes the enhancer I region was mutated. Most of these mutations resulted in decreased transcriptional activity. The strongest effects were detectable in strains with mutations in the hepatocyte nuclear factor 3 and 4 (HNF3 and HNF4) binding sites of the enhancer I core domain. Replication-competent HBV constructs containing these mutations demonstrated up to 10-fold-reduced levels of virus replication. Before liver transplantation, when the mutant strains were detected in the patients' sera, low HBV DNA levels were found. After transplantation and reinfection with a wild-type virus, the levels of replication were up to 240-fold higher. Our results show that mutations in the enhancer I region of HBV have a major impact on HBV replication. These mutations may also determine the switch from high to low levels of viral replication which is frequently observed during chronic HBV infection.
Intrahepatic Induction of Alpha/Beta Interferon Eliminates Viral RNA-Containing Capsids in Hepatitis B Virus Transgenic Mice
Authors: Wieland Stefan F. , Guidotti Luca G. , Chisari Francis V.
Published: July 2002

We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by stimuli that induce alpha/beta interferon (IFN-α/β) in the liver. The present study was done to identify the step(s) in HBV replication that is affected by this cytokine in transgenic mice treated with the IFN-α/β inducer polyinosinic-polycytidylic acid [poly(I-C)]. Here we show that the pool of cytoplasmic HBV pregenomic RNA (pgRNA)-containing capsids is reduced 10-fold within 9 h after poly(I-C) administration, while there is no change in the abundance of HBV mRNA or in the translational status of cytoplasmic HBV transcripts. In addition, we show that the pool of HBV DNA-containing capsids is not reduced to the same degree until at least 15 h posttreatment, and we show that virus export is not accelerated and the half-life of virions in the serum is unchanged. These results indicate that IFN-α/β triggers intracellular events that either inhibit the assembly of pgRNA-containing capsids or accelerate their degradation, and that maturation and secretion of virus is responsible for clearance of HBV capsids and their cargo of replicative intermediates from the cytoplasm of the hepatocyte.
Induction and Inhibition of Apoptosis by Pseudorabies Virus in the Trigeminal Ganglion during Acute Infection of Swine
Authors: Alemañ Nuria , Quiroga Marı́a Isabel , López-Peña Mónica , Vázquez Sonia , Guerrero Florentina H. , Nieto José M.
Published: July 2002

We examined the ability of pseudorabies virus (PRV) to induce and suppress apoptosis in the trigeminal ganglion during acute infection of its natural host. Eight pigs were intranasally inoculated with a virulent field strain of PRV, and at various early times after inoculation, the trigeminal ganglia were assessed histologically. PRV-infected cells were detected by use of immunohistochemistry and in situ hybridization, and apoptosis was identified by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Light and electron microscopy was also used for morphological studies. Apoptosis was readily detected among infiltrating immune cells that were located surrounding PRV-infected neurons. The majority of PRV-infected neurons did not show morphological or histochemical evidence of apoptosis, even including those neurons that were surrounded by numerous inflammatory cells and exhibited profound pathological changes. However, neuronal virus-induced apoptosis also occurred but at a sporadic low level. These findings suggest that PRV is able to block apoptosis of infected trigeminal ganglionic neurons during acute infection of swine. Furthermore, our results also suggest that apoptosis of infiltrating inflammatory cells may represent an important viral mechanism of immune evasion.
Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly of Virus-Like Particles Composed of Simian Virus 40 VP1 Capsid Protein
Authors: Ishizu Ken-Ichiro , Watanabe Hajime , Han Song-Iee , Kanesashi Shin-Nosuke , Hoque Mainul , Yajima Hiroaki , Kataoka Kohsuke , Handa Hiroshi
Published: July 2002

The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.
Sustained Dysfunction of Antiviral CD8+T Lymphocytes after Infection with Hepatitis C Virus
Authors: Gruener Norbert H. , Lechner Franziska , Jung Maria-Christina , Diepolder Helmut , Gerlach Tilman , Lauer Georg , Walker Bruce , Sullivan John , Phillips Rodney , Pape Gerd R. , Klenerman Paul
Published: July 2002

Hepatitis C virus (HCV) sets up persistent infection in the majority of those exposed. It is likely that, as with other persistent viral infections, the efficacy of T-lymphocyte responses influences long-term outcome. However, little is known about the functional capacity of HCV-specific T-lymphocyte responses induced after acute infection. We investigated this by using major histocompatibility complex class I-peptide tetrameric complexes (tetramers), which allow direct detection of specific CD8+T lymphocytes ex vivo, independently of function. Here we show that, early after infection, virus-specific CD8+T lymphocytes detected with a panel of four such tetramers are abnormal in terms of their synthesis of antiviral cytokines and lytic activity. Furthermore, this phenotype is commonly maintained long term, since large sustained populations of HCV-specific CD8+T lymphocytes were identified, which consistently had very poor antiviral cytokine responses as measured in vitro. Overall, HCV-specific CD8+T lymphocytes show reduced synthesis of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) after stimulation with either mitogens or peptides, compared to responses to Epstein-Barr virus and/or cytomegalovirus. This behavior of antiviral CD8+T lymphocytes induced after HCV infection may contribute to viral persistence through failure to effectively suppress viral replication.
High Numbers of Viral RNA Copies in the Central Nervous System of Mice during Persistent Infection with Theiler's Virus
Authors: Trottier Mark , Kallio Pat , Wang Wei , Lipton Howard L.
Published: July 2002

The low-neurovirulence Theiler's murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 × 109) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.
Requirements for the Induction of Interleukin-6 by Herpes Simplex Virus-Infected Leukocytes
Authors: Paludan Søren R.
Published: July 2002

Cytokines play important roles in the clearance of herpes simplex virus (HSV) infections and in virus-induced immunopathology. One cytokine known to contribute to resistance against HSV is interleukin-6 (IL-6). Here we have investigated virus-cell interactions responsible for IL-6 induction by HSV in leukocytes. Both HSV type 1 and type 2 are potent inducers of IL-6, and this phenomenon is augmented in the presence of gamma interferon. The ability to induce IL-6 is dependent on de novo protein synthesis and is sensitive to UV irradiation of the virus. Virus mutants lacking the virion-transactivating protein VP16 or any of the immediate-early proteins ICP0, ICP4, or ICP27 displayed unaltered capacities to induce IL-6. However, wild-type virus was unable to induce IL-6 in a macrophage cell line overexpressing a mutant of double-stranded RNA-activated protein kinase (PKR). This suggests a role for PKR in HSV-induced IL-6 expression. HSV infection led to enhanced binding to the κB, CRE, and AP-1 sites of the IL-6 promoter, and inhibitors against NF-κB and the p38 kinase strongly reduced accumulation of IL-6 mRNA in infected cells. Moreover, macrophage cell lines expressing dominant negative mutants of IκBα and p38 responded to HSV-1 infection with reduced IL-6 expression compared to the control-vector-transfected cell line. The results show that induction of IL-6 by HSV in leukocytes is dependent on PKR and cellular signaling through NF-κB and a p38-dependent pathway.
Morbillivirus Infection of the Mouse Central Nervous System Induces Region-Specific Upregulation of MMPs and TIMPs Correlated to Inflammatory Cytokine Expression
Authors: Khuth Seng-Thuon , Akaoka Hideo , Pagenstecher Axel , Verlaeten Olivier , Belin Marie-Françoise , Giraudon Pascale , Bernard Arlette
Published: July 2002

Viral infection of the central nervous system (CNS) can result in perturbation of cell-to-cell communication involving the extracellular matrix (ECM). ECM integrity is maintained by a dynamic balance between the synthesis and proteolysis of its components, mainly as a result of the action of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). An MMP/TIMP imbalance may be critical in triggering neurological disorders, in particular in virally induced neural disorders. In the present study, a mouse model of brain infection using a neurotropic strain of canine distemper virus (CDV) was used to study the effect of CNS infection on the MMP/TIMP balance and cytokine expression. CDV replicates almost exclusively in neurons and has a unique pattern of expression (cortex, hypothalamus, monoaminergic nuclei, hippocampus, and spinal cord). Here we show that although several mouse brain structures were infected, they exhibited a differential pattern in terms of MMP, TIMP, and cytokine expression, exemplified by (i) a large increase in pro-MMP9 levels, in particular in the hippocampus, which occurred mainly in neurons and was associated with in situ gelatinolytic activity, (ii) specific and significant upregulation of MT1-MMP mRNA expression in the cortex and hypothalamus, (iii) an MMP/TIMP imbalance, suggested by the upregulation of TIMP-1 mRNA in the cortex, hippocampus, and hypothalamus and of TIMP-3 mRNA in the cortex, and (iv) a concomitant region-specific large increase in expression of Th1-like cytokines, such as gamma interferon, tumor necrosis factor alpha, and interleukin 6 (IL-6), contrasting with weaker induction of Th2-like cytokines, such as IL-4 and IL-10. These data indicate that an MMP/TIMP imbalance in specific brain structures, which is tightly associated with a local inflammatory process as shown by the presence of immune infiltrating cells, differentially impairs CNS integrity and may contribute to the multiplicity of late neurological disorders observed in this viral mouse model.
A Cellular J-Domain Protein Modulates Polyprotein Processing and Cytopathogenicity of a Pestivirus
Authors: Rinck G. , Birghan C. , Harada T. , Meyers G. , Thiel H.-J. , Tautz N.
Published: July 2002

Pestiviruses are positive-strand RNA viruses closely related to human hepatitis C virus. Gene expression of these viruses occurs via translation of a polyprotein, which is further processed by cellular and viral proteases. Here we report the formation of a stable complex between an as-yet-undescribed cellular J-domain protein, a member of the DnaJ-chaperone family, and pestiviral nonstructural protein NS2. Accordingly, we termed the cellular protein Jiv, for J-domain protein interacting with viral protein. Jiv has the potential to induce intransone specific processing step in the viral polyprotein, namely, cleavage of NS2-3. Efficient generation of its cleavage product NS3 has previously been shown to be obligatory for the cytopathogenicity of the pestiviruses. Regulated expression of Jiv in cells infected with noncytopathogenic bovine viral diarrhea virus disclosed a direct correlation between the intracellular level of Jiv, the extent of NS2-3 cleavage, and pestiviral cytopathogenicity.
Differential Infection of Polarized Epithelial Cell Lines by Sialic Acid-Dependent and Sialic Acid-Independent Rotavirus Strains
Authors: Ciarlet Max , Crawford Sue E. , Estes Mary K.
Published: July 2002

Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence ofN-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-μm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [3H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium, but SA-dependent and SA-independent rotavirus strains infect polarized epithelial cells by different mechanisms, which may be relevant for pathogenesis and selection of vaccine strains. Finally, rotavirus-induced alterations of the epithelial barrier and paracellular permeability suggest that common mechanisms of pathogenesis may exist between viral and bacterial pathogens of the intestinal tract.
Mucosal Transmission and Induction of Simian AIDS by CCR5-Specific Simian/Human Immunodeficiency Virus SHIV
SF162P3
Authors: Harouse Janet M. , Gettie Agegnehu , Eshetu Tadesse , Tan Rei Chin How , Bohm Rudolf , Blanchard James , Baskin Gary , Cheng-Mayer Cecilia
Published: July 2002

Nonhuman primate models are increasingly used in the screening of candidate AIDS vaccine and immunization strategies for advancement to large-scale human trials. The predictive value of such macaque studies is largely dependent upon the fidelity of the model system in mimicking human immunodeficiency virus (HIV) type 1 infection in terms of viral transmission, replication, and pathogenesis. Herein, we describe the efficient mucosal transmission of a CCR5-specific chimeric simian/human immunodeficiency virus, SHIV
SF162P3
. Female rhesus macaques were infected with SHIV
SF162P3
after a single atraumatic application to the cervicovaginal mucosa. The disease course of SHIV
SF162P3
-infected monkeys is similar and as varied as natural HIV infection in terms of viral replication, gradual loss of CD4
+
peripheral blood mononuclear cells, and the development of simian AIDS-defining opportunistic infections. The SHIV
SF162P3
/macaque model should facilitate direct preclinical assessment of HIV vaccine strategies in addition to antiviral compounds directed towards envelope target cell interactions. Furthermore, this controlled model provides the setting to investigate immunologic responses and putative host-specific susceptibility factors that alter viral transmission and subsequent disease progression.
Exposure of Hepatitis C Virus (HCV) RNA-Positive Recipients to HCV RNA-Positive Blood Donors Results in Rapid Predominance of a Single Donor Strain and Exclusion and/or Suppression of the Recipient Strain
Authors: Laskus Tomasz , Wang Lian-Fu , Radkowski Marek , Vargas Hugo , Nowicki Marek , Wilkinson Jeffrey , Rakela Jorge
Published: July 2002

We have analyzed three cases of hepatitis C virus (HCV)-infected recipients who received blood from HCV-infected donors. Two recipients were exposed to two different HCV RNA-positive donors, and one was exposed to a single donor. All parental genomes from the actual infecting units of blood and the recipients were defined, and their presence in the follow-up serum samples was determined using sensitive strain-specific assays. The strain from one of the donors was found to predominate in all recipients' serum samples collected throughout the follow-up period of 10 to 30 months. In two recipients exposed to two infected donors, the strain from the second donor was occasionally found at very low level. However, the original recipients' strains were not detected. Our observations show that HCV-infected individuals can be superinfected with different strains, and this event may lead to eradication or suppression of the original infecting strain. Furthermore, our findings demonstrate that simultaneous exposure to multiple HCV strains may result in concomitant infection by more than one strain, although a single strain could rapidly establish its dominance. The results of the present study suggest the existence of competition among infecting HCV strains which determines the ultimate outcome of multiple HCV exposure.
Egress of Alphaherpesviruses: Comparative Ultrastructural Study
Authors: Granzow Harald , Klupp Barbara G. , Fuchs Walter , Veits Jutta , Osterrieder Nikolaus , Mettenleiter Thomas C.
Published: July 2002

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of thetrans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.
Role of the Cytoplasmic Domain of the β-Subunit of Integrin αvβ6 in Infection by Foot-and-Mouth Disease Virus
Authors: Miller Laura C. , Blakemore Wendy , Sheppard Dean , Atakilit Amha , King Andrew M. Q. , Jackson Terry
Published: July 2002

Field isolates of foot-and-mouth disease virus (FMDV) are believed to use RGD-dependent integrins as cellular receptors in vivo. Using SW480 cell transfectants, we have recently established that one such integrin, αvβ6, functions as a receptor for FMDV. This integrin was shown to function as a receptor for virus attachment. However, it was not known if the αvβ6 receptor itself participated in the events that follow virus binding to the host cell. In the present study, we investigated the effects of various deletion mutations in the β6 cytoplasmic domain on infection. Our results show that although loss of the β6 cytoplasmic domain has little effect on virus binding, this domain is essential for infection, indicating a critical role in postattachment events. The importance of endosomal acidification in αvβ6-mediated infection was confirmed by experiments showing that infection could be blocked by concanamycin A, a specific inhibitor of the vacuolar ATPase.