ABSTRACT
The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry the blaCMY-2 gene. In the present study, the distribution of these four known blaCMY-2-carrying plasmids among 35 ceftriaxone-resistant Salmonella isolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates were Salmonella enterica serotype Newport, 10 were Salmonella enterica serotype Typhimurium, 1 was Salmonella enterica serotype Agona, and 1 was Salmonella enterica serotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surrounding blaCMY-2 was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surrounding blaCMY-5 from the Klebsiella oxytoca plasmid pTKH11. These findings are consistent with a model in which blaCMY-2 has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multiple Salmonella chromosomal backbones.
Subject(s)
DNA, Bacterial/analysis , Plasmids/genetics , Salmonella/genetics , beta-Lactamases/genetics , Bacterial Typing Techniques , Base Sequence , Blotting, Southern , DNA Primers , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Gene Transfer, Horizontal , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , Replicon/geneticsABSTRACT
Salmonellae are ubiquitous throughout nature where they infect or colonize humans, animals and even insects worldwide. The genus is comprised of a number of different antigenically distinct members, many of which have a particular niche within nature. Infection in animals and humans can be endemic or epidemic. Many nations have established extensive surveillance systems to track Salmonella infections and disrupt epidemic spread. Most of these surveillance projects rely on traditional serotype and phage type analyses to identify trends and potential outbreaks. Many clinical outbreaks cluster among a few serotypes so further discrimination is often needed. Molecular epidemiological techniques have been used to enhance surveillance and discriminate outbreak strains within these common serotypes. The institution of these techniques has led to enhanced detection of outbreaks worldwide. Molecular techniques used for Salmonella surveillance are described and comparisons of different molecular techniques are outlined. Overall, traditional serotype surveillance in association with one or several molecular typing techniques, especially chromosomal restriction fragment analysis with pulsed-field gel electrophoresis, appears to provide the most reproducible and comparable discrimination of epidemiologically-linked isolates at this time.
Subject(s)
Molecular Epidemiology/methods , Salmonella Infections/epidemiology , Salmonella Infections/genetics , Salmonella/classification , Salmonella/isolation & purification , Salmonella/genetics , Species SpecificityABSTRACT
Escherichia coli is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described Salmonella isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like beta-lactamase, CMY-2. In the present study, 59 of 377 E. coli isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human E. coli from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An ampC gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human E. coli isolates harboring the CMY-2 gene. The ampC genes from 10 animal and human E. coli isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of E. coli. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described Salmonella CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in Salmonella and E. coli isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated Salmonella and E. coli, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans.
Subject(s)
Escherichia coli/genetics , Gene Transfer, Horizontal/genetics , Salmonella/genetics , beta-Lactamases/genetics , Animals , Anti-Bacterial Agents/pharmacology , Cattle , Escherichia coli/drug effects , Humans , Microbial Sensitivity Tests , Molecular Epidemiology , Phenotype , Plasmids/genetics , Salmonella/drug effects , SwineABSTRACT
Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Cystic Fibrosis/microbiology , Drug Resistance, Multiple/physiology , Alcaligenes/drug effects , Amino Acid Sequence , Blood Proteins/pharmacology , Burkholderia cepacia/drug effects , Cathelicidins , Drug Synergism , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Peptides/pharmacology , Pseudomonas aeruginosa/drug effects , Sequence Homology, Amino Acid , Sputum/chemistry , Stenotrophomonas maltophilia/drug effects , Time FactorsABSTRACT
To evaluate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains among species of Enterobacteriaceae, a microdilution susceptibility test was performed with strains of Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella species that were isolated as part of the SENTRY project. The highest percentage of ESBL phenotype (defined as a minimum inhibitory concentration [MIC] > or =2 microg/mL for ceftazidime, ceftriaxone, or aztreonam) was detected among K. pneumoniae strains from Latin America (45%), followed by those from the Western Pacific region (25%), Europe (23%), the United States (8%), and Canada (5%). P. mirabilis and E. coli strains for which MICs of extended-spectrum cephalosporins or monobactams were elevated also were more prominent in Latin America. Testing with ceftazidime revealed more isolates with elevated MICs than did testing with ceftriaxone or aztreonam. ESBL strains showed high levels of co-resistance to aminoglycosides, tetracycline, trimethoprim-sulfamethoxazole, and ciprofloxacin. Imipenem remains highly effective against ESBL strains. Organisms expressing an ESBL are widely distributed worldwide, although prevalence rates are significantly higher in certain geographic regions.
Subject(s)
Cephalosporins/pharmacology , beta-Lactam Resistance/genetics , beta-Lactamases/genetics , Aztreonam/pharmacology , Ceftazidime/pharmacology , Ceftriaxone/pharmacology , Drug Resistance, Multiple , Electrophoresis, Gel, Pulsed-Field , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Escherichia coli/drug effects , Europe , Asia, Eastern , Klebsiella pneumoniae/drug effects , Latin America , Microbial Sensitivity Tests , North America , Phenotype , Proteus mirabilis/drug effects , Ribotyping , Salmonella/drug effects , beta-Lactamases/isolation & purificationABSTRACT
Salmonella spp. are important food-borne pathogens that are demonstrating increasing antimicrobial resistance rates in isolates obtained from food animals and humans. In this study, 10 multidrug-resistant, cephalosporin-resistant Salmonella isolates from bovine, porcine, and human sources from a single geographic region were identified. All isolates demonstrated resistance to cephamycins and extended-spectrum cephalosporins as well as tetracycline, chloramphenicol, streptomycin, and sulfisoxazole. Molecular epidemiological analyses revealed eight distinct chromosomal DNA patterns, suggesting that clonal spread could not entirely explain the distribution of this antimicrobial resistance phenotype. However, all isolates encoded an AmpC-like beta-lactamase, CMY-2. Eight isolates contained a large nonconjugative plasmid that could transform Escherichia coli. Transformants coexpressed cephalosporin, tetracycline, chloramphenicol, streptomycin, and sulfisoxazole resistances. Plasmid DNA revealed highly related restriction fragments though plasmids appeared to have undergone some evolution over time. Multidrug-resistant, cephalosporin-resistant Salmonella spp. present significant therapeutic problems in animal and human health care and raise further questions about the association between antimicrobial resistance, antibiotic use in animals, and transfer of multidrug-resistant Salmonella spp. between animals and man.
Subject(s)
Cephalosporin Resistance , Salmonella Infections, Animal/microbiology , Salmonella Infections/microbiology , Salmonella/drug effects , Salmonella/genetics , beta-Lactamases/biosynthesis , Animals , Animals, Domestic , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Drug Resistance, Multiple , Electrophoresis, Gel, Pulsed-Field , Humans , Isoelectric Focusing , Microbial Sensitivity Tests , Molecular Epidemiology , Plasmids/genetics , Polymorphism, Restriction Fragment Length , Reverse Transcriptase Polymerase Chain Reaction , beta-Lactamases/geneticsABSTRACT
The beta-lactamases from 403 Moraxella (Branhamella) catarrhalis clinical isolates obtained during 1994-1995 and 1997-1998 U.S. multicenter surveillance studies were characterized by isoelectric focusing. The overall prevalences of the BRO-1 and BRO-2 enzymes among beta-lactamase-positive isolates were estimated to be 97.5 and 2.5%, respectively. The minimum inhibitory concentrations (MICs) of ampicillin for all BRO-2-producing isolates were =1 microg/ml; however, numerous beta-lactamase-positive isolates for which the ampicillin MICs were =1 microg/ml produced the BRO-1 enzyme (88. 1%).
Subject(s)
Moraxella catarrhalis/enzymology , beta-Lactamases/metabolism , Humans , Isoelectric Focusing , Microbial Sensitivity Tests , Moraxella catarrhalis/genetics , Moraxella catarrhalis/isolation & purification , Multicenter Studies as Topic , beta-Lactamases/geneticsSubject(s)
Klebsiella Infections/microbiology , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/isolation & purification , beta-Lactamases/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Microbial , Drug Resistance, Multiple , Electrophoresis, Gel, Pulsed-Field , Humans , Isoelectric Focusing , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests , Russia/epidemiology , Substrate Specificity , beta-Lactamases/chemistry , beta-Lactamases/genetics , beta-LactamsABSTRACT
Urinary tract infection (UTI) is common and involves pathogens with changing susceptibility patterns. The SENTRY Antimicrobial Surveillance Program evaluates international pathogen incidence patterns to detect and manage the emergence of resistant strains. We describe the antimicrobial resistance patterns among 1617 pathogens recovered from UTIs during the third-quarter of 1997 in North America (United States and Canada), as part of this worldwide program. The isolates were tested against more than 50 antimicrobial agents (20 reported) by reference broth microdilution methods, and selected isolates were characterized by pulsed-field gel electrophoresis (PFGE) and automated ribotyping. The five most frequently isolated species were Escherichia coli (48.6%), Enterococcus spp. (13.7%), Klebsiella spp. (12.0%), Pseudomonas aeruginosa (6.2%), and Enterobacter spp. or Proteus mirabilis (3.8% each). For each nation, imipenem and cefepime produced the widest spectrum of coverage among the beta-lactams and amikacin was best among the aminoglycosides. For Gram-negative species, high resistance among beta-lactam antimicrobial agents was noted especially for various penicillins against E. coli (37.9% to 42.8%) and for the cephalosporins tested against enterococci (99.4% and 100%). Approximately 7.0% of enterococci in the USA were vancomycin-resistant (88% with Van A). P. aeruginosa provided the most consistent levels of resistance, but the following agents were most active against these organisms: amikacin (96.6 to 97.4% susceptible), tobramycin (89.5 to 100.0%), piperacillin/tazobactam (89.5 to 100.0%), piperacillin (89.5 to 96.6%), imipenem (89.7 to 92.1%), cefepime (77.6 to 89.7%), and ceftazidime (82.9 to 86.2%). E. coli (2.2 to 2.7%), K. pneumoniae (6.2 to 6.4%), and a single Enterobacter cloacae strain produced extended-spectrum beta-lactamases; and five other Enterobacter spp. were likely to have expressed chromosomally mediated (Amp C) Stably derepressed cephalosporinases with associated resistance to ceftazidime (16.7 to 21.2% resistance). These data demonstrated that several UTI isolates in SENTRY hospitals have high levels of resistance to various classes of antimicrobial agents with little evidence of clonal dissemination.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Infections/microbiology , Gram-Negative Bacteria/drug effects , Urinary Tract Infections/microbiology , Drug Resistance, Microbial , Hospitals , Humans , Microbial Sensitivity Tests , North AmericaABSTRACT
During 1997, a total of 4,267 nosocomial and community-acquired bloodstream infections due to gram-negative organisms were reported from SENTRY hospitals in Canada (8 sites), the United States (30 sites), and Latin America (10 sites). Escherichia coli was the most common isolate (41% of all gram-negative isolates), followed by Klebsiella species (17.9%), Pseudomonas aeruginosa (10.6%), and Enterobacter species (9.4%). For all gram-negative isolates combined, the most active antimicrobials tested were meropenem, imipenem, and cefepime. The quinolones levofloxacin (MIC90, 2 microg/mL), ciprofloxacin (MIC90, 1 microg/mL), gatifloxacin (MIC90, 2 microg/mL), sparfloxacin (MIC90, 2 microg/mL), and trovafloxacin (MIC90, 2 microg/mL) were also active against most isolates. Bloodstream infection isolates from Latin America were uniformly more resistant to all classes of antimicrobial agents tested than were isolates from Canada or the United States. Resistance phenotypes consistent with extended-spectrum beta-lactamase production were also most common among E. coli and Klebsiella species from Latin America. Further investigation of the reasons for regional differences in resistance patterns is needed, as is ongoing surveillance to detect resistance trends and to guide antimicrobial use.
Subject(s)
Bacteremia/epidemiology , Bacteremia/microbiology , Cross Infection/epidemiology , Drug Resistance, Microbial , Gram-Negative Bacteria/isolation & purification , Gram-Negative Bacterial Infections/epidemiology , Canada/epidemiology , Cross Infection/microbiology , Female , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/microbiology , Humans , Incidence , Latin America/epidemiology , Male , Microbial Sensitivity Tests , Population Surveillance , United States/epidemiologyABSTRACT
Twenty-three multi-resistant strains of Escherichia coli were isolated at a single hospital in Beijing, China between January 1997 and May 1998. All isolates produced extended spectrum beta-lactamases (ESBLs) as detected by the double disk synergy test and the Etest ESBL strip (AB BIODISK, Solna Sweden). Additional antimicrobial susceptibility testing showed that most isolates were resistant to gentamicin, tobramycin, tetracycline, trimethoprim/sulfamethoxazole, ciprofloxacin, and cefepime. All isolates remained susceptible to imipenem with MICs of < or = 0.5 microgram/ml. The isolates each produced several beta-lactamases (range 1-4 enzymes/strain) with pI values ranging from 5.2-8.4. Molecular epidemiologic typing revealed four ribotypes and eight pulsed field gel electrophoresis (PFGE) patterns with subgroups among the 23 isolates. Clusters of isolates with the same DNA type were observed as follows (ribotype/PFGE): Wards A (242-5/2, and 242-5/3a), B (242-5/4), and C (880-1/1a). Moreover, similar molecular types were observed in patients from two or more different wards. Further use of isoelectric focusing results and co-resistance patterns produced evidence of potential nosocomial dissemination of strains in only two instances (two identical strains on one ward and two identical strains on different wards). There were also strong similarities in beta-lactamase pIs and co-resistances among many of the strains throughout this medical center. These data document the wide genetic diversity among E. coli producing ESBLs, and a potential for nosocomial spread of these highly resistant organisms requiring increasingly more sophisticated molecular-based techniques and local interventions.
Subject(s)
Escherichia coli Infections/microbiology , Escherichia coli/classification , Escherichia coli/enzymology , beta-Lactamases/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Typing Techniques , China , Deoxyribonuclease EcoRI/metabolism , Electrophoresis, Gel, Pulsed-Field , Escherichia coli/drug effects , Escherichia coli/isolation & purification , Genotype , Humans , Isoelectric Focusing , Microbial Sensitivity Tests , Phenotype , Restriction Mapping , beta-Lactam Resistance , beta-LactamsABSTRACT
Modified MicroScan gram-positive MIC no. 8 panels (PM-8) were analyzed for their improved ability to detect vancomycin resistance (VR) and high-level aminoglycoside resistance (HLAR) in enterococci. A validation study design that utilized selected challenge strains, recent clinical isolates, and reproducibility experiments in a multicenter format was selected. Three independent medical centers compared the commercial panels to reference broth microdilution panels (RBM) and Synergy Quad Agar (QA). Resistance was verified by demonstration of VR and HLAR genes by PCR tests. The study was conducted in three phases. (i) In the challenge phase (CP), two well-characterized sets of enterococci were obtained from the Centers for Disease Control and Prevention; one set contained 50 isolates for VR testing and one contained 48 isolates for HLAR testing. In addition, a set of 47 well-characterized isolates representing diverse geographic areas, obtained from earlier national surveillance studies, was tested at the University of Iowa College of Medicine (UICM). (ii) In the efficacy phase (EP), each laboratory tested 50 recent, unique clinical isolates by all methods. (iii) In the reproducibility Phase (RP), each laboratory tested the same 10 strains by all methods in triplicate on three separate days. All isolates from the EP were sent to the UICM for molecular characterization of vanA, -B, -C1, -C2-3, and HLAR genes. In the CP, the ranking of test methods by error rates (in parentheses; very major and major errors combined, versus PCR results) were as follows: for high-level streptomycin resistance (HLSR), QA (12.0%) > PM-8 (5.2%) > RBM (1.6%); for high-level gentamicin resistance (HLGR), RBM (3.7%) > PM-8 (3.1%) > QA (2.6%); and for VR, RBM = QA (3.0%) > PM-8 (1.2%). In the EP, agreement between all methods and the reference PCR result was 98.0% for HLSR, 99.3% for HLGR, and 98. 6% for VR. In the RP, the percentages of results +/- 1 log2 dilution of the all-participant mode were as follows: for VR, 100% (PM-8), 98.9% (QA), and 90.0% (RBM); for HLSR, 99.6% (RBM), 98.5% (PM-8), and 82.2% (QA); and for HLGR, 99.6% (RBM), 99.3% (PM-8), and 98.1% (QA). The ability of the PM-8 to detect VR and HLAR in enterococci was comparable to those for reference susceptibility and molecular PCR methods and was considered acceptable for routine clinical laboratory use.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus/drug effects , Gram-Positive Bacteria/drug effects , Microbial Sensitivity Tests/methods , Microbial Sensitivity Tests/standards , Vancomycin/pharmacology , Drug Resistance, Microbial , Enterococcus/genetics , Enterococcus/isolation & purification , Gentamicins/pharmacology , Gram-Positive Bacteria/genetics , Humans , Laboratories/standards , Quality Control , Reference Values , Reproducibility of Results , Streptomycin/pharmacology , United StatesABSTRACT
Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted.
Subject(s)
Antigens, Viral/biosynthesis , Hepatitis A Virus, Human/immunology , Hepatitis A/diagnosis , Radioimmunoassay , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/immunology , Cell Line , Enzyme-Linked Immunosorbent Assay , Hepatitis A/immunology , Hepatitis A Antigens , Hepatitis A Virus, Human/growth & development , Humans , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunology , Sensitivity and Specificity , Vaccinia virus/geneticsABSTRACT
The papillomavirus E2 protein is required for viral transcriptional regulation and replication. The E2 protein has a modular structure with two highly conserved domains, a sequence-specific DNA-binding and dimerization domain and a conserved N-terminus which is important for transcriptional transactivation, replication, and interaction with the E1 protein to determine which specific amino acids or regions in the N-terminus were important for the replication or transactivation functions. Single amino acid substitutions were created at highly conserved, highly charged amino acids in the HPV 11 E2 N-terminus. Each amino acid was mutated to a nonpolar alanine residue or a similarly charged amino acid. The mutated E2 proteins were analyzed for their abilities to support transcriptional transactivation and transient DNA replication and to enhance binding of E1 to the origin of replication. Single amino acid substitutions were identified which were defective for either the replication or transactivation functions, which demonstrated that the replication and transactivation functions within the N-terminus are separable. In several cases different amino acid substitutions at the same site had variable effects on transcription or replication, highlighting the importance of hydrophobic interactions or side chain structure at each site. The replication function appeared to correlate with the ability of E2 to enhance binding of E1 to the origin of replication though these studies also suggest that other functions performed by the E2 protein may be important for replication.
Subject(s)
Alanine/metabolism , Oncogene Proteins, Viral/metabolism , Papillomaviridae/metabolism , Transcriptional Activation , Virus Replication , Alanine/genetics , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Conserved Sequence , DNA Replication , DNA-Binding Proteins/metabolism , Gene Expression , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Papillomaviridae/physiology , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription, Genetic , Tumor Cells, Cultured , Viral Proteins/metabolismABSTRACT
The bovine papillomavirus E2 transactivator protein enhances the ability of the E1 protein to bind to the viral origin of replication which contains an E1 binding site flanked by two E2 binding sites. To determine which regions and functions of the E2 protein are important for this cooperative interaction, a series of mutated E2 proteins were assayed for their ability to enhance E1 origin-specific binding. Cooperative origin binding required at least one E2 DNA binding site, an intact functional E2 DNA binding domain, and an intact transactivation domain. The hinge region of the E2 proteins was dispensable for this activity. To further examine the role of the E2 C-terminal domain, a series of chimeric proteins were generated that substituted the yeast GAL4 DNA binding domain for the E2 DNA binding domain. These chimeric proteins were able to cooperatively bind to a hybrid origin that contained GAL4 binding sites in place of the E2 binding sites. These studies indicate that the E2 transactivation domain is sufficient for interaction with the E1 protein and that the E2 DNA binding domain is required for interaction with origin DNA sequences.
Subject(s)
DNA-Binding Proteins/metabolism , Replication Origin , Trans-Activators/metabolism , Viral Proteins/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , Chlorocebus aethiops , DNA, Viral/metabolism , Defective Viruses/metabolism , Molecular Sequence Data , Protein Binding , Transcriptional ActivationABSTRACT
Hepatitis A virus (HAV) has an immunodominant neutralization antigenic site. By using a panel of monoclonal antibodies targeted against the HAV neutralization antigenic site, it was shown that three epitopes within this site are present on 14S subunits (pentamers of the structural unit). In contrast, two other epitopes within this site are formed upon assembly of 14S subunits into capsids. Thus, the epitopes recognized by these two monoclonal antibodies are formed either by a conformational change in the antigenic site or by the juxtaposition of epitope fragments present on different 14S subunits during assembly of 14S into 70S particles. Both 14S and 70S particles elicited HAV-neutralizing antibodies in mice; thus, these particles may be useful for HAV vaccine development.
Subject(s)
Antigens, Viral/immunology , Capsid/immunology , Epitopes/immunology , Hepatovirus/immunology , Antibodies, Monoclonal , Antibodies, Viral/biosynthesis , Antibody Formation , Antigens, Viral/genetics , Capsid/genetics , Hepatitis A Antigens , Hepatovirus/genetics , Hepatovirus/ultrastructure , Neutralization Tests , Protein Conformation , Recombinant Proteins/immunology , Vaccinia virus/geneticsABSTRACT
Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA-binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable.
Subject(s)
Bovine papillomavirus 1/physiology , DNA-Binding Proteins/metabolism , Genes, Viral , Protein-Tyrosine Kinases/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Transcriptional Activation , Viral Proteins/metabolism , Virus Replication , Animals , Base Sequence , Bovine papillomavirus 1/genetics , Cell Line , DNA-Binding Proteins/genetics , Haplorhini , Molecular Sequence Data , Mutagenesis , Plasmids , Protein-Tyrosine Kinases/genetics , Restriction Mapping , Sequence Deletion , Trans-Activators/genetics , Viral Proteins/geneticsABSTRACT
Studies conducted over the past 45 years have shown that immunoglobulin (IG) prevents 80%-90% of cases of hepatitis A when administered before exposure or shortly thereafter. Protection is short lived and requires early diagnosis and timely administration of IG to contacts. Inactivated and attenuated hepatitis A virus (HAV) vaccines have recently been developed and should be available for clinical use within the next few years. Evaluation of antibodies to HAV in IG and in IG recipients provides one method of determining the immunogenicity of HAV vaccines. The role of IG in the prevention of hepatitis A is reviewed with emphasis on the relationship of antibody response following IG administration to the efficacy of HAV vaccines.
Subject(s)
Hepatitis A/prevention & control , Immunization, Passive , HumansABSTRACT
Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.