bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Alginate and Its O Acetylation in Formation of
Pseudomonas aeruginosa
Microcolonies and Biofilms
Authors: Nivens David E. , Ohman Dennis E. , Williams Jessica , Franklin Michael J.
Published: July 2002

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of
Pseudomonas aeruginosa
on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm
−1
. All strains produced biofilms that absorbed IR radiation near 1,650 cm
−1
(amide I), 1,550 cm
−1
(amide II), 1,240 cm
−1
(PO stretching, C—O—C stretching, and/or amide III vibrations), 1,100 to 1,000 cm
−1
(C—OH and P—O stretching) 1,450 cm
−1
, and 1,400 cm
−1
. The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm
−1
(C—OH stretching of alginate) and 1,250 cm
−1
(C—O stretching of the
O
-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 μm, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 μm in depth. In contrast, FRD1 produced microcolonies that were approximately 40 μm in depth. An
algJ
mutant strain that produced alginate lacking
O
-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the
algJ
mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for
P. aeruginosa
biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in
P. aeruginosa
biofilm architecture.
Essential Thioredoxin-Dependent Peroxiredoxin System from
Helicobacter pylori
: Genetic and Kinetic Characterization
Authors: Baker Laura M. S. , Raudonikiene Ausra , Hoffman Paul S. , Poole Leslie B.
Published: July 2002

Helicobacter pylori
, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed
ahpC
to be essential, suggesting a critical role for AhpC in defending
H. pylori
against oxygen toxicity. Characterization of the
ahpC
promoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue of
ahpF
, which encodes the dedicated AhpC reductase in most eubacteria, was found in the
H. pylori
genome. Instead, homologues of
Escherichia coli
thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for
H. pylori
AhpC. A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates [insulin and 5,5′-dithiobis(2-nitrobenzoic acid)]. AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite
V
max
and

K
m

values) but rapid enough (at 1 × 10
5
to 2 × 10
5
M
−1
s
−1
) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides composed of little more than the cysteinyl (Cys49) active site.
H. pylori
AhpC was not reduced by
Salmonella typhimurium
AhpF and was slightly more active with
E. coli
TrxR and Trx1 than was
S. typhimurium
AhpC, demonstrating the specialized catalytic properties of this peroxiredoxin.
Molecular Characterization of the PceA Reductive Dehalogenase of
Desulfitobacterium
sp. Strain Y51
Authors: Suyama Akiko , Yamashita Masaki , Yoshino Sadazo , Furukawa Kensuke
Published: July 2002

The tetrachloroethene (PCE) reductive dehalogenase (encoded by the
pceA
gene and designated PceA dehalogenase) of
Desulfitobacterium
sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to
cis
-1,2-dichloroethene at a specific activity of 113.6 nmol · min
−1
 · mg of protein
−1
. The apparent

K
m

values for PCE and TCE were 105.7 and 535.3 μM, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The
pceA
gene of
Desulfitobacterium
sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in
Escherichia coli
for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.
The Enigmatic
Escherichia coli fadE
Gene Is
yafH
Authors: Campbell John W. , Cronan John E.
Published: July 2002

The identity of the gene encoding acyl coenzyme A dehydrogenase is a major remaining mystery of the
Escherichia coli
fatty acid degradation (
fad
) regulon. Our prior genome array analyses showed that transcription of the
yafH
gene is controlled by the FadR regulatory protein. We now report direct experimental proof that
yafH
and
fadE
are the same gene.
Diversity of Strains of
Salmonella enterica
Serotype Enteritidis from English Poultry Farms Assessed by Multiple Genetic Fingerprinting
Authors: Liebana Ernesto , Garcia-Migura Lourdes , Breslin Mark F. , Davies Robert H. , Woodward Martin J.
Published: July 2002

Reliable and sufficiently discriminative methods are needed for differentiating individual strains of
Salmonella enterica
serotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism was
Pst
I-
Sph
I ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of
Xba
I-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins of
Salmonella
serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.
Culture with BACTEC Peds Plus/F Bottle Compared with Conventional Methods for Detection of Bacteria in Synovial Fluid
Authors: Hughes John G. , Vetter Emily A. , Patel Robin , Schleck Cathy D. , Harmsen Scott , Turgeant L. Thomas , Cockerill Franklin R.
Published: July 2002

An evaluation was undertaken to determine the utility of the BACTEC Peds Plus/F bottle and the BACTEC 9240 instrument (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) for the detection of clinically significant microorganisms in synovial fluid specimens. The Peds Plus/F bottle was used because in our laboratory the quantity of synovial fluid available for culture is frequently in the range of 0.5 to 3.0 ml. The culture results obtained with the Peds Plus/F bottle were compared to those obtained by a conventional agar plate method for a total of 805 synovial fluid specimens. Microbial growth was produced by 74 cultures (9.2%) from 60 patients, yielding a total of 77 microorganisms. Organisms were classified as pathogens (
n
= 62), contaminants (
n
= 12), or indeterminate (
n
= 3) on the basis of a review of the patients' medical histories. Culture using BACTEC Peds Plus/F bottle detected statistically significantly more pathogens overall (62 versus 51 pathogens [
P
= 0.001]) and statistically fewer contaminants overall (1 versus 11 contaminants [
P
= 0.006]) than culture by the agar plate method. These results indicate the superior performance of the BACTEC Peds Plus/F bottle over the conventional agar plate method for the detection of clinically significant microorganisms from synovial fluid specimens.
Differences in Clinical and Laboratory Diagnostic Characteristics of Penicilliosis Marneffei in Human Immunodeficiency Virus (HIV)- and Non-HIV-Infected Patients
Authors: Wong S. S. Y. , Wong K. H. , Hui W. T. , Lee S. S. , Lo J. Y. C. , Cao L. , Yuen K. Y.
Published: July 2002

We compared the clinical and laboratory features of human immunodeficiency virus (HIV)- and non-HIV-infected patients with penicilliosis marneffei. HIV-infected patients had a higher incidence of fungemia. A total of 85.7% of the HIV-negative patients had underlying diseases including hematologic malignancies or had received therapy with corticosteroids or cytotoxic agents. By a
Penicillium marneffei
-specific mannoprotein Mp1p enzyme-linked immunosorbent assay, serum antigen titers were found to be higher in HIV-positive patients, whereas serum antibody levels were found to be higher in HIV-negative patients.
Consensus and Variable Region PCR Analysis of
Helicobacter pylori
3′ Region of
cagA
Gene in Isolates from Individuals with or without Peptic Ulcer
Authors: Rota Cláudia Augustin , Pereira-Lima Júlio C. , Blaya Carolina , Nardi Nance Beyer
Published: July 2002

The clinical outcome of
Helicobacter pylori
infection may be associated with the
cagA
bacterial genotype. To investigate the
cagA
status of
H. pylori
-infected patients and the relationship between
cagA
and peptic ulcer disease, gastric biopsy specimens from 103 Caucasian patients in Brazil were analyzed by PCR. Since allelic variation in
cagA
exists and distinct
H. pylori
subgenotypes may circulate in different regions, PCR using primers for a variable 3′ region of the
cagA
gene according to a Japanese methodology and for a consensus
cagA
3′ region used in Western methods was used for
cagA
detection.
cagA
was present in 53 (71%) of 75
H. pylori
-positive cases when analyzed by the consensus region method and was associated with duodenal ulcer disease (
P
= 0.02), but not with gastric ulcer (
P
= 0.26), when compared to patients with duodenitis or gastritis. The variable region PCR method was able to detect 43 (57%)
cagA
-positive cases within the same group of
H. pylori
-positive patients and showed three subtypes of
cagA
(A, B/D, and C) that were not associated with clinical outcome. However, in 8 (18%) of the cases, more than one subtype was present, and an association between patients with multiple subtypes and disease outcome was observed when compared to patients with isolated subtypes (
P
= 0.048).
cagA
was a marker of
H. pylori
strains for duodenal ulcer disease in our population, and in spite of the differences in the 3′ region of the
cagA
gene, the Japanese methodology was able to detect the
cagA
status in most cases. The presence of multiple subgenotypes of
cagA
was associated with gastric ulcer.
Enzyme-Linked Immunosorbent Assay Using a Virus Type-Specific Peptide Based on a Subdomain of Envelope Protein E
rns
for Serologic Diagnosis of Pestivirus Infections in Swine
Authors: Langedijk J. P. M. , Middel W. G. J. , Meloen R. H. , Kramps J. A. , de Smit J. A.
Published: July 2002

Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein E
rns
were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure of the E
rns
protein, and the peptide was selected for its probable independent folding and good exposure, which would make it a good candidate for an antigenic peptide to be used in a diagnostic test. A solid-phase peptide ELISA which was cross-reactive for several types of pestivirus antibodies and which can be used for the general detection of pestivirus antibodies was developed. To identify type-specific pestivirus antibodies, a liquid-phase peptide ELISA, with a labeled, specific classical swine fever virus (CSFV) peptide and an unlabeled bovine viral diarrhea virus peptide to block cross-reactivity, was developed. Specificity and sensitivity of the liquid-phase peptide ELISA for CSFV were 98 and 100%, respectively. Because the peptide is a fragment of the E
rns
protein, it can be used to differentiate between infected and vaccinated animals when a vaccine based on the E2 protein, which is another pestivirus envelope protein, is used.
Expression and Self-Assembly in Baculovirus of Porcine Enteric Calicivirus Capsids into Virus-Like Particles and Their Use in an Enzyme-Linked Immunosorbent Assay for Antibody Detection in Swine
Authors: Guo Mingzhang , Qian Yuan , Chang Kyeong-Ok , Saif Linda J.
Published: July 2002

Porcine enteric calicivirus (PEC) causes diarrhea and intestinal lesions in pigs. PEC strain Cowden grows to low to moderate titers in cell culture but only with the addition of intestinal contents from uninfected gnotobiotic pigs (W. T. Flynn and L. J. Saif, J. Clin. Microbiol. 26:206–212, 1988; A. V. Parwani, W. T. Flynn, K. L. Gadfield, and L. J. Saif, Arch. Virol. 120:115–122, 1991). Cloning and sequence analysis of the PEC Cowden full-length genome revealed that it is most closely related genetically to the human Sapporo-like viruses. In this study, the complete PEC capsid gene was subcloned into the plasmid pBlueBac4.5 and the recombinant baculoviruses were identified by plaque assay and PCR. The PEC capsid protein was expressed in insect (Sf9) cells inoculated with the recombinant baculoviruses, and the recombinant capsid proteins self- assembled into virus-like particles (VLPs) that were released into the cell supernatant and purified by CsCl gradient centrifugation. The PEC VLPs had the same molecular mass (58 kDa) as the native virus capsid and reacted with pig hyperimmune and convalescent-phase sera to PEC Cowden in enzyme-linked immunosorbent assay (ELISA) and Western blotting. The PEC capsid VLPs were morphologically and antigenically similar to the native virus by immune electron microscopy. High titers (1:102,400 to 204,800) of PEC-specific antibodies were induced in guinea pigs inoculated with PEC VLPs, suggesting that the VLPs could be useful for future candidate PEC vaccines. A fixed-cell ELISA and VLP ELISA were developed to detect PEC serum antibodies in pigs. For the fixed-cell ELISA, Sf9 cells were infected with recombinant baculoviruses expressing PEC capsids, followed by cell fixation with formalin. For the VLP ELISA, the VLPs were used for the coating antigen. Our data indicate that both tests were rapid, specific, and reproducible and might be used for large-scale serological investigations of PEC antibodies in swine.
Comparative Study Using Amplified Fragment Length Polymorphism Fingerprinting, PCR Genotyping, and Phenotyping To Differentiate
Campylobacter fetus
Strains Isolated from Animals
Authors: Wagenaar Jaap A. , van Bergen Marcel A. P. , Newell Diane G. , Grogono-Thomas Rose , Duim Birgitta
Published: July 2002

A collection of
Campylobacter fetus
strains, including both
C. fetus
subsp.
fetus
and
C. fetus
subsp.
venerealis
, were phenotypically identified to the subspecies level and genotypically typed by PCR and amplified fragment length polymorphism (AFLP) analysis. Phenotypic subspecies determination methods were unreliable. Genotyping of the strains by PCR and AFLP showed a clear discrimination between the two subspecies.
Characterization of Mutations in the
rpoB
Gene Associated with Rifampin Resistance in
Rhodococcus equi
Isolated from Foals
Authors: Fines Marguerite , Pronost Stephane , Maillard Karine , Taouji Said , Leclercq Roland
Published: July 2002

Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by
Rhodococcus equi
. Frequently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rif
r
)-determining regions of 12 rifampin-resistant
R. equi
strains isolated from three foals and of mutants selected in vitro from
R. equi
ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible
R. equi
strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (
Escherichia coli
numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 μg/ml), whereas His526Asp conferred high-level resistance (rifampin MIC, 128 μg/ml) in the 7 remaining strains. In strains from the two other foals, His526Asp and Ser531Leu mutations were found to be associated with high-level and low-level resistance, respectively. The in vitro mutants, highly resistant to rifampin, harbored His526Tyr and His526Arg substitutions. As described in other bacterial genera, His526, Ser531, and Asp516 are critical residues for rifampin resistance in
R. equi
, and the resistance levels are dependent on both the location and the nature of the substitution.
Multicenter Comparison of Three Different Analytical Systems for Evaluation of DNA Banding Patterns from
Cryptococcus neoformans
Authors: Cardinali Gianluigi , Martini Alessandro , Preziosi Roberta , Bistoni Francesco , Baldelli Franco
Published: July 2002

The enormous improvement of molecular typing techniques for epidemiological and clinical studies has not always been matched by an equivalent effort in applying optimal criteria for the analysis of both phenotypic and molecular data. In spite of the availability of a large collection of statistical and phylogenetic methods, the vast majority of commercial packages are limited by using only the unweighted pair group method with arithmetic mean algorithm to construct trees and by considering electrophoretic pattern only as migration distances. The latter method has serious drawbacks when different runs (separate gels) of the same molecular analysis are to be compared. This work presents a multicenter comparison of three different systems of banding pattern analysis on random amplified polymorphic DNA, (GACA)
4
, and contour-clamped homogeneous electric field patterns from strains of
Cryptococcus neoformans
var.
neoformans
isolated in different clinical and geographical situations and a standard
Saccharomyces cerevisiae
strain employed as an outgroup. The systems considered were evaluated for their actual ability to(i) recognize identities, (ii) define complete differences (i.e., the ability to place
S. cerevisiae
out of the
C. neoformans
cluster), and (iii) estimate the extent of similarity among different strains. The ability to cluster strains according to the patient from which they were isolated was also evaluated. The results indicate that different algorithms do indeed produce divergent trees, both in overall topology and in clustering of individual strains, thus suggesting that care must be taken by individual investigators to use the most appropriate procedure and by the scientific community in defining a consensus system.
Simian Immunodeficiency Viruses of Diverse Origin Can Use CXCR4 as a Coreceptor for Entry into Human Cells
Authors: Owen Sherry M. , Masciotra Silvina , Novembre Frank , Yee JoAnn , Switzer William M. , Ostyula Moses , Lal Renu B.
Published: July 2002

Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [
n
= 6]), stumptail (SIVstm [
n
= 1]), mandrill (SIVmnd [
n
= 1]), and African green (SIVagm [
n
= 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5
+/+
donor, and seven of eight isolates tested also infected CCR5
−/−
PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.
Enhanced Binding of Antibodies to Neutralization Epitopes following Thermal and Chemical Inactivation of Human Immunodeficiency Virus Type 1
Authors: Grovit-Ferbas K. , Hsu J. F. , Ferbas J. , Gudeman V. , Chen I. S. Y.
Published: July 2002

Inactivation of viral particles is the basis for several vaccines currently in use. Initial attempts to use simian immunodeficiency virus to model a killed human immunodeficiency virus type 1 (HIV-1) vaccine were unsuccessful, and limited subsequent effort has been directed toward a systematic study of the requirements for a protective killed HIV-1 vaccine. Recent insights into HIV-1 virion and glycoprotein structure and neutralization epitopes led us to revisit whether inactivated HIV-1 particles could serve as the basis for an HIV-1 vaccine. Our results indicate that relatively simple processes involving thermal and chemical inactivation can inactivate HIV-1 by at least 7 logs. For some HIV-1 strains, significant amounts of envelope glycoproteins are retained in high-molecular-weight fractions. Importantly, we demonstrate retention of each of three conformation-dependent neutralization epitopes. Moreover, reactivity of monoclonal antibodies directed toward these epitopes is increased following treatment, suggesting greater exposure of the epitopes. In contrast, treatment of free envelope under the same conditions leads only to decreased antibody recognition. These inactivated virions can also be presented by human dendritic cells to direct a cell-mediated immune response in vitro. These data indicate that a systematic study of HIV-1 inactivation, gp120 retention, and epitope reactivity with conformation-specific neutralizing antibodies can provide important insights for the development of an effective killed HIV-1 vaccine.
Isolation and Characterization of Monoclonal Antibodies That Inhibit Hepatitis C Virus NS3 Protease
Authors: Ueno Takamasa , Misawa Satoru , Ohba Yoichi , Matsumoto Mitsuhiro , Mizunuma Makiko , Kasai Nobuhiro , Tsumoto Kouhei , Kumagai Izumi , Hayashi Hideya
Published: July 2002

A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (

K
i

= 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependent
cis
processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent
trans
processing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.
Pathogenicity of Hantaan Virus in Newborn Mice: Genetic Reassortant Study Demonstrating that a Single Amino Acid Change in Glycoprotein G1 Is Related to Virulence
Authors: Ebihara Hideki , Yoshimatsu Kumiko , Ogino Michiko , Araki Koichi , Ami Yasushi , Kariwa Hiroaki , Takashima Ikuo , Li Dexin , Arikawa Jiro
Published: July 2002

Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mu11E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-1 Ile to mu11E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mu11E10-derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.
Establishment of a Rescue System for Canine Distemper Virus
Authors: Gassen Uta , Collins Fergal M. , Duprex W. Paul , Rima Bert K.
Published: July 2002

Canine distemper virus
(CDV) has been rescued from a full-length cDNA clone. Besides
Measles virus
(MV) and
Rinderpest virus
, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.
Tumor Necrosis Factor Alpha-Deficient, but Not Interleukin-6-Deficient, Mice Resist Peripheral Infection with Scrapie
Authors: Mabbott Neil A. , Williams Alun , Farquhar Christine F. , Pasparakis Manolis , Kollias Giorgos , Bruce Moira E.
Published: July 2002

In most peripheral infections of rodents and sheep with scrapie, infectivity is found first in lymphoid tissues and later in the central nervous system (CNS). Cells within the germinal centers (GCs) of the spleen and lymph nodes are important sites of extraneural replication, from which infection is likely to spread to the CNS along peripheral nerves. Here, using immunodeficient mice, we investigate the identity of the cells in the spleen that are important for disease propagation. Despite possessing functional T and B lymphocytes, tumor necrosis factor alpha-deficient (TNF-α−/−) mice lack GCs and follicular dendritic cell (FDC) networks in lymphoid tissues. In contrast, lymphoid tissues of interleukin-6-deficient (IL-6−/−) mice possess FDC networks but have impaired GCs. When the CNSs of TNF-α−/−, IL-6−/−, and wild-type mice were directly challenged with the ME7 scrapie strain, 100% of the mice were susceptible, developing disease after closely similar incubation periods. However, when challenged peripherally (intraperitoneally), most TNF-α−/−mice failed to develop scrapie up to 503 days postinjection. All wild-type and IL-6−/−mice succumbed to disease approximately 300 days after the peripheral challenge. High levels of scrapie infection and the disease-specific isomer of the prion protein, PrPSc, were detectable in spleens from challenged wild-type and IL-6−/−mice but not from TNF-α−/−mice. Histopathological analysis of spleen tissue demonstrated heavy PrP accumulations in direct association with FDCs in challenged wild-type and IL-6−/−mice. No PrPScaccumulation was detected in spleens from TNF-α−/−mice. We conclude that, for the ME7 scrapie strain, mature FDCs are critical for replication in lymphoid tissues and that in their absence, neuroinvasion following peripheral challenge is impaired.
Viral DNA Synthesis Defects in Assembly-Competent Rous Sarcoma Virus CA Mutants
Authors: Cairns Tina M. , Craven Rebecca C.
Published: July 2002

The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle.
The Vaccinia Virus A33R Protein Provides a Chaperone Function for Viral Membrane Localization and Tyrosine Phosphorylation of the A36R Protein
Authors: Wolffe Elizabeth J. , Weisberg Andrea S. , Moss Bernard
Published: July 2002

The products of the A33R and A36R genes of vaccinia virus are incorporated into the membranes of intracellular enveloped virions (IEV). When extracts of cells that had been infected with vaccinia virus and labeled with H
3
32
PO
4
were immunoprecipitated with antibodies against the A33R protein, two prominent bands were resolved. The moderately and more intensely labeled bands were identified as phosphorylated A33R and A36R proteins, respectively. The immunoprecipitated complex contained disulfide-bonded dimers of A33R protein that were noncovalently linked to A36R protein. Biochemical analysis indicated that the two proteins were phosphorylated predominantly on serine residues, with lesser amounts on threonines. The A36R protein was also phosphorylated on tyrosine, as determined by specific binding to an anti-phosphotyrosine antibody. Serine phosphorylation and A33R-A36R protein complex formation occurred even when virus assembly was blocked at an early stage with the drug rifampin. Tyrosine phosphorylation was selectively reduced in cells infected with F13L or A34R gene deletion mutants that were impaired in the membrane-wrapping step of IEV formation. In addition, tyrosine phosphorylation was specifically inhibited in cells infected with an A33R deletion mutant that still formed IEV. Immunofluorescence and immunoelectron microscopy indicated that in the absence of the A33R protein, the A36R protein was localized in Golgi membranes but not in IEV. In the absence of the A36R protein, however, the A33R protein still localized to IEV membranes. These studies together with others suggest that the A33R protein guides the A36R protein to the IEV membrane, where it subsequently becomes tyrosine phosphorylated as a signal for actin tail formation.
Extrachromosomal Recombinant Adeno-Associated Virus Vector Genomes Are Primarily Responsible for Stable Liver Transduction In Vivo
Authors: Nakai Hiroyuki , Yant Stephen R. , Storm Theresa A. , Fuess Sally , Meuse Leonard , Kay Mark A.
Published: July 2002

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 × 10
11
to 3.0 × 10
11
vector genomes per mouse (
n
= 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than ∼10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.
DNA Binding and Modulation of Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Garber Alexander C. , Shu Marla A. , Hu Jianhong , Renne Rolf
Published: July 2002

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is highly expressed in these malignancies and has been shown to play an important role in episomal maintenance, presumably by binding to a putative oriP. In addition, LANA modulates cellular and viral gene expression and interacts with the cellular tumor suppressors p53 and retinoblastoma suppressor protein. Many of these features are reminiscent of Epstein-Barr virus nuclear antigens (EBNAs), a family of six proteins expressed during latency. EBNA-1 is required for episome maintenance, binds to oriP, and strongly activates transcription from two promoters, including its own. We have previously shown that LANA can transactivate its own promoter and therefore asked whether LANA, like EBNA-1, activates transcription by direct binding to DNA. By using recombinant LANA expressed from vaccinia virus vectors for electrophoretic mobility shift assays, we found that LANA does not bind to its own promoter. In contrast, LANA binds specifically to sequences containing an imperfect 20-bp palindrome in the terminal repeat (TR) of KSHV. We further show that the C-terminal domain of LANA is sufficient for site-specific DNA binding. Unlike EBNA-1, which activates transcription through binding of oriP, we found that LANA inhibits transcription from a single TR binding site. A multimerized TR as found in the viral genome results in strong transcriptional suppression when linked to a heterologous promoter. These data suggest that LANA, although fulfilling functions similar to those of EBNA-1, does so by very different mechanisms.
Efficient c-kit Receptor-Targeted Gene Transfer to Primary Human CD34-Selected Hematopoietic Stem Cells
Authors: Zhong Qiu , Oliver Peter , Huang Weitao , Good David , La Russa Vincent , Zhang Zili , Cork John R. , Veith Robert Woody , Theodossiou Chris , Kolls Jay K. , Schwarzenberger Paul
Published: July 2002

We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit
+
human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.
Induction of Cell Death in Human Immunodeficiency Virus-Infected Macrophages and Resting Memory CD4 T Cells by TRAIL/Apo2L
Authors: Lum Julian J. , Pilon André A. , Sanchez-Dardon Jaime , Phenix Barbara N. , Kim John E. , Mihowich Jennifer , Jamison Keri , Hawley-Foss Nanci , Lynch David H. , Badley Andrew D.
Published: July 2002

Because the persistence of human immunodeficiency virus (HIV) in cellular reservoirs presents an obstacle to viral eradication, we evaluated whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induces apoptosis in such reservoirs. Lymphocytes and monocyte-derived macrophages (MDM) from uninfected donors do not die following treatment with either leucine zipper human TRAIL (LZhuTRAIL) or agonistic anti-TRAIL receptor antibodies. By contrast, such treatment induces apoptosis of in vitro HIV-infected MDM as well as peripheral blood lymphocytes from HIV-infected patients, including CD4+CD45RO+HLA-DR−lymphocytes. In addition, LZhuTRAIL-treated cells produce less viral RNA and p24 antigen than untreated controls. Whereas untreated cultures produce large amounts of HIV RNA and p24 antigen, of seven treated CD4+CD45RO+HLA-DR−cell cultures, viral RNA production was undetectable in all, p24 antigen was undetectable in six, and proviral DNA was undetectable in four. These data demonstrate that TRAIL induces death of cells from HIV-infected patients, including cell types which harbor latent HIV reservoirs.
Identification of Six Putative Novel Human Papillomaviruses (HPV) and Characterization of Candidate HPV Type 87
Authors: Menzo Stefano , Monachetti Alessia , Trozzi Caterina , Ciavattini Andrea , Carloni Guido , Varaldo Pietro E. , Clementi Massimo
Published: July 2002

Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (
cand
HPV87) by the Reference Center for Human Papillomavirus.
cand
HPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that the
cand
HPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and
cand
HPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of
cand
HPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.
Hantavirus Nucleocapsid Protein Oligomerization
Authors: Alfadhli Ayna , Love Zac , Arvidson Brian , Seeds Joshua , Willey Jessica , Barklis Eric
Published: July 2002

Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome. The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein. The N protein is a 420- to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood. We have undertaken a biochemical and genetic analysis of N protein oligomerization. Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers. Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer. We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions. Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion. These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils. Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates.
Degenerate Immunogenicity of an HLA-A2-Restricted Hepatitis B Virus Nucleocapsid Cytotoxic T-Lymphocyte Epitope That Is Also Presented by HLA-B51
Authors: Thimme Robert , Chang Kyong-Mi , Pemberton Janell , Sette Alessandro , Chisari Francis V.
Published: July 2002

The recent identification of hepatitis B virus (HBV) epitopes restricted by multiple HLA alleles has greatly expanded the epitope repertoire available for T-cell-mediated therapeutic vaccine development. The HLA-B51-restricted peptide HBc19-27 is particularly interesting because it is located entirely within the HLA-A2-restricted HBc18-27 epitope. Here we show that HLA-B51-restricted cytotoxic T lymphocytes specific for HBc19-27 from a patient with acute HBV infection were also able to lyse HLA-B51-positive target cells pulsed with HBc18-27 and to produce gamma interferon when stimulated by that peptide, implying that HBc18-27 can be presented by HLA-B51 as well as by HLA-A2. These results demonstrate the concept of degenerate immunogenicity across HLA class supertype boundaries in a human viral disease setting. In addition, they could facilitate the development of an epitope-based therapeutic vaccine to terminate chronic HBV infection that could provide a broad and diverse population coverage with a single peptide.
Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection of Mice: Proliferation of Cell Clones with Integrated HTLV-1 Provirus in Lymphoid Organs
Authors: Tanaka Masakazu , Sun Binlian , Fang Jianhua , Nitta Takayuki , Yoshida Toshinori , Kohtoh Sayaka , Kikukawa Hiroko , Hanai Shuji , Uchida Kazuhiko , Miwa Masanao
Published: July 2002

Human T-cell leukemia virus type 1 (HTLV-1) is suggested to cause adult T-cell leukemia after 40 to 50 years of latency in a small percentage of carriers. However, little is known about the pathophysiology of the latent period and the reservoir organs where polyclonal proliferation of cells harboring integrated provirus occurs. The availability of animal models would be useful to analyze the latent period of HTLV-1 infection. At 18 months after HTLV-1 infection of C3H/HeJ mice inoculated with the MT-2 cell line, which is an HTLV-1-producing human T-cell line, HTLV-1 provirus was detected in spleen DNA from eight of nine mice. No more than around 100 proviruses were found per 10
5
spleen cells. Cellular sequences flanking the 3′ long terminal repeat (LTR) and the clonalities of the cells which harbor integrated HTLV-1 provirus were analyzed by linker-mediated PCR. The results showed that the flanking sequences are of mouse genome origin and that polyclonal proliferation of the spleen cells harboring integrated HTLV-1 provirus had occurred in three mice. A sequence flanking the 5′ LTR was isolated from one of the mice and revealed the presence of a 6-nucleotide duplication of cellular sequences, consistent with typical retroviral integration. Moreover, PCR was performed on DNA from infected tissues, with LTR primers and primers derived from seven novel flanking sequences of the three mice. Data revealed that the expected PCR products were found from lymphatic tissues of the same mouse, suggesting that the lymphatic tissues were the reservoir organs for the infected and proliferating cell clones. The mouse model described here should be useful for analysis of the carrier state of HTLV-1 infection in humans.