bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Characterization of cLV25, a
Bacteroides fragilis
Chromosomal Transfer Factor Resembling Multiple
Bacteroides
sp. Mobilizable Transposons
Authors: Bass Kathleen A. , Hecht David W.
Published: July 2002

Horizontal DNA transfer contributes significantly to the dissemination of antibiotic resistance genes in
Bacteroides fragilis
. To further our understanding of DNA transfer in
B. fragilis
, we isolated and characterized a new transfer factor, cLV25. cLV25 was isolated from
B. fragilis
LV25 by its capture on the nonmobilizable
Escherichia coli-Bacteroides
shuttle vector pGAT400ΔBglII. Similar to other
Bacteroides
sp. transfer factors, cLV25 was mobilized in
E. coli
by the conjugative plasmid R751. Using Tn
1000
mutagenesis and deletion analysis of cLV25, two mobilization genes,
bmgA
and
bmgB
, were identified, whose predicted proteins have similarity to DNA relaxases and mobilization proteins, respectively. In particular, BmgA and BmgB were homologous to MocA and MocB, respectively, the two mobilization proteins of the
B. fragilis
mobilizable transposon Tn
4399
. A
cis
-acting origin of transfer (
oriT
) was localized to a 353-bp region that included nearly all of the intergenic region between
bmgB
and
orf22
and overlapped with the 3′ end of
orf22
. This
oriT
contained a putative
nic
site sequence but showed no significant similarity to the
oriT
regions of other transfer factors, including Tn
4399
. Despite the lack of sequence similarity between the
oriT
s of cLV25 and Tn
4399
, a mutation in the cLV25 putative DNA relaxase,
bmgA
, was partially complemented by Tn
4399
. In addition to the functional cross-reaction with Tn
4399
, a second distinguishing feature of cLV25 is that predicted proteins have similarity to proteins encoded not only by Tn
4399
but by several
Bacteroides
sp. transfer factors, including NBU1, NBU2, CTnDOT, Tn
4555
, and Tn
5520
.
Borrelia burgdorferiSensu Lato andEhrlichiaspp. inIxodesTicks from Southern Norway
Authors: Jenkins Andrew , Kristiansen Bjørn-Erik , Allum Anne-Gry , Aakre Randi Kersten , Strand Linda , Kleveland Ellen Johanne , van de Pol Ingrid , Schouls Leo
Published: July 2002

We report the results of a study of the prevalence ofEhrlichiaandBorreliaspecies in 341 questingIxodes ricinusticks from two locations in southern Norway. The prevalences ofBorrelia burgdorferisensu lato andEhrlichiaspp. were, respectively, 16 and 11.5% at site 1 and 17 and 6% at site 2. Prevalence and species composition ofBorreliaandEhrlichiavaried with location and date of collection. The dominantBorreliaspecies at both sites wasBorrelia afzelii, followed byBorrelia burgdorferisensu stricto.Borrelia gariniiwas found in only a single tick. The dominant member of theEhrlichiagroup was a recently describedEhrlichia-like organism related to the monocytic ehrlichiae. Variants ofEhrlichia phagocytophilaand the agent of human granulocytic ehrlichiosis were also found. The highest prevalences forB. afzelii,B. burgdorferisensu stricto, and theEhrlichia-like organism were observed in May.B. afzeliiwas most prevalent in females, less prevalent in nymphs, and least prevalent in males, while the prevalence ofEhrlichiawas highest in nymphs, lower in females, and least in males. Double infections withB. afzeliiandB. burgdorferisensu stricto and withB. afzeliiand theEhrlichia-like organism were significantly overrepresented. Tick densities were highest in May, when densities of more than 200 ticks/100 m2were observed, and declined during the summer months to densities as low as 20 ticks/100 m2. We conclude that estimates of the prevalence of tick-borne bacteria are sensitive to the choice of date and site for collection of ticks. This is the first study of tick-borneBorreliaandEhrlichiain Norway and the lowest reportedB.gariniiprevalence in Northern Europe. The prevalence of theEhrlichia-like organism is described for the first time in questing ticks.
Rapid Differentiation of “
Mycobacterium canettii
” from Other
Mycobacterium tuberculosis
Complex Organisms by PCR-Restriction Analysis of the
hsp65
Gene
Authors: Goh Khye Seng , Legrand Eric , Sola Christophe , Rastogi Nalin
Published: July 2002

A total of 102 isolates of the
Mycobacterium tuberculosis
complex, including available “
M. canettii
” isolates, were studied by PCR-restriction analysis of a 441-bp fragment of the
hsp65
gene. PRA upon
Hha
I enzyme digestion (GCGC) allowed easy differentiatiation of “
M. canettii
” from other members of the
M. tuberculosis
complex (three bands of 260, 105, and 60 bp for “
M. canetti
,” compared to four bands of 185, 105, 75, and 60 bp for other members of the
M. tuberculosis
complex). Sequencing of the 441-bp
hsp65
fragment of “
M. canettii
” isolates showed the disappearance of an
Hha
I site at position 235 due to a C-to-T transition that corresponded to position 631 of the homologous
hsp65
gene of
M. tuberculosis
H37Rv. Considering that “
M. canettii
” may also exist as a stable rough morphotype, we suggest that the true number of “
M. canettii
” isolates may be underestimated in clinical microbiology laboratories.
Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (TaqMan) Assays for Dengue Virus
Authors: Callahan Johnny D. , Wu Shuenn-Jue L. , Dion-Schultz Amanda , Mangold Beverly E. , Peruski Leonard F. , Watts Douglas M. , Porter Kevin R. , Murphy Gerald R. , Suharyono Wuryadi , King Chwan-Chuen , Hayes Curtis G. , Temenak Joseph J.
Published: July 2002

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.
Evaluation of Typing of
Vibrio
parahaemolyticus
by Three PCR Methods Using Specific Primers
Authors: Wong Hin-Chung , Lin Chih-Hsueh
Published: July 2002

Vibrio
parahaemolyticus
is a halophilic bacterium frequently involved in human outbreaks of seafood-associated gastroenteritis. For epidemiological purposes, different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen; however, these methods are mostly labor-intensive and time-consuming. In this work, we designed and evaluated three rapid PCR typing methods for this pathogen using primers designed on the basis of the following specific sequences: conserved ribosomal gene spacer sequence (RS), repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive intergenic consensus sequence (ERIC). Typing patterns and clustering analysis indicated that these methods apparently differentiated
V
.
parahaemolyticus
strains from reference strains of interspecific
Escherichia
coli
,
V
.
cholerae
, and
V
.
vulnificus
and were also valuable in subspecies typing of this pathogen. Forty domestic strains of
V
.
parahaemolyticus
, representing a wide range of PFGE patterns, were grouped into 15, 27, and 27 patterns, with discrimination indexes of 0.91, 0.97, and 0.98, by RS-, REP-, and ERIC-PCR, respectively. The discriminative abilities of these PCR methods closely approached or even exceeded those of PFGE and ribotyping. REP-PCR is preferable to ERIC-PCR because of the greater reproducibility of its fingerprints, while RS-PCR may be a practical method because it generates fewer amplification bands and patterns than the alternatives.
Antimicrobial Resistance and Bacterial Identification Utilizing a Microelectronic Chip Array
Authors: Westin Lorelei , Miller Carolyn , Vollmer Dana , Canter David , Radtkey Ray , Nerenberg Michael , O'Connell James P.
Published: July 2002

Species-specific bacterial identification of clinical specimens is often limited to a few species due to the difficulty of performing multiplex reactions. In addition, discrimination of amplicons is time-consuming and laborious, consisting of gel electrophoresis, probe hybridization, or sequencing technology. In order to simplify the process of bacterial identification, we combined anchored in situ amplification on a microelectronic chip array with discrimination and detection on the same platform. Here, we describe the simultaneous amplification and discrimination of six gene sequences which are representative of different bacterial identification assays:
Escherichia coli gyrA
,
Salmonella gyrA
,
Campylobacter gyrA
,
E. coli parC
,
Staphylococcus mecA,
and
Chlamydia
cryptic plasmid. The assay can detect both plasmid and transposon genes and can also discriminate strains carrying antibiotic resistance single-nucleotide polymorphism mutations. Finally, the assay is similarly capable of discriminating between bacterial species through reporter-specific discrimination and allele-specific amplification. Anchored strand displacement amplification allows multiplex amplification and complex genotype discrimination on the same platform. This assay simplifies the bacterial identification process greatly, allowing molecular biology techniques to be performed with minimal processing of samples and practical experience.
Helicobacter pylori
Infection in an Urban African Population
Authors: Fernando Neluka , Holton John , Zulu Isaac , Vaira Dino , Mwaba Peter , Kelly Paul
Published: July 2002

We have studied 221 adults drawn from an impoverished urban population with high human immunodeficiency virus (HIV) seroprevalence (35%) to determine the prevalence of gastroduodenal pathology and its relationship to serological markers of
Helicobacter pylori
virulence proteins and other potential environmental and immunological determinants of disease including HIV infection. Eighty-one percent were
H. pylori
seropositive, and 35% were HIV seropositive. Urban upbringing and low CD4 count were associated with a reduced likelihood of
H. pylori
seropositivity, as was current
Ascaris
infection, in keeping with recent evidence from an animal model. One hundred ninety-one adults underwent gastroduodenoscopy, and 14 had gastroduodenal pathology. Mucosal lesions were a major cause of abdominal pain in this population. While the majority of patients with gastroduodenal pathology (12 of 14) were seropositive for
H. pylori
, none were seropositive for HIV. Smoking was associated with increased risk of macroscopic pathology, and a history of
Mycobacterium bovis
BCG immunization was associated with reduced risk. Antibodies to
H. pylori
lipopolysaccharide were associated with pathology. HIV infection was associated with protection against mucosal lesions, suggesting that fully functional CD4 lymphocytes may be required for the genesis of gastroduodenal pathology.
Definition of a Divergent Epitope That Allows Differential Detection of Early Protein p41 from Human Herpesvirus 6 Variants A and B
Authors: Xu Yunhe , Linde Annika , Dahl Helena , Winberg Gosta
Published: July 2002

The human herpesvirus 6 (HHV-6) early protein, p41, encoded by the U27 gene has been detected in oligodendrocytes of multiple sclerosis (MS) patients by using a monoclonal antibody (MAb to p41/38). We here report the antigenic epitope of HHV-6 p41 recognized by this MAb. First, we established that the MAb to p41/38 recognizes a nuclear antigen in HHV-6A strain GS-infected cells but not in HHV-6B strain Z29-infected cells. Secondly, we compared the reactivity of the MAb to p41/38 to that of another p41-specific MAb (MAb to p41) on immunoblots with purified p41-glutathione
S
-transferase fusion protein from strains GS and Z29 and GS- and Z29-infected-cell lysates. The two MAbs were tested in an enzyme-linked immunosorbent assay against a panel of synthetic peptides covering the amino acid substitutions between the GS- and Z29-derived p41 proteins, as determined by DNA sequencing of our cloned isolates of the U27 gene. The MAb to p41/38 reacted specifically with a peptide comprising p41 residues 321 to 340 from strain GS. The critical residue in this peptide was serine 328, as the substitution S328N in the Z29 strain rendered the corresponding peptide nonreactive. The p41 S328 marker was present in three of three HHV-6A strains, while four of four sequenced p41 genes from HHV-6B strains had N328. Our findings are of value for the interpretation of previous findings of p41 expression in brains of MS patients and may allow a more detailed analysis of the role of HHV-6 variants in other disorders.
Evaluation of Candida ID, a New Chromogenic Medium for Fungal Isolation and Preliminary Identification of Some Yeast Species
Authors: Fricker-Hidalgo H. , Orenga S. , Lebeau B. , Pelloux H. , Brenier-Pinchart M. P. , Ambroise-Thomas P. , Grillot R.
Published: July 2002

Candida ID, a new chromogenic medium, allows identification of
Candida albicans
(blue colonies) and preliminary identification into a group of four species (pink colonies). In comparison with Albicans ID2 and Sabouraud gentamicin chloramphenicol on 446 fungal strains, Candida ID allowed the isolation of more species than Albicans ID 2 (95.5% versus 91.2%).
Development and Evaluation of Detection Systems for Staphylococcal Exfoliative Toxin A Responsible for Scalded-Skin Syndrome
Authors: Ladhani Shamez , Robbie Scott , Garratt Richard C. , Chapple Daniel S. , Joannou Christopher L. , Evans Robert W.
Published: July 2002

Staphylococcal scalded-skin syndrome is usually diagnosed clinically by its characteristic exfoliating rash. Isolation of
Staphylococcus aureus
from the patient further supports the diagnosis. Several detection systems have been developed to determine whether the isolated strain produces exfoliative toxin, but none are routinely available in hospital laboratories. In a novel approach, we used computer models to predict the structure of the exfoliative toxins based on other serine proteases and to identify surface epitopes for the production of antibodies that specifically bound the exfoliative toxin A (ETA) serotype. Several rapid immunologically based diagnostic tests for ETA were developed with these antibodies and compared with existing systems. Our results showed that Western blot analysis using these antibodies was in complete correlation with PCR, which has been validated against the “gold standard” mouse model. On the other hand, the double-antibody enzyme-linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion assay gave unacceptably high false-positive results due to interference by staphylococcal protein A. This problem was successfully overcome by the development of a F(ab′)
2
fragment ELISA, which was rapid and reproducible and was as sensitive and specific as PCR and Western blot analysis. The F(ab′)
2
fragment ELISA is superior to existing diagnostic systems because it is quantitative, which may be related to the severity of the condition, and can detect amounts of exfoliative toxin in the picogram range directly from serum. This is the first detection system with the potential to confirm the diagnosis of staphylococcal scalded-skin syndrome from a routine blood test within 3 h of presentation.
Basis of the Superiority of Cefoperazone Amphotericin Teicoplanin for Isolating
Campylobacter upsaliensis
from Stools
Authors: Byrne Catherine , Doherty Deirdre , Mooney Adele , Byrne Margaret , Woodward David , Johnson Wendy , Rodgers Frank , Bourke Billy
Published: July 2002

The optimum method for isolating
Campylobacter upsaliensis
from stools has not been clearly defined. In a preliminary study, cefoperazone amphotericin teicoplanin (CAT) selective medium isolated six
C. upsaliensis
strains which were not detected using modified cefoperazone charcoal deoxycholate (mCCDA). In order to identify the factors that underlie the superiority of CAT over mCCDA for isolating
C. upsaliensis
, we examined the effect of incubation time and antibiotic content of culture media on the growth of
C. upsaliensis
isolates using semiquantitative methods. The recovery of a subgroup of
C. upsaliensis
isolates from seeded stool specimens was also evaluated. Differences in growth of
C. upsaliensis
on CAT and mCCDA were modest and were not explained by the antibiotic profiles of the two media. Recovery of
C. upsaliensis
from spiked human feces on CAT was superior to that on mCCDA at lower concentrations of organisms (10
3
CFU/ml). We conclude that although CAT is more suitable than mCCDA for the isolation of
C. upsaliensis
from stools, the superiority of CAT for detecting this organism is not accounted for by the antibiotic composition of the medium.
Pneumococcal Carriage in Children in The Netherlands: a Molecular Epidemiological Study
Authors: Bogaert Debby , Engelen Marlene N. , Timmers-Reker Anja J. M. , Elzenaar Kees P. , Peerbooms Paul G. H. , Coutinho Roel A. , de Groot Ronald , Hermans Peter W. M.
Published: July 2002

In 1999, Engelen and coworkers investigated colonization in Amsterdam among 259 children attending 16 day-care centers (DCCs) and among 276 children who did not attend day-care centers (NDCCs). A 1.6- to 3.4-fold increased risk for nasopharyngeal colonization was observed in children attending DCCs compared with NDCC children, while no difference in antibiotic resistance was found between groups. The serotype and genotype distributions of 305 nasopharyngeal
Streptococcus pneumoniae
isolates of the latter study were investigated. The predominant serotypes in both the DCC and the NDCC groups included 19F (19 and 18%, respectively), 6B (14 and 16%, respectively), 6A (13 and 7%, respectively), 23F (9 and 7%, respectively), and 9V (7 and 7%, respectively). The theoretical vaccine coverage of the 7-valent conjugate vaccine was 59% for the DCC children and 56% for the NDCC group. Genetic analysis of the pneumococcal isolates revealed 75% clustering among pneumococci isolated from DCC attendees versus 50% among the NDCC children. The average pneumococcal cluster size in the DCC group was 3.8 and 4.6 isolates for two respective sample dates (range, 2 to 13 isolates per cluster), while the average cluster size for the NDCC group was 3.0 (range, 2 to 6 isolates per cluster). Similar to observations made in other countries, these results indicate a higher risk for horizontal spread of pneumococci in Dutch DCCs than in the general population. This study emphasizes the importance of molecular epidemiological monitoring before, during, and after implementation of pneumococcal conjugate vaccination in national vaccination programs for children.
Phylogeny-Based Rapid Identification of Mycoplasmas and Ureaplasmas from Urethritis Patients
Authors: Yoshida Takashi , Maeda Shin-Ichi , Deguchi Takashi , Ishiko Hiroaki
Published: July 2002

Some strains of mycoplasmas and ureaplasmas (family
Mycoplasmataceae
) are associated with nongonococcal urethritis (NGU) or other genitourinary infections. We have developed a rapid and reliable method of identifying the presence and prevalence of mycoplasmas and ureaplasmas in men with NGU. This method is based on the amplification of a part of the 16S rRNA gene by PCR and phylogenetic analysis. A portion of the 16S rRNA gene from 15 prototype strains was amplified with a set of common primers, and their nucleotides were sequenced. The nucleotide sequence of the V4 and V5 regions was analyzed by the neighbor-joining method. The 15 prototype strains were grouped into three distinct clusters, allowing us to clearly segregate the strains into distinct lineages. To determine the prevalence of these pathogens among patients with NGU, this protocol was tested with 148 urine samples. Amplifications were observed for 42 samples, and their nucleotide sequences were analyzed along with those of the 15 prototype strains. The phylogenetic tree thus constructed indicated that 15 of the 42 formed a cluster with
Mycoplasma genitalium
. Among the remaining specimens, 2 formed a cluster with
Mycoplasma hominis
, 19 with
Ureaplasma urealyticum
, and 5 with
Ureaplasma parvum
; the remaining sample contained both
M. genitalium
and
U. urealyticum
. This phylogeny-based identification of mycoplasmas and ureaplasmas provides not only a powerful tool for rapid diagnosis but also the basis for etiological studies of these pathogens.
Molecular Identification of Enterovirus by Analyzing a Partial VP1 Genomic Region with Different Methods
Authors: Palacios G. , Casas I. , Tenorio A. , Freire C.
Published: July 2002

VP1 is the most suitable region for use in the identification of enterovirus. Although VP1 sequencing methods may vary, it is necessary to agree on a common strategy of sequence analysis. Identification of a strain type may be achieved by three different approaches: pairwise sequence alignment, multiple-sequence alignment, and phylogenetic inference. Other methods are also available, but they are not simple enough to be performed at a virology laboratory. The performances of these methods were evaluated with nucleotide and protein sequences obtained from 32 original samples, 8 enterovirus isolates, and 64 GenBank sequences. Pairwise sequence alignment methods had very different results. The DNASTAR package identified only 28.8% of enterovirus strains, while the Genetics Computer Group package identified 50.0 or 72.1% of enterovirus strains when nucleotide or amino acid sequences were analyzed, respectively. Multiple-sequence alignment methods identified 94.2% (Clustal W program) or 92.3% (Pileup program) of the enterovirus strains, while the phylogenetic method increased this rate to 99.0%. Comparative evaluation of these analysis methods showed that the Clustal W program (version 1.81), a freely available multiple-sequence alignment program, presented one of the best performances when used with the correct criteria. Other commercial and expensive programs did not achieve the same performances, making them less suitable for molecular typing of enteroviruses. Finally, although phylogenetic inference is the most demanding method in terms of knowledge of the user, it remained the best option analyzed.
Modifications That Stabilize Human Immunodeficiency Virus Envelope Glycoprotein Trimers in Solution
Authors: Yang Xinzhen , Florin Lauren , Farzan Michael , Kolchinsky Peter , Kwong Peter D. , Sodroski Joseph , Wyatt Richard
Published: July 2002

The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.
Identification of Critical Amino Acid Residues in Human Immunodeficiency Virus Type 1 IN Required for Efficient Proviral DNA Formation at Steps prior to Integration in Dividing and Nondividing Cells
Authors: Tsurutani Naomi , Kubo Makoto , Maeda Yosuke , Ohashi Takashi , Yamamoto Naoki , Kannagi Mari , Masuda Takao
Published: July 2002

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is thought to have several putative roles at steps prior to integration, such as reverse transcription and nuclear transport of the preintegration complex (PIC). Here, we investigated new functional aspects of HIV-1 IN in the context of the viral replication cycle through point mutagenesis of Ser, Thr, Tyr, Lys, and Arg residues conserved in IN, some of which are located at possible phosphorylation sites. Our results showed that mutations of these Ser or Thr residues had no effect on reverse transcription and nuclear transport of PIC but had a slight effect on integration. Of note, mutations in the conserved KRK motif (amino acids 186 to 189), proposed previously as a putative nuclear localization signal (NLS) of HIV-1 IN, did not affect the karyophilic property of HIV-1 IN as shown by using a green fluorescent protein fusion protein expression system. Instead, these KRK mutations resulted in an almost complete lack of viral gene expression due to the failure to complete reverse transcription. This defect was complemented by supplying wild-type IN in
trans
, suggesting a
trans
-acting function of the KRK motif of IN in reverse transcription. Mutation at the conserved Tyr 143 (Y143G) resulted in partial impairment of completion of reverse transcription in monocyte-derived macrophages (MDM) but not in rhabdomyosarcoma cells. Similar effects were obtained by introducing a stop codon in the
vpr
gene (ΔVpr), and additive effects of both mutations (Y143G plus ΔVpr) were observed. In addition, these mutants did not produce two-long terminal repeat DNA, a surrogate marker for nuclear entry, in MDM. Thus, the possible impairment of Y143G might occur during the nuclear transport of the PIC. Taken together, our results identified new functional aspects of the conserved residues in HIV-1 IN: i) the KRK motif might have a role in efficient reverse transcription in both dividing and nondividing cells but not in the NLS function; ii) Y143 might be an important residue for maintaining efficient proviral DNA formation in nondividing cells.
Structure-Function Analysis of Hepatitis C Virus Envelope-CD81 Binding
Authors: Petracca Roberto , Falugi Fabiana , Galli Giuliano , Norais Nathalie , Rosa Domenico , Campagnoli Susanna , Burgio Vito , Di Stasio Enrico , Giardina Bruno , Houghton Michael , Abrignani Sergio , Grandi Guido
Published: July 2002

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a

K
d

of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10
−9
M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.
Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus
Authors: Mitnaul Lyndon J. , Matrosovich Mikhail N. , Castrucci Maria R. , Tuzikov Alexander B. , Bovin Nikolai V. , Kobasa Darwyn , Kawaoka Yoshihiro
Published: July 2002

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.
Recovery of Pathogenic Measles Virus from Cloned cDNA
Authors: Takeda Makoto , Takeuchi Kaoru , Miyajima Naoko , Kobune Fumio , Ami Yasushi , Nagata Noriyo , Suzaki Yuriko , Nagai Yoshiyuki , Tashiro Masato
Published: July 2002

Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700–705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773–5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.
Detection and Quantification of CD4+T Cells with Specificity for a New Major Histocompatibility Complex Class II-Restricted Influenza A Virus Matrix Protein Epitope in Peripheral Blood of Influenza Patients
Authors: Linnemann Thomas , Jung Günther , Walden Peter
Published: July 2002

FVFTLTVPS was identified as the core sequence of a new major histocompatibility complex class II-restricted T-cell epitope of influenza virus matrix protein. Epitope-specific CD4+T cells were detected in the peripheral blood of patients with frequencies of up to 0.94%, depending on the number of additional terminal amino acids.
Severe Murine Lung Immunopathology Elicited by the Pathogenic Equine Herpesvirus 1 Strain RacL11 Correlates with Early Production of Macrophage Inflammatory Proteins 1α, 1β, and 2 and Tumor Necrosis Factor Alpha
Authors: Smith Patrick M. , Zhang Yunfei , Grafton Warren D. , Jennings Stephen R. , O'Callaghan Dennis J.
Published: July 2002

The CBA mouse model was used to investigate the immunopathology induced in the lung by the pathogenic equine herpesvirus 1 (EHV-1) strain RacL11 in comparison to infection with the attenuated vaccine candidate strain KyA. Intranasal infection with KyA resulted in almost no inflammatory infiltration in the lung. In contrast, infection with the pathogenic RacL11 strain induced a severe alveolar and interstitial inflammation, consisting primarily of lymphocytes, macrophages, and neutrophils. Infection with either EHV-1 strain resulted in the accumulation of similar numbers and ratios of CD4 and CD8 T lymphocytes in the lung and bronchoalveolar lavage (BAL) fluid. Further analysis of these T-cell populations revealed identical EHV-1-specific cytotoxic T-lymphocyte responses. RNase protection analysis of RNA isolated from the BAL fluid of RacL11-infected mice on day 3 postinfection revealed much higher levels of RNA specific for macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and MIP-2 than were observed for KyA-infected mice. Furthermore, significantly higher levels of transcripts specific for tumor necrosis factor alpha were induced on day 3 postinfection with RacL11 compared with KyA. These findings suggest that the early production of proinflammatory beta chemokines plays a major role in the severe, most often lethal, respiratory inflammatory response induced by the pathogenic EHV-1 strain RacL11.
ICP0 Induces the Accumulation of Colocalizing Conjugated Ubiquitin
Authors: Everett Roger D.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 is a general activator of viral gene expression which stimulates the initiation of lytic infection and reactivation from quiescence and latency. The importance of ICP0 to the biology of HSV-1 infection has stimulated interest in its mode of action. Previous studies have reported its interactions with other viral regulatory molecules, with the translation apparatus, with cyclin D3, and with a ubiquitin-specific protease. It has been demonstrated that ICP0 is able to induce the proteasome-dependent degradation of a number of cellular proteins, including components of centromeres and small nuclear substructures known as ND10 or PML nuclear bodies. ICP0 has a RING finger zinc-binding domain which is essential for its functions. In view of several recent examples of other RING finger proteins which modulate the stability of specific target proteins by acting as components of E3 ubiquitin ligase complexes, this study has explored whether ICP0 might operate via a similar mechanism. Evidence that the foci of accumulated ICP0 in transfected and infected cells contain enhanced levels of conjugated ubiquitin is presented. This effect was dependent on the RING finger region of ICP0, and comparison of the properties of a number of ICP0 mutants revealed an excellent correlation between previously established functions of ICP0 and its ability to induce concentrations of colocalizing conjugated ubiquitin. These results strongly support the hypothesis that a major factor in the mechanism by which ICP0 influences virus infection is its ability to induce the degradation of specific cellular targets by interaction with the ubiquitin-proteasome pathway.
Efficacy and Safety Studies of a Recombinant Chimeric Respiratory Syncytial Virus FG Glycoprotein Vaccine in Cotton Rats
Authors: Prince Gregory A. , Capiau Carine , Deschamps Marguerite , Fabry Luc , Garçon Nathalie , Gheysen Dirk , Prieels Jean-Paul , Thiry Georges , Van Opstal Omer , Porter David D.
Published: July 2002

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.
Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway
Authors: Badou Abdallah , Bennasser Yamina , Moreau Marc , Leclerc Catherine , Benkirane Monsef , Bahraoui Elmostafa
Published: July 2002

The clinical manifestations observed in human immunodeficiency virus type 1 (HIV-1)-infected patients are primarily due to the capacity of the virus and its components to inactivate the immune system. HIV-1 Tat protein could participate in this immune system disorder. This protein is secreted by infected cells of HIV-infected patients and is free in the plasma, where it can interact and be taken up by both infected and noninfected cells. In asymptomatic patients infected by HIV-1, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with disease progression to AIDS. In the present work, we tested the capacity of Tat to induce IL-10 production by peripheral blood monocytes of healthy donors. The results show that Tat causes the production of IL-10 in a dose- and stimulation time-dependent manner. Investigations of the mechanisms involved in signal transduction show that (i) the calcium pathway is not or only slightly involved in Tat-induced IL-10 production, (ii) the protein kinase C pathway plays an essential role, and (iii) monocyte stimulation by Tat results in the intranuclear translocation of transcription factor NF-κB and in the induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2; activation of these two potential substrates of protein kinase C is required for the production of IL-10. Finally, our results suggest that the effect of Tat is exerted at the membrane level and that the active domain is located within N-terminal residues 1 to 45. This production of IL-10 induced by Tat could participate in the progression of HIV infection to AIDS.
Resistance of Native, Oligomeric Envelope on Simian Immunodeficiency Virus to Digestion by Glycosidases
Authors: Means Robert E. , Desrosiers Ronald C.
Published: July 2002

Stocks of simian immunodeficiency virus (SIV) from the supernatants of infected cell cultures were used to examine the sensitivity of envelope glycoprotein gp120 to enzymatic deglycosylation and the effects of enzyme treatment on infectivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis revealed little or no change in the mobility of virion-associated gp120 after digestion with high concentrations ofN-glycosidase F, endoglycosidase F, endoglycosidase H, and endo-β-galactosidase. Soluble gp120, which was not pelletable after the enzymatic reaction, was sensitive to digestion by the same enzymes within the same reaction mix and was only slightly less sensitive than gp120 that had been completely denatured by boiling in the presence of SDS and β-mercaptoethanol. Digestion by three of the seven glycosidases tested significantly changed the infectivity titer compared to that of mock-treated virus. Digestion by endo-β-galactosidase increased infectivity titers by about 2.5-fold, and neuraminidase from Newcastle disease virus typically increased infectivity titers by 8-fold. Most or all of the increase in infectivity titer resulting from treatment with neuraminidase could be accounted for by effects on the virus, not the cells; SIV produced in the presence of the sialic acid analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid also exhibited increased infectivity, and the effects could not be duplicated by neuraminidase treatment of cells. Digestion with mannosidase reduced infectivity by fivefold. Our results indicate that carbohydrates on native oligomeric gp120 as it exists on the surface of virus particles are largely occluded and are refractory to digestion by glycosidases. Furthermore, the sialic acid residues at the ends of carbohydrate side chains significantly reduce the inherent infectivity of SIV.
Sialic Acid Species as a Determinant of the Host Range of Influenza A Viruses
Authors: Suzuki Yasuo , Ito Toshihiro , Suzuki Takashi , Holland Robert E. , Chambers Thomas M. , Kiso Makoto , Ishida Hideharu , Kawaoka Yoshihiro
Published: July 2002

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the α2,6(SAα2,6Gal) linkage, while equine viruses favor SAα2,3Gal. However, whether a difference in relative abundance of specific SA species (
N
-acetylneuraminic acid [NeuAc] and
N
-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcα2,6Gal but not NeuAcα2,3Gal or NeuGcα2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcα2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcα2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.
Orf Virus Encodes a Novel Secreted Protein Inhibitor of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2
Authors: Deane David , McInnes Colin J. , Percival Ann , Wood Ann , Thomson Jackie , Lear Andrea , Gilray Janice , Fleming Stephen , Mercer Andrew , Haig David
Published: July 2002

The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with aKdof 369 pM and ovine IL-2 with aKdof 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.
Characterization of a Panel of Insertion Mutants in Human Cytomegalovirus Glycoprotein B
Authors: Singh Jasbir , Compton Teresa
Published: July 2002

Glycoprotein B (gB; gpUL55) of human cytomegalovirus (HCMV) plays a critical role in virus entry and cell-to-cell spread of infection. To define the structure-function relationships in gB, a panel of linker-insertion mutations was generated throughout the coding region. This strategy yielded a panel of 22 mutants with four amino acid insertions and 3 large truncation mutants. Assessment of the mutant proteins' biosynthetic properties and folding patterns analyzed in context with predicted secondary features revealed novel insights into gB's structure and trafficking properties. All of the insertion mutants were able to assemble into oligomers, suggesting that oligomerization is tolerant of small insertions and/or that multiple regions of the protein may be involved. Computer algorithm predictions of gB's secondary structure indicate that the furin-recognized cleavage site falls within an exposed loop. This loop may be particularly sensitive to structural alterations, since insertions upstream and downstream of the cleavage site rendered the mutant proteins cleavage defective. In addition, a strong correlation existed between terminal folding and cleavage of gB. Interestingly, terminal folding was not correlated with delivery to the cell surface but may influence the rate of transport to the cell surface. Nine mutants, containing insertions in both the extracellular and intracellular portions of gB, retained wild-type structural properties. This panel of characterized gB mutants, the first of this type for an HCMV protein, will be a useful tool in dissecting the role of gB during HCMV infection.
The c-mycLocus Is a Common Integration Site in Type B Retrovirus-Induced T-Cell Lymphomas
Authors: Rajan Lakshmi , Broussard Dana , Lozano Mary , Lee Chun G. , Kozak Christine A. , Dudley Jaquelin P.
Published: July 2002

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell leukemias. TBLV insertions near the c-mycgene were detectable in 2 of 30 tumors tested, whereas 80% of the tumors showed c-mycoverexpression. TBLV insertions on chromosome 15 (including a newly identified locus,Pad7) may cause c-mycoverexpression bycis-acting effects at a distance.
Therapeutic Effect of Anti-Macrophage Inflammatory Protein 2 Antibody on Influenza Virus-Induced Pneumonia in Mice
Authors: Sakai Shinya , Kawamata Hiroshi , Mantani Naoki , Kogure Toshiaki , Shimada Yutaka , Terasawa Katsutoshi , Sakai Takeshi , Imanishi Nobuko , Ochiai Hiroshi
Published: July 2002

We investigated the effect of anti-macrophage inflammatory protein 2 immunoglobulin G (aMIP-2 IgG) on the progression of influenza virus-induced pneumonia in mice. When mice were infected with a mouse lung-adapted strain of influenza A/PR/8/34 virus by intranasal inoculation, neutrophil counts in the bronchoalveolar lavage fluid (BALF) increased in parallel with the kinetics of MIP-2 production, which peaked 2 days after infection. After intracutaneous injection of a dose of 10 or 100 μg of aMIP-2 IgG once a day on days 0 and 1, neutrophil counts in BALF on day 2 were reduced to 49 or 37%, respectively, of the value in the control infected mice administered anti-protein A IgG. The antibody administration also improved lung pathology without affecting virus replication. Furthermore, by prolonged administration with a higher or lower dose for up to 5 days, body weight loss became slower and finally 40% of mice in both treatment groups survived potentially lethal pneumonia. These findings suggest that MIP-2-mediated neutrophil infiltration during the early phase of infection might play an important role in lung pathology. Thus, MIP-2 was considered to be a novel target for intervention therapy in potentially lethal influenza virus pneumonia in mice.