bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplex LightCycler PCR Assay for Detection and Differentiation of
Bordetella pertussis
and
Bordetella parapertussis
in Nasopharyngeal Specimens
Authors: Sloan Lynne M. , Hopkins Marlene K. , Mitchell P. Shawn , Vetter Emily A. , Rosenblatt Jon E. , Harmsen W. Scott , Cockerill Franklin R. , Patel Robin
Published: July 2002

A rapid real-time multiplex PCR assay for detecting and differentiating
Bordetella pertussis
and
Bordetella parapertussis
in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS
481
and IS
1001
of
B. pertussis
and
B. parapertussis
, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for
Bordetella
culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9
B. pertussis
and 3
B. parapertussis
) and 68 specimens were negative by all methods. Three other specimens were positive for
B. pertussis
by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for
B. pertussis
by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS
481
) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (
n
= 96): 44 specimens were positive and 41 specimens were negative for
B. pertussis
with both PCR methods. Nine specimens were positive for
B. pertussis
by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for
B. pertussis
by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (
P
= 0.0654). The insertion sequence IS
481
is also present in
Bordetella holmesii
; specimens containing
B
.
holmesii
may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of
B
.
pertussis
and
B
.
parapertussis
in nasopharyngeal specimens.
Multiserotype Enzyme-Linked Immunosorbent Assay as a Diagnostic Aid for Periodontitis in Large-Scale Studies
Authors: Pussinen P. J. , Vilkuna-Rautiainen T. , Alfthan G. , Mattila K. , Asikainen S.
Published: July 2002

Periodontitis is a common chronic oral infection caused by gram-negative bacteria, including
Actinobacillus actinomycetemcomitans
and
Porphyromonas gingivalis
. Periodontitis evokes inflammatory host response locally in the periodontium but also systemically. The systemic humoral antibody response against oral pathogens can conveniently be measured by an immunoassay. The aim of the study was to measure serum immunoglobulin G class antibodies against
A. actinomycetemcomitans
and
P. gingivalis
by an enzyme-linked immunosorbent assay (ELISA) in which mixtures of several serotypes of the pathogens were used as antigens to avoid biasing of the results in favor of a particular strain. For
A. actinomycetemcomitans
the antigen consisted of six strains representing serotypes a, b, c, d, and e and one nonserotypeable strain. In the
P. gingivalis
ELISA, antigens representing serotypes a, b, and c were used. Serum samples from 90 subjects, including 35 samples from patients with diagnosed periodontitis, 10 samples from periodontally healthy controls, and 45 samples from randomly selected apparently healthy volunteers (referred to as “healthy subjects”), were tested. For both pathogens the antibody levels (means ± standard deviations) of the patients—expressed as area under the dilution curve—were significantly higher than those for healthy controls or healthy subjects, with values for
A. actinomycetemcomitans
and
P. gingivalis
, respectively, as follows: patients, 22.60 ± 9.94 mm
2
and 26.72 ± 11.13 mm
2
; healthy controls, 9.99 ± 3.92 mm
2
and 6.90 ± 3.38 mm
2
; and healthy subjects, 16.85 ± 6.67 mm
2
and 8.51 ± 4.23 mm
2
. The serotype mixture ELISA is suitable for measuring antibodies against periodontal pathogens in large epidemiological studies in order to evaluate the role of periodontitis as a risk factor for other diseases.
Use of 16S rRNA Sequencing for Identification of
Actinobacillus ureae
Isolated from a Cerebrospinal Fluid Sample
Authors: Whitelaw A. C. , Shankland I. M. , Elisha B. G.
Published: July 2002

Actinobacillus ureae
, previously
Pasteurella ureae
, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to
A. ureae
. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene.
Enzyme-Linked Immunosorbent Assay Based on Soluble Promastigote Antigen Detects Immunoglobulin M (IgM) and IgG Antibodies in Sera from Cases of Visceral and Cutaneous Leishmaniasis
Authors: Ryan Jeffrey R. , Smithyman Anthony M. , Rajasekariah G-Halli , Hochberg Lisa , Stiteler John M. , Martin Samuel K.
Published: July 2002

Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. In areas where it is nonendemic, global travel and increased incidence of the disease in human immunodeficiency virus and intravenous-drug user populations are also causes for concern. The unavailability of rapid and reliable tests for diagnosis of the various leishmaniases makes patient management difficult. We have developed an enzyme-linked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis. These practical assays are based on soluble antigens from promastigotes cultivated in a protein-free medium. In preliminary studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controls were tested. Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. Serological tests for the various clinical manifestations of leishmaniasis could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity.
Laccase and Melanization in Clinically Important
Cryptococcus
Species Other than
Cryptococcus
neoformans
Authors: Ikeda Reiko , Sugita Takashi , Jacobson Eric S. , Shinoda Takako
Published: July 2002

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in
Cryptococcus neoformans
. Since isolations of
Cryptococcus
species other than
C.
neoformans
from clinical specimens have been increasing, we examined the laccase activities of
C. albidus
,
C. laurentii
,
C. curvatus
, and
C. humicola
. Incubation of cells with epinephrine produced adrenochrome color in
C. albidus
,
C. laurentii
, and
C. curvatus
but not in
C. humicola
. Activity was always less than in
C. neoformans
. Laccase was detected in the soluble fractions of disrupted
C. albidus
,
C. laurentii
, and
C. curvatus
cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from
C. albidus
,
C. laurentii
, and
C. curvatus
migrated more slowly than that from
C. neoformans
. One strain of
C. curvatus
exhibited two melanin bands. Thus, several clinically emerging
Cryptococcus
species express laccase and can synthesize melanin.
Quantitative Detection of
Mycoplasma genitalium
from First-Pass Urine of Men with Urethritis and Asymptomatic Men by Real-Time PCR
Authors: Yoshida Takashi , Deguchi Takashi , Ito Masayasu , Maeda Shin-Ichi , Tamaki Masayoshi , Ishiko Hiroaki
Published: July 2002

We developed a TaqMan-based real-time PCR assay for quantifying
Mycoplasma genitalium
. This assay is able to specifically quantify concentrations of the
M. genitalium
16S rRNA gene ranging from 10
7
to 10 copies/reaction. Using the TaqMan assay, we quantified the
M. genitalium
16S rRNA gene in first-pass urine of men with urethritis and asymptomatic men who were positive for
M. genitalium
by PCR- and phylogeny-based assay. Of 130 men with gonococcal urethritis (GU), five were positive for
M. genitalium
. The mycoplasma load for each specimen was <5 × 10 copies/ml. Of 84 men with chlamydial non-GU (CNGU), seven were positive for
M. genitalium
. One man had an
M. genitalium
load of <5 × 10 copies/ml, and six men had loads ranging from 1.1 × 10
7
to 2.7 × 10
2
copies/ml. Of 86 men with nonchlamydial NGU (NCNGU), 17 were positive for
M. genitalium
. The mycoplasma loads for these men ranged from 3.3 × 10
6
to 2.3 × 10
2
copies/ml. Of 76 asymptomatic men, only two were positive for
M. genitalium
. For these men, the loads were 2 × 10
2
and <5 × 10 copies/ml. The patients with NGU had significantly higher concentrations of
M. genitalium
in their first-pass urine than did men with GU (
P
< 0.01) or asymptomatic men (
P
< 0.05). In addition,
M. genitalium
loads were significantly higher in men with NCNGU than those in asymptomatic men (
P
< 0.05). The quantitative assessment of
M. genitalium
loads by the TaqMan assay will provide useful information for understanding the pathogenicity of this mycoplasma in the urogenital tract.
Comparison of Second- and Third-Generation Enzyme Immunoassays for Detecting Antibodies to Hepatitis C Virus
Authors: Abdel-Hamid Mohamed , El-Daly Mai , El-Kafrawy Sherif , Mikhail Nabiel , Strickland G. Thomas , Fix Alan D.
Published: July 2002

Supplemental assays, such as recombinant immunoblot assays (RIBA), are used to confirm detection of antibodies to hepatitis C virus (HCV). However, due to their expense, they are not widely used in developing countries. The purpose of our study was to compare the results of second- and third-generation (G2 and G3, respectively) enzyme immunoassays (EIAs) and to resolve discordant results by using a supplemental assay to assess the reliability of G2 and G3 EIAs to confirm anti-HCV antibody-positive results. We performed both G2 and G3 EIAs for anti-HCV antibodies on 1,134 serum samples collected during the 2nd year of a longitudinal community-based study in Egypt; 35 samples with discordant results were tested by Abbott Laboratories Micro-Particle Immunoassay (M-EIA) and RIBA. Viremia was determined with an in-house nested reverse transcriptase PCR (RT-PCR) to detect HCV RNA. Concordance between the two assays (G2/G3) was 96.9%; 87 (7.7%) samples were positive and 1,012 (89.2%) were negative by both assays. For 17 samples, the discordant results were G2 assay negative and G3 assay positive, and for 18 samples, the discordant results were G2 assay positive and G3 assay negative. Among the 17 G2 assay-negative and G3 assay-positive samples, 15 were M-EIA positive and 7 were PCR positive. Among the 18 G2 assay-positive and G3 assay-negative samples, 2 were M-EIA positive and none were PCR positive. RIBA results from 24 discordant samples showed 87.5% agreement with the G3 EIA, 12.5% agreement with the G2 EIA, and 95.8% agreement with M-EIA. Eleven samples were indeterminate by RIBA and excluded from this analysis. Based on RIBA results, the sensitivity of the G3 EIA was 99%, compared to 89.8% for the G2 EIA, while the specificity of the G3 EIA was 99.8%, compared to 98.9% for the G2 EIA. These results show that the reliability of the G3 EIA in screening these sera is excellent, and the G3 assay can be used in the absence of supplemental tests where resources are limited. RIBA appears not to have advantages over the less expensive M-EIA screening assay. The main disadvantage of RIBA is the occurrence of indeterminate results, especially among problematic samples. Samples giving discordant results in multiple assays are often indeterminate with the RIBA.
PreservCyt Transport Medium Used for the ThinPrep Pap Test Is a Suitable Medium for Detection of
Chlamydia trachomatis
by the COBAS Amplicor CT/NG Test: Results of a Preliminary Study and Future Implications
Authors: Bianchi Anne , Moret François , Desrues Jean-Marc , Champenois Thierry , Dervaux Yves , Desvouas Orlane , Oursin André , Quinzat Dominique , Dachez Roger , Bathelier Christian , Ronsin Christophe
Published: July 2002

The commercial COBAS Amplicor CT/NG test (Roche Diagnostic Systems, Meylan, France) is a sensitive and specific method for detection of
Chlamydia trachomatis
infections. This test currently consists of using a nucleic acid amplification method to detect
C. trachomatis
in first-void urine specimens and in endocervical swabs collected in 2-sucrose-phosphate (2SP) transport medium. We conducted a prospective study to determine whether the automated COBAS Amplicor CT/NG test can detect
C. trachomatis
in cervical specimens collected in PreservCyt transport medium (ThinPrep Pap Test; Cytyc Corporation, Boxborough, Mass.). PreservCyt medium is used to preserve cervical samples before the preparation of ThinPrep slides. We collected 1,000 cervical specimens from young women (age range, 15 to 25 years) during routine Pap smear tests. Only specimens with normal cytology and in which the gynecologist found no clinical evidence of urogenital infections were selected. The samples were stored in PreservCyt transport medium at 15 to 20°C.
C. trachomatis
was detected in 22 of the 1,000 cervical specimens that had been stored in PreservCyt. To confirm the positive samples, the test was repeated on new endocervical swab specimens collected in 2SP transport medium. Only 9 of the 22 positive patients agreed to undergo this control, but all 9 retested positive. To evaluate the influence of storage conditions on the sensitivity of the
C. trachomatis
PCR test, all of the positive samples were stored at 15 to 20°C in PreservCyt transport medium and were retested every 2 weeks for 6 weeks.
C. trachomatis
was successfully amplified from all 22 specimens for the whole 6-week period. The prevalence of
C. trachomatis
infection was 2.2% in our study population. These results demonstrate that PreservCyt transport medium is a suitable transport medium for detection of
C. trachomatis
by the COBAS Amplicor CT/NG test. The ThinPrep Pap Test may enable gynecologists to monitor for both cervical lesions and
C. trachomatis
infections with a single endocervical specimen.
Genotyping of Hepatitis C Virus Types 1, 2, 3, and 4 by a One-Step LightCycler Method Using Three Different Pairs of Hybridization Probes
Authors: Schröter Matthias , Zöllner Bernhard , Schäfer Peter , Landt Olfert , Lass Ulrich , Laufs Rainer , Feucht Heinz-Hubert
Published: July 2002

Determination of hepatitis C virus (HCV) genotypes has become increasingly important during the last years for prediction of the clinical course and the outcome of antiviral therapy. Therefore, numerous different methods have been developed to enable HCV genotyping. However, many of them are very laborious and expensive, leading to limited usage in daily routine diagnostics. We have established a method which combines the speed of the new LightCycler technology with the use of amplification products generated for diagnostic quantitative HCV RNA determination. Differentiation of HCV genotypes is performed with these amplicons in a single step by using fluorophore-labeled hybridization probes. Although currently only two different acceptor fluorophores are available for the LightCycler, types 1, 2, 3, and 4, which are by far the prevailing HCV genotypes in Europe and the United States, can be distinguished. Genotypes of specimens from 190 chronically HCV-infected patients were determined by the LightCycler method and compared with the results of nucleotide sequencing. Concordant results were obtained for all samples. This new method offers a fast and convenient possibility to determine the quantitative HCV RNA load and the genotype in large-scale settings within about 4 h.
Initiation of DNA Replication within
oriP
Is Dispensable for Stable Replication of the Latent Epstein-Barr Virus Chromosome after Infection of Established Cell Lines
Authors: Norio Paolo , Schildkraut Carl L. , Yates John L.
Published: July 2002

The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called
oriP
, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of
oriP
, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of
oriP
, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the
oriP
region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of
oriP
is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of
oriP
. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.
Characteristics of Bursal T Lymphocytes Induced by Infectious Bursal Disease Virus
Authors: Kim In-Jeong , You Seung Kwon , Kim Hyungee , Yeh Hung-Yeuh , Sharma Jagdev M.
Published: July 2002

Infectious bursal disease virus (IBDV) is an avian lymphotropic virus that causes immunosuppression. When specific-pathogen-free chickens were exposed to a pathogenic strain of IBDV (IM), the virus rapidly destroyed B cells in the bursa of Fabricius. Extensive viral replication was accompanied by an infiltration of T cells in the bursa. We studied the characteristics of intrabursal T lymphocytes in IBDV-infected chickens and examined whether T cells were involved in virus clearance. Flow cytometric analysis of single-cell suspensions of the bursal tissue revealed that T cells were first detectable at 4 days postinoculation (p.i.). At 7 days p.i., 65% of bursal cells were T cells and 7% were B cells. After virus infection, the numbers of bursal T cells expressing activation markers Ia and CD25 were significantly increased (P< 0.03). In addition, IBDV-induced bursal T cells produced elevated levels of interleukin-6-like factor and nitric oxide-inducing factor in vitro. Spleen and bursal cells of IBDV-infected chickens had upregulated gamma interferon gene expression in comparison with virus-free chickens. In IBDV-infected chickens, bursal T cells proliferated in vitro upon stimulation with purified IBDV in a dose-dependent manner (P< 0.02), whereas virus-specific T-cell expansion was not detected in the spleen. Cyclosporin A treatment, which reduced the number of circulating T cells and compromised T-cell mitogenesis, increased viral burden in the bursae of IBDV-infected chickens. The results suggest that intrabursal T cells and T-cell-mediated responses may be important in viral clearance and promoting recovery from infection.
Isolation of Transformation Suppressor Genes by cDNA Subtraction: Lumican Suppresses Transformation Induced by v-
src
and v-K-
ras
Authors: Yoshioka Naohisa , Inoue Hirokazu , Nakanishi Kazuyoshi , Oka Kiyomasa , Yutsudo Masuo , Yamashita Atsuko , Hakura Akira , Nojima Hiroshi
Published: July 2002

We have reported that suppressive factors for transformation by viral oncogenes are expressed in primary rat embryo fibroblasts (REFs). To identify such transformation suppressor genes, we prepared a subtracted cDNA library by using REFs and a rat normal fibroblast cell line, F2408, and isolated 30 different cDNA clones whose mRNA expression was markedly reduced in F2408 cells relative to that in REFs. We referred to these as TRIF (transcript reduced in F2408) clones. Among these genes, we initially tested the suppressor activity for transformation on three TRIF genes, TRIF1 (neuronatin), TRIF2 (heparin-binding growth-associated molecule), and TRIF3 (lumican) by focus formation assay and found that lumican inhibited focus formation induced by activated H-
ras
in F2408 cells. Colony formation in soft agar induced by v-K-
ras
or v-
src
was also suppressed in F2408 clones stably expressing exogenous lumican without disturbing cell proliferation. Tumorigenicity in nude mice induced by these oncogenes was also suppressed in these lumican-expressing clones. These results indicate that lumican has the ability to suppress transformation by v-
src
and v-K-
ras
.
The r144 Major Histocompatibility Complex Class I-Like Gene of Rat Cytomegalovirus Is Dispensable for both Acute and Long-Term Infection in the Immunocompromised Host
Authors: Beisser Patrick S. , Kloover Jeroen S. , Grauls Gert E. L. M. , Blok Marinus J. , Bruggeman Cathrien A. , Vink Cornelis
Published: July 2002

The rat cytomegalovirus (RCMV) r144 gene encodes a polypeptide homologous to major histocompatibility complex class I heavy chains. To study the role of r144 in virus replication, an RCMV r144 null mutant strain (RCMVΔr144) was generated. This strain replicated with efficiency similar to that of wild-type (WT) RCMV in vitro. Additionally, WT RCMV and RCMVΔr144 were found not to differ in their replication characteristics in vivo. First, the survival rate was similar among groups of immunosuppressed rats infected with either RCMVΔr144 or WT RCMV. Second, the dissemination of virus did not differ in either RCMVΔr144- or WT RCMV-infected, immunosuppressed rats, either in the acute phase of infection or approximately 1 year after infection. These data indicate that the RCMV r144 gene is essential neither for virus replication in the acute phase of infection nor for long-term infection in immunocompromised rats. Interestingly, in a local infection model in which footpads of immunosuppressed rats were inoculated with virus, a significantly higher number of infiltrating macrophage cells as well as of CD8+T cells was observed in WT RCMV-infected paws than in RCMVΔr144-infected paws. This suggests that r144 might function in the interaction with these leukocytes in vivo.
Interactions between Equine Cyclin T1, Tat, and TAR Are Disrupted by a Leucine-to-Valine Substitution Found in Human Cyclin T1
Authors: Taube Ran , Fujinaga Koh , Irwin Dan , Wimmer Jörg , Geyer Matthias , Peterlin B. Matija
Published: July 2002

Transcriptional transactivators (Tat) from human immunodeficiency and equine infectious anemia viruses (HIV and EIAV) interact with their transactivation response elements (TAR) to increase the rates of viral transcription. Whereas the human cyclin T1 is required for the binding of Tat to TAR from HIV, it is unknown how Tat from EIAV interacts with its TAR. Furthermore, Tat from EIAV functions in equine and canine cells but not in human cells. In this study, we present sequences of cyclins T1 from horse and dog and demonstrate that their N-terminal 300 residues rescue the transactivation of Tat from EIAV in human cells. Although human and equine cyclins T1 bind to this Tat, only the equine cyclin T1 supports the binding of Tat to TAR from EIAV. Finally, a reciprocal exchange of the valine for the leucine at position 29 in human and equine cyclins T1, respectively, renders the human cyclin T1 active and the equine cyclin T1 inactive for Tat transactivation from EIAV. Thus, the collaboration between a specific cyclin T1 and Tat for their high-affinity interaction with TAR is a common theme of lentiviral transactivation.
In Vivo Analysis of Human T-Cell Leukemia Virus Type 1 Reverse Transcription Accuracy
Authors: Mansky Louis M.
Published: July 2002

Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (
neo
) gene, a bacterial origin of DNA replication, and the
lacZ
α peptide gene region (the mutational target). The vector was replicated by
trans
-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 × 10
−6
mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.
Immunogenicity of an Anti-Clade B Feline Immunodeficiency Fixed-Cell Virus Vaccine in Field Cats
Authors: Matteucci Donatella , Poli Alessandro , Mazzetti Paola , Sozzi Sabrina , Bonci Francesca , Isola Patrizia , Zaccaro Lucia , Giannecchini Simone , Calandrella Michela , Pistello Mauro , Specter Steven , Bendinelli Mauro
Published: July 2002

Attempts at vaccine development for feline immunodeficiency virus (FIV) have been extensive, both because this is a significant health problem for cats and because FIV may be a useful vaccine model for human immunodeficiency virus. To date, only modest success, producing only short-term protection, has been achieved for vaccine trials in controlled laboratory settings. It is unclear how relevant such experiments are to prevention of natural infection. The current study used a vaccine that employs cell-associated FIV-M2 strain fixed with paraformaldehyde. Subject cats were in a private shelter where FIV was endemic, a prevalence of 29 to 58% over an 8-year observation period. Cats roamed freely from the shelter through the surrounding countryside but returned for food and shelter. After ensuring that cats were FIV negative, they were immunized using six doses of vaccine over a 16-month period and observed for 28 months after the initiation of immunization. Twenty-six cats (12 immunized and 14 nonimmunized controls) were monitored for a minimum of 22 months. Immunized cats did not experience significant adverse effects from immunization and developed both antibodies and cellular immunity to FIV, although individual responses varied greatly. At the conclusion of the study, 0 of 12 immunized cats had evidence of FIV infection, while 5 of 14 control cats were infected. Thus, the vaccine was safe and immunogenic and did not transmit infection. Furthermore, vaccinated cats did not develop FIV infection in a limited clinical trial over an extended time period. Thus, the data suggest that a fixed, FIV-infected cell vaccine has potential for preventing natural FIV infection in free-roaming cats.
Cellular Receptor Traffic Is Essential for Productive Duck Hepatitis B Virus Infection
Authors: Breiner Klaus M. , Schaller Heinz
Published: July 2002

We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.
Role of Maternal Antibody in Natural Infection of
Peromyscus maniculatus
with Sin Nombre Virus
Authors: Borucki Monica K. , Boone John D. , Rowe Joan E. , Bohlman Marlene C. , Kuhn Edward A. , DeBaca Robert , St. Jeor Stephen C.
Published: July 2002

Data from naturally infected deer mice (
Peromyscus maniculatus
) were used to investigate vertical transmission of Sin Nombre virus (SNV) and SNV-specific antibody. The antibody prevalence in juvenile mice (14 g or less) was inversely proportional to the mass of the animal, with juvenile deer mice weighing less than 11 g most likely to be antibody positive (26.9%) and juvenile mice weighing between 13 and 14 g least likely to be antibody positive (12.9%). Although a significant sex bias in seropositivity was detected in adult deer mice, no significant sex bias in seropositivity was detected in juvenile animals. Ten juvenile deer mice were identified that had initially tested positive for SNV-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA) but had subsequently tested negative when recaptured as adults. SNV RNA was detected by reverse transcriptase PCR (RT-PCR) in the blood of ELISA-positive adult deer mice but not in the blood of ELISA-positive juveniles. One of the juvenile mice initially tested negative for SNV RNA but later tested positive when recaptured as an ELISA-positive adult. The RT-PCR results for that individual correlated with the disappearance and then reappearance of SNV-specific IgG, indicating that the presence of SNV RNA at later time points was due to infection with SNV via horizontal transmission. SNV-specific antibody present in both ELISA-positive juvenile and adult mice was capable of neutralizing SNV. Additionally, our data indicate that SNV is not transmitted vertically.
Feline Immunodeficiency Virus Vif Localizes to the Nucleus
Authors: Chatterji Udayan , Grant Chris K. , Elder John H.
Published: July 2002

Monoclonal antibodies prepared against recombinant Vif derived from the 34TF10 strain of feline immunodeficiency virus (FIV) were used to assess the expression and localization of Vif in virus-infected cells. Analyses by Western blotting and by immunoprecipitation from cells infected with FIV-34TF10 revealed the presence of a single 29-kDa species specific for virus-infected cells. Confirmation of antibody specificity was also performed by specific immunoprecipitation of in vitro-transcribed and -translated recombinant Vif. Localization experiments were also performed on virus-infected cells, using different fixation procedures. Results for methanol fixation protocols similar to those reported for localization of human immunodeficiency virus (HIV) Vif showed a predominant cytoplasmic localization for FIV Vif, very similar to localization of HIV type 1 Vif and virtually identical to the localization observed for the Gag antigens of the virus. However, with milder fixation procedures that used 2% formaldehyde at 4°C, FIV Vif was strongly evident in the nucleus. The localization was distinct from the nuclear localization noted with Rev and did not involve the nucleolus. Attempts to show colocalization or coprecipitation of Vif with Gag antigens were unsuccessful. In addition, Vif was not detected in purified FIV virions. The results are consistent with the notion that the primary role of Vif in virus infection initiates in the nucleus.
Attenuation Markers of a Candidate Dengue Type 2 Vaccine Virus, Strain 16681 (PDK-53), Are Defined by Mutations in the 5′ Noncoding Region and Nonstructural Proteins 1 and 3
Authors: Butrapet Siritorn , Huang Claire Y.-H. , Pierro Dennis J. , Bhamarapravati Natth , Gubler Duane J. , Kinney Richard M.
Published: July 2002

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5′ noncoding region (5′NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK
2
cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10
7.3
PFU/ml or greater in LLC-MK
2
cells. The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5′NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5′NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype. Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.
Cell-to-Cell Spread of Wild-Type Herpes Simplex Virus Type 1, but Not of Syncytial Strains, Is Mediated by the Immunoglobulin-Like Receptors That Mediate Virion Entry, Nectin1 (PRR1/HveC/HIgR) and Nectin2 (PRR2/HveB)
Authors: Cocchi Francesca , Menotti Laura , Dubreuil Patrice , Lopez Marc , Campadelli-Fiume Gabriella
Published: July 2002

The immunoglobulin-like receptors that mediate entry of herpes simplex virus type 1 (HSV-1) into human cells were found to mediate the direct cell-to-cell spread of wild-type virus. The receptors here designated Nectin1α and -δ and Nectin2α were originally designated HIgR, PRR1/HveC, and PRR2α/HveB, respectively. We report the following. (i) Wild-type HSV-1 spreads from cell to cell in J cells expressing nectin1α or nectin1δ but not in parental J cells that are devoid of entry receptors. A monoclonal antibody to nectin1, which blocks entry, also blocked cell-to-cell spread in nectin1-expressing J cells. Moreover, wild-type virus did not spread from a receptor-positive to a receptor-negative cell. (ii) The antibody to nectin1 blocked transmission of wild-type virus in a number of human cell lines, with varying efficiencies, suggesting that nectin1 is the principal mediator of wild-type virus spread in a variety of human cell lines. (iii) Nectin1 did not mediate cell fusion induced by the syncytial strains HSV-1(MP) and HFEM-syn. (iv) Nectin2α could serve as a receptor for spread of a mutant virus carrying the L25P substitution in glycoprotein D, but not of wild-type virus, in agreement with its ability to mediate entry of the mutant but not of wild-type virus.
Reevaluation of Amino Acid Variability of the Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein and Prediction of New Discontinuous Epitopes
Authors: Yamaguchi-Kabata Yumi , Gojobori Takashi
Published: July 2002

To elucidate the evolutionary mechanisms of the human immunodeficiency virus type 1 gp120 envelope glycoprotein at the single-site level, the degree of amino acid variation and the numbers of synonymous and nonsynonymous substitutions were examined in 186 nucleotide sequences for gp120 (subtype B). Analyses of amino acid variabilities showed that the level of variability was very different from site to site in both conserved (C1 to C5) and variable (V1 to V5) regions previously assigned. To examine the relative importance of positive and negative selection for each amino acid position, the numbers of synonymous and nonsynonymous substitutions that occurred at each codon position were estimated by taking phylogenetic relationships into account. Among the 414 codon positions examined, we identified 33 positions where nonsynonymous substitutions were significantly predominant. These positions where positive selection may be operating, which we call putative positive selection (PS) sites, were found not only in the variable loops but also in the conserved regions (C1 to C4). In particular, we found seven PS sites at the surface positions of the α-helix (positions 335 to 347 in the C3 region) in the opposite face for CD4 binding. Furthermore, two PS sites in the C2 region and four PS sites in the C4 region were detected in the same face of the protein. The PS sites found in the C2, C3, and C4 regions were separated in the amino acid sequence but close together in the three-dimensional structure. This observation suggests the existence of discontinuous epitopes in the protein's surface including this α-helix, although the antigenicity of this area has not been reported yet.
Contingent Neutrality in Competing Viral Populations
Authors: Quer Josep , Hershey Christine L. , Domingo Esteban , Holland John J. , Novella Isabel S.
Published: July 2002

The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465–471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5°C. Thus, the “neutral fitness” of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations.
Different Levels of T-Cell Receptor Triggering Induce Distinct Functions in Hepatitis B and Hepatitis C Virus-Specific Human CD4+T-Cell Clones
Authors: Diepolder Helmut M. , Gruener Norbert H. , Gerlach J. Tilman , Jung Maria-Christina , Wierenga Eddy A. , Pape Gerd R.
Published: July 2002

CD4+T cells play a major role in the host defense against viruses and intracellular microbes. During the natural course of such an infection, specific CD4+T cells are exposed to a wide range of antigen concentrations depending on the body compartment and the stage of disease. While epitope variants trigger only subsets of T-cell effector functions, the response of virus-specific CD4+T cells to various concentrations of the wild-type antigen has not been systematically studied. We stimulated hepatitis B virus core- and hepatitis C virus NS3-specific CD4+T-cell clones which had been isolated from patients with acute hepatitis during viral clearance with a wide range of specific antigen concentrations and determined the phenotypic changes and the induction of T-cell effector functions in relation to T-cell receptor internalization. A low antigen concentration induced the expression of T-cell activation markers and adhesion molecules in CD4+T-cell clones in the absence of cytokine secretion and proliferation. The expression of CD25, HLA-DR, CD69, and intercellular cell adhesion molecule 1 increased as soon as T-cell receptor internalization became detectable. A 30- to 100-fold-higher antigen concentration, corresponding to the internalization of 20 to 30% of T-cell receptor molecules, however, was required for the induction of proliferation as well as for gamma interferon and interleukin-4 secretion. These data indicate that virus-specific CD4+T cells can respond to specific antigen in a graded manner depending on the antigen concentration, which may have implications for a coordinate regulation of specific CD4+T-cell responses.
A Single Amino Acid Mutation in the
Carnation Ringspot Virus
Capsid Protein Allows Virion Formation but Prevents Systemic Infection
Authors: Sit Tim L. , Haikal Patrick R. , Callaway Anton S. , Lommel Steven A.
Published: July 2002

A
Carnation ringspot virus
(CRSV) variant (1.26) was identified that accumulates virions but is incapable of forming a systemic infection. The 1.26 capsid protein gene possesses a Ser→Pro mutation at amino acid 282. Conversion of 1.26 amino acid 282 to Ser restored systemic infection, while the reciprocal mutation in wild-type CRSV abolished systemic infection. Similar mutations introduced into the related
Red clover necrotic mosaic
virus capsid protein gene failed to induce the packaging but nonsystemic movement phenotype. These results provide additional support for the theory that virion formation is necessary but not sufficient for systemic movement with the dianthoviruses.
Papillomavirus Pseudovirus: a Novel Vaccine To Induce Mucosal and Systemic Cytotoxic T-Lymphocyte Responses
Authors: Shi Wei , Liu Jianzhong , Huang Yujun , Qiao Liang
Published: July 2002

Intestinal mucosa is a portal for many infectious pathogens. Systemic immunization, in general, does not induce a cytotoxic T-lymphocyte (CTL) response at the mucosal surface. Because papillomavirus (PV) naturally infects mucosa and skin, we determined whether PV pseudovirus, i.e., PV-like particles in which unrelated DNA plasmids are packaged, could generate specific mucosal immunity. We found that the pseudovirus that encoded the lymphocytic choriomeningitis virus gp33 epitope induced a stronger CTL response than a DNA vaccine (plasmid) encoding the same epitope given systemically. The virus-like particles that were used to make the pseudoviruses provided an adjuvant effect for induction of CTLs by the DNA vaccine. The PV pseudovirus pseudoinfected mucosal and systemic lymphoid tissues when administered orally. Oral immunization with the pseudovirus encoding human PV type 16 mutant E7 induced mucosal and systemic CTL responses. In comparison, a DNA vaccine encoding E7, when given orally, did not induce a CTL response in intestinal mucosal lymphoid tissue. Further, oral immunization with the human PV pseudovirus encoding E7 protected mice against mucosal challenge with an E7-expressing bovine PV pseudovirus. Thus, PV pseudovirus can be used as a novel vaccine to induce mucosal and systemic CTL responses.
Human Immunodeficiency Virus Type 1 Entry into Macrophages Mediated by Macropinocytosis
Authors: Maréchal Valérie , Prevost Marie-Christine , Petit Caroline , Perret Emmanuelle , Heard Jean-Michel , Schwartz Olivier
Published: July 2002

Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.