bluuebunny/crossref_metadata_2025 huggingface dataset:
Origin of
Cryptococcus neoformans
var.
neoformans
Diploid Strains
Authors: Cogliati Massimo , Esposto Maria C. , Clarke David L. , Wickes Brian L. , Viviani Maria A.
Published: July 2002

The basidiomycetous yeast
Cryptococcus neoformans
is an important human fungal pathogen. Two varieties,
C. neoformans
var.
neoformans
and
C. neoformans
var.
gattii
, have been identified. Both are heterothallic with two mating types,
MATa
and
MATα.
Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene UniversitàMilano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)
4
primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (
MFa
and
MFα
) as probes identified 36 of the 49 diploid isolates as
MATa/α
. The results of pheromone gene sequencing showed that two allelic
MFα
genes exist and are distinct for serotypes A and D. In contrast, the
MFa
gene sequence was conserved in both serotype alleles. Amplification of serotype-specific
STE20
alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the
C. neoformans
population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.
Diagnostic Examination of Human Intestinal Spirochetosis by Fluorescent In Situ Hybridization for
Brachyspira aalborgi
,
Brachyspira pilosicoli
, and Other Species of the Genus
Brachyspira
(
Serpulina
)
Authors: Jensen T. K. , Boye M. , Ahrens P. , Korsager B. , Teglbjærg P. S. , Lindboe C. F. , Møller K.
Published: July 2002

Human intestinal spirochetosis, characterized by end-on attachment of densely packed spirochetes to the epithelial surface of the large intestines as a fringe has been associated with the weakly beta-hemolytic spirochetes
Brachyspira aalborgi
and
Brachyspira
(
Serpulina
)
pilosicoli
. In this study, fluorescent in situ hybridization with oligonucleotide probes targeting 16S or 23S rRNA of
B. aalborgi, B. pilosicoli
, and the genus
Brachyspira
was applied to 40 sections of formalin-fixed, paraffin-embedded intestinal biopsy specimens from 23 Danish and 15 Norwegian patients with histologic evidence of intestinal spirochetosis. Five biopsy specimens from patients without intestinal spirochetosis and three samples from pigs with experimental
B. pilosicoli
colitis were examined as well. In addition, the 16S ribosomal DNAs of two clinical isolates of
B. aalborgi
were sequenced, and a PCR procedure was developed for the identification of
B. aalborgi
in cultures. The genotypic characteristics of the two clinical isolates showed very high (99.5%) similarity with two existing isolates, the type strain of
B. aalborgi
and a Swedish isolate. Hybridization with the
Brachyspira
genus-specific probe revealed a brightly fluorescing fringe of spirochetes on the epithelia of 39 biopsy specimens, whereas 1 biopsy specimen was hybridization negative. The spirochetes in biopsy specimens from 13 Danish and 8 Norwegian patients (55.3%) were identified as
B. aalborgi
. The spirochetes in the biopsy specimens from the other 17 patients hybridized only with the
Brachyspira
probe, possibly demonstrating the involvement of as-yet-uncharacterized
Brachyspira
spirochetes in human intestinal spirochetosis.
Evaluation of a Latex Agglutination Test (MRSA-Screen) for Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci
Authors: Louie L. , Majury A. , Goodfellow J. , Louie M. , Simor A. E.
Published: July 2002

The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84
mecA
-positive strains and 116
mecA
-negative strains) consisting of 108
Staphylococcus epidermidis
, 37
S. saprophyticus
, 15
S. haemolyticus
, 11
S. hominis
, 10
S. capitis
, 10
S. warneri
, and 3
S. lugdunensis
species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 μg/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the
mecA
gene was used as the “gold standard.” The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.
Evaluation of a PCR/DNA Probe Colorimetric Membrane Assay for Identification of
Campylobacter
spp. in Human Stool Specimens
Authors: Collins Evelyn , Glennon Maura , Hanley Shirley , Murray Anne-Marie , Cormican Martin , Smith Terry , Maher Majella
Published: July 2002

DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the
Campylobacter
genus,
Campylobacter jejuni
, and
Campylobacter coli
in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for
C. jejuni
. The assay is rapid and simple and can be applied to stool specimens for the detection of
Campylobacter
.
Rapid PCR–Single-Strand Conformation Polymorphism Method To Differentiate and Estimate Relative Abundance of
Pneumocystis carinii
Special Forms Infecting Rats
Authors: Nahimana Aimable , Cushion Melanie T. , Blanc Dominique S. , Hauser Philippe M.
Published: July 2002

A rapid method that uses PCR–single-strand conformation polymorphism analysis of the intron of the nuclear 26S rRNA gene was shown to differentiate the two
Pneumocystis carinii
special forms that infect rats,
P. carinii
f. sp.
carinii
and
P. carinii
f. sp.
ratti
. The method also provides a means for estimation of the relative abundance of the two special forms in the case of a coinfected rat. The results suggest that the method described will help to further standardize the immunosuppressed rat model of
P. carinii
infection and, thus, contribute to a better understanding of
P. carinii
infection in humans.
Detection of
Treponema denticola
in Atherosclerotic Lesions
Authors: Okuda Katsuji , Ishihara Kazuyuki , Nakagawa Taneaki , Hirayama Akihiko , Inayama Yoshiyuki , Okuda Kenji
Published: July 2002

We examined 26 atherosclerotic lesions and 14 nondiseased aorta specimens to detect the periodontopathogenic part of the bacterial 16S rRNA locus by PCR.
Treponema denticola
sequence of the 16S rRNA locus was found in 6 out of 26 DNA samples (23.1%) from the formalin-fixed, paraffin-embeded atherosclerotic lesions obtained during surgery but not in any of the 14 nondiseased aorta samples from deceased persons. Utilizing immunofluorescence microscopy, we observed aggregated antigenic particles reacting with rabbit antiserum against
T. denticola
in thin sections of the PCR-positive samples, but we could not detect any reacting particles in the PCR-negative thin sections.
High-Throughput Detection of West Nile Virus RNA
Authors: Shi Pei-Yong , Kauffman Elizabeth B. , Ren Ping , Felton Andy , Tai Jennifer H. , Dupuis Alan P. , Jones Susan A. , Ngo Kiet A. , Nicholas David C. , Maffei Joseph , Ebel Gregory D. , Bernard Kristen A. , Kramer Laura D.
Published: July 2002

The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.
Specific Detection and Prevalence of
Helicobacter heilmannii
-Like Organisms in the Human Gastric Mucosa by Fluorescent In Situ Hybridization and Partial 16S Ribosomal DNA Sequencing
Authors: Trebesius K. , Adler K. , Vieth M. , Stolte M. , Haas R.
Published: July 2002

Gastric infection with
Helicobacter heilmannii
(previously known as
Gastrospirillum hominis
) is invariably linked with the presence of chronic gastritis and the risk of developing low-grade mucosa-associated lymphoid tissue lymphoma in humans. In contrast to
Helicobacter pylori
, various
H. heilmannii
species colonize the stomachs of domestic animals, which might be a reservoir for transmission to humans (zoonosis). To identify the number and prevalence of different
H. heilmanni
types in humans, we analyzed 89 gastric biopsy samples histologically identified as
H. heilmannii
positive by fluorescence in situ hybridization. Of these gastric specimens, 84 (94.4%) contained a single
H. heilmannii
type. In five samples, however, two different
H. heilmannii
types were detected. The most prevalent species in monoinfected samples is
H. heilmannii
type 1, found in 78.5% (66 of 84) of the specimens, followed by a novel
H. heilmannii
-like organism (HHLO), HHLO type 4, identified in 9.6% (8 of 84) of tissue sections.
H. heilmannii
type 2 and a further HHLO type not described before, type 3, were found in 8.3% (7 of 84) and 1.2% (1 of 84) of the monoinfected samples, respectively. Additionally, HHLO type 5 with a 16S ribosomal DNA sequence identical to that of
Helicobacter salomonis
was found with a prevalence of 2.4% (2 of 89). Thirteen of these biopsy samples were also investigated by a PCR approach developed for this study that allows a
Helicobacter
-specific amplification of a variable portion of the 16S rRNA gene and subsequent sequencing. In total, five different types of HHLOs could be identified within these samples. We conclude that humans can be infected by at least five different HHLO types, which presumably have their origin in animal species like dogs, cats, and pigs.
Isolation of Dobrava Virus from
Apodemus flavicollis
in Greece
Authors: Papa Anna , Nemirov Kirill , Henttonen Heikki , Niemimaa Jukka , Antoniadis Antonis , Vaheri Antti , Plyusnin Alexander , Vapalahti Olli
Published: July 2002

Dobrava virus (DOBV) carried by
Apodemus flavicollis
is the causative agent of severe hemorrhagic fever with renal syndrome (HFRS). DOBV was isolated from an
A. flavicollis
mouse trapped in northeastern Greece. This is the third DOBV cell culture isolate in the world, clustering together with other Greek DOBV sequences from HFRS patients and rodents.
Misidentification and Diagnostic Delay Caused by a False-Positive Amplified
Mycobacterium tuberculosis
Direct Test in an Immunocompetent Patient with a
Mycobacterium celatum
Infection
Authors: Tjhie Jeroen H. T. , van Belle Arne F. , Dessens-Kroon Mirjam , van Soolingen Dick
Published: July 2002

The Gen-Probe amplified
Mycobacterium tuberculosis
direct test can give discrepant results directly in respiratory or cultured samples from patients infected with
Mycobacterium celatum
, leading to inappropriate therapy for, in our case, an immunocompetent patient.
Correlations between Molecular Subtyping and Serotyping of
Listeria monocytogenes
Authors: Nadon C. A. , Woodward D. L. , Young C. , Rodgers F. G. , Wiedmann M.
Published: July 2002

To define relationships between
Listeria monocytogenes
genetic lineages, ribotypes, and serotypes, 235
L. monocytogenes
isolates were characterized by serotyping and automated
Eco
RI ribotyping. Genetic lineage predicted the following serovar clusters: lineage I, comprising serotypes 1/2b, 3b, 3c, and 4b; lineage II, comprising serotypes 1/2a, 1/2c, and 3a; and lineage III, comprising serotypes 4a and 4c. Some
Eco
RI ribotypes contained multiple serotypes; a subset of these isolates was further differentiated with
Pvu
II ribotyping. Of the 12 resultant
Eco
RI-
Pvu
II combination types, only 4 contained multiple serotypes, demonstrating the potential of ribotyping for serotype prediction.
Rapid Pulsed-Field Gel Electrophoresis Method for Group B Streptococcus Isolates
Authors: Benson Jeffrey A. , Ferrieri Patricia
Published: July 2002

We developed a rapid pulsed-field gel electrophoresis (PFGE) method that required 3 days to complete, an improvement over the standard method that required as many as 8 days. The accuracy and reproducibility of the rapid method were verified by analysis of DNA band sizes of our control group B streptococcus isolate. The rapid method was superior to the standard method, providing more precise molecular sizing and gels of higher image quality. The reproducibility of rapid PFGE substantiated its value and continued use.
Detection of
Chlamydia trachomatis
by Isothermal Ramification Amplification Method: a Feasibility Study
Authors: Zhang Wandi , Cohenford Menashi , Lentrichia Brian , Isenberg Henry D. , Simson Elkin , Li Hengjin , Yi Jizu , Zhang David Y.
Published: July 2002

Chlamydia trachomatis
is the leading cause of sexually transmitted disease in the United States. Effective screening for this agent can facilitate prompt treatment and prevent its sequelae. The recent introduction of liquid-based cytology has made possible the simultaneous screening of cervical intraepithelial lesions and detection of
C. trachomatis
in a single collection vial. In this study we determined whether cytological fluid could support DNA-based amplification for the detection of
C. trachomatis
. Three methods were compared, including ramification amplification (RAM), real-time PCR with molecular beacon, and Abbott’s ligase chain reaction (LCx). RAM is a novel, recently introduced, isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. Our results show that RAM can detect as few as 10
C. trachomatis
elementary bodies in less than 2 h, comparable to results with real-time PCR. Thirty clinical specimens collected in PreservCyt solution were tested by LCx, real-time PCR, and RAM. Among 30 specimens, 15 were positive by PCR and LCx and 14 were positive by RAM. One specimen missed by RAM had an inadequate amount of residual cellular material. Our results show that nucleic acid amplification methods can serve to detect
C. trachomatis
and presumably other sexually transmitted agents in cytological fluid and that the RAM assay can be an alternative to PCR and LCx because of its simplicity and isothermal amplification.
Typing of
Pasteurella multocida
Isolated from Pigs with and without Porcine Dermatitis and Nephropathy Syndrome
Authors: Lainson F. A. , Aitchison K. D. , Donachie W. , Thomson J. R.
Published: July 2002

Porcine dermatitis and nephropathy syndrome (PDNS) is a sporadic, usually fatal disease of growing and finishing pigs that has been recognized in many pig-producing countries.
Pasteurella multocida
strains isolated from 15 pigs with PDNS and 51 pigs without PDNS were characterized by capsule and somatic antigen typing, random amplified polymorphic DNA (RAP-D) typing, and restriction analysis of genomic DNA using pulsed-field gel electrophoresis (PFGE). While capsular, somatic, and RAP-D typing did not discriminate PDNS isolates from non-PDNS isolates, all of the isolates from PDNS cases showed an identical
Apa
I PFGE restriction pattern. This pattern was also found in a high proportion (36%) of
P. multocida
strains isolated from non-PDNS cases. Isolation of a single variant of
P. multocida
from tissues of pigs with PDNS warrants further investigation into the possible role of these bacteria in the etiology of the disease.
Association of
Bacteroides forsythus
and a Novel
Bacteroides
Phylotype with Periodontitis
Authors: Leys Eugene J. , Lyons Sharon R. , Moeschberger Melvin L. , Rumpf Robert W. , Griffen Ann L.
Published: July 2002

Chronic periodontitis is a common infectious disease in the adult population. The etiology is clearly bacterial, and a small number of bacterial species have been consistently associated with periodontitis, including
Bacteroides forsythus
and
Porphyromonas gingivalis
. Comparatively little attention has been paid to the identification of health-associated and potentially beneficial bacterial species that may reside in the gingival sulcus. The purpose of the present study was to examine the relationship of the presence of
B. forsythus
and a newly identified
Bacteroides
phylotype, oral clone BU063, to periodontal health status. The study was accomplished with a set of samples that were collected from subjects with periodontitis and healthy controls. These samples had previously been analyzed for the presence of
P. gingivalis
. An oral sampling strategy that included every tooth and a PCR-based detection method were used to maximize detection sensitivity. The presence of
B. forsythus
in the oral cavity was strongly associated with periodontitis, and its nearest genetic neighbor, oral clone BU063, was associated with oral health (
P
< 0.0001 for both). Colonization with
P. gingivalis
was independent of the presence of either
Bacteroides
species, but the two
Bacteroides
species were found together less often than would be expected by chance (
P
< 0.0001). This suggests the presence of a specific exclusionary mechanism between the two
Bacteroides
species. Comparisons between these two organisms may prove useful for studies that determine how
B
.
forsythus
functions in the disease process. In addition, oral clone BU063 deserves further study as a possible preventive or therapeutic intervention for periodontitis.
Timing of Inoculation of the Pouch Makes No Difference in Increased Detection of
Trichomonas vaginalis
by the InPouch TV Method
Authors: Barenfanger Joan , Drake Cheryl , Hanson Chad
Published: July 2002

The InPouch TV is a method which combines a wet preparation and a culture method to detect
Trichomonas vaginalis.
The top portion of the InPouch TV essentially functions as a slide to be examined under the microscope. If the initial examination is negative, the specimen is pushed down into the bottom pouch, which serves as a broth for cultivation. The issue of timing has not been specifically addressed for optimal processing. To assess the effect of timing on the inoculation of the bottom pouch, we conducted a study designed to determine which procedure had better sensitivity, that of delaying inoculation of the bottom pouch until the initial examination on the top pouch is performed (method A) or that of immediately inoculating the bottom pouch (method B). In addition, we compared the sensitivity of the InPouch TV to that of the traditional wet mount. Fifty of 498 specimens were positive. Methods A and B had identical results: 31 specimens were initially positive regardless of transit time, and incubation yielded another 19 positives. The wet preparation detected 36 positive specimens. The sensitivities of the methods were 100% for the InPouch TV (including examination on receipt and after incubation) and 72% for the traditional wet mount. In conclusion, the InPouch TV method is more sensitive than the traditional method and no detectable differences were observed with timing of the inoculation of the top or bottom pouch.
Use of Phylogenetic Analysis of Hepatitis C Virus (HCV) Hypervariable Region 1 Sequences To Trace an Outbreak of HCV in an Autodialysis Unit
Authors: Halfon Philippe , Roubicek Christa , Gerolami Victoria , Quentin Yves , Khiri Hacene , Pepe Gérad , Berland Yvon
Published: July 2002

Hemodialysis patients are at high risk of infection by hepatitis C virus (HCV). The aim of this study was to investigate an HCV outbreak that occurred in an autodialysis unit by using epidemiological and molecular methods. Seroconversion to HCV antibody (anti-HCV) was observed in two patients over an 18-month period; two other patients had previously been recorded as anti-HCV positive. All four patients involved in the outbreak were tested for HCV RNA, and hepatitis C genotype determination was accomplished by a reverse hybridization assay. Furthermore, part of hypervariable region 1 (HVR1) of the hepatitis C genome was amplified and sequenced in samples from all HCV RNA-positive patients. Phylogenetic analysis of the nucleotide sequences obtained was carried out in order to investigate any possible epidemiological linkages among patients. The nucleotide sequences of the HVR1 regions of both newly infected patients were found to be identical to sequences of samples from previously recorded anti-HCV-positive original patients, suggesting that they were infected by the same isolate. Molecular and epidemiological analysis suggested that nosocomial patient-to-patient transmission was the most likely explanation for the virus spread in the autodialysis unit under study.
Recovery of an Unusual Fusogenic Herpes Simplex Virus Type 2 Strain from a Clinical Specimen
Authors: Johnson F. Brent
Published: July 2002

A highly unusual herpes simplex virus type 2 strain, strain Burr, was isolated from a female genital tract clinical specimen. This virus induced remarkably rapid and extensive syncytium formation in Vero cells involving hundreds of cells but was less fusion active in HEp-2 cells, MRC-5 cells, and mink lung cells. Virus-infected cells produced the glycoproteins gB, gC, gD and gE.
Performance Characteristics of VIDAS and Directigen Respiratory Syncytial Virus (RSV) Antigen Detection Assays and Culture for the Identification of RSV in Respiratory Specimens
Authors: Ribes Julie A. , Seabolt John Pat , Overman Sue B.
Published: July 2002

In a comparison of the Directigen and VIDAS respiratory syncytial virus antigen detection assays with viral culture, the sensitivity, specificity, positive and negative predictive values, and testing efficiency were 86, 93.1, 82.7, 94.6, and 91.2% for Directigen; 96.1, 90.8, 80.3, 98.3, and 92.3% for VIDAS; and 88.2, 100, 100, 95.7, and 96.8% for viral culture, respectively.
Potential Application of Nonstructural Protein NS1 Serotype-Specific Immunoglobulin G Enzyme-Linked Immunosorbent Assay in the Seroepidemiologic Study of Dengue Virus Infection: Correlation of Results with Those of the Plaque Reduction Neutralization Test
Authors: Shu Pei-Yun , Chen Li-Kuang , Chang Shu-Fen , Yueh Yi-Yun , Chow Ling , Chien Li-Jung , Chin Chuan , Yang Hui-Hua , Lin Ting-Hsiang , Huang Jyh-Hsiung
Published: July 2002

An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping.
Detection of West Nile Virus Antigen in Mosquitoes and Avian Tissues by a Monoclonal Antibody-Based Capture Enzyme Immunoassay
Authors: Hunt Ann R. , Hall Roy A. , Kerst Amy J. , Nasci Roger S. , Savage Harry M. , Panella Nicholas A. , Gottfried Kristy L. , Burkhalter Kristen L. , Roehrig John T.
Published: July 2002

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10
2.1
to 10
3.7
PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (
n
= 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10
3
PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.
Antimicrobial Susceptibility of
Neisseria gonorrhoeae
Isolated in Bangladesh (1997 to 1999): Rapid Shift to Fluoroquinolone Resistance
Authors: Rahman Motiur , Sultan Zafar , Monira Shirajum , Alam Ashraful , Nessa Khairun , Islam Sonia , Nahar Shamsun , Shama-A-Waris  , Alam Khan Shahnewaz , Bogaerts Jozef , Islam Nazrul , Albert John
Published: July 2002

Periodic monitoring of antimicrobial susceptibility of
Neisseria gonorrhoeae
is essential for early detection of emergence of drug resistance. A total of 343 gonococcal strains isolated from high-risk and general populations in Bangladesh from 1997 to 1999 were studied. The MICs of penicillin, tetracycline, ciprofloxacin, ceftriaxone, and spectinomycin for the isolates were determined by the agar dilution method. Of the isolates from 1997, 9% were resistant (MIC ≥ 1.0 μg/ml) to ciprofloxacin, while 41 and 49% of the isolates from 1998 and 1999, respectively, were resistant to ciprofloxacin. Of the
N. gonorrhoeae
isolates from 1998 and 1999, 1.2 and 3.6%, respectively, both were penicillinase producing and displayed plasmid-mediated tetracycline resistance.
Analysis of Urinary
Escherichia coli
Isolates for Ability To Produce Shiga Toxin
Authors: Johnson James R. , Jerome Catherine , Boster Daniel R. , Stapleton Ann E. , Tarr Phillip I.
Published: July 2002

The frequency of Shiga toxin (Stx)-producing
Escherichia coli
(STEC) in the urinary tract, which can precipitate the hemolytic-uremic syndrome, is unknown. We tested 597 urinary
E. coli
isolates by Stx immunoassay and found no STEC. The routine screening of urinary
E. coli
for the ability to produce Stx is not warranted.
Detection of Methicillin Resistance in Primary Blood Culture Isolates of Coagulase-Negative Staphylococci by PCR, Slide Agglutination, Disk Diffusion, and a Commercial Method
Authors: Hussain Zafar , Stoakes Luba , John Michael A. , Garrow Shaunalee , Fitzgerald Viivi
Published: July 2002

The methicillin resistance of 363 coagulase-negative staphylococci isolated from blood cultures was determined by a slide latex agglutination (LA) test for penicillin-binding protein 2a (PBP 2a), the presence of the
mecA
gene by PCR, disk diffusion, and Vitek. LA was performed on primary cultures, and PBP 2a expression was induced by placing an oxacillin disk in the primary inoculum. Compared to the PCR results, LA was the most sensitive and specific in the detection of methicillin resistance. Without induction, LA failed to detect 50% of
mecA
-positive strains grown on two different media.
Comparison of Pulsed-Field Gel Electrophoresis and PCR Analysis of Polymorphisms on the
mec
Hypervariable Region for Typing Methicillin-Resistant
Staphylococcus aureus
Authors: Senna J. P. M. , Pinto C. A. , Carvalho L. P. S. , Santos D. S.
Published: July 2002

Two hundred fifty-four methicillin-resistant
Staphylococcus aureus
(MRSA) strains typed by pulsed-field gel electrophoresis (PFGE) were tested by PCR for the
mec
-associated hypervariable region (HVR-PCR) to determine their number of direct repeat units (DRUs). Eight different groups of repeats were found among the MRSA strains and compared to 28 pulsotypes classified by PFGE. Some MRSA strains belonging to the same pulsotype showed different numbers of DRUs. HVR-PCR was rapid, easy to perform, and reproducible and has the ability to obtain an unambiguous positive result for each isolate analyzed. However, this technique shows a discriminatory power inferior to that of PFGE. We conclude that PFGE is a more reliable method of typing MRSA than HVR-PCR.
Infection of Red Deer, Cattle, and Humans with
Mycobacterium bovis
subsp.
caprae
in Western Austria
Authors: Prodinger Wolfgang M. , Eigentler Angelika , Allerberger Franz , Schönbauer Michael , Glawischnig Walter
Published: July 2002

Twelve cases of
Mycobacterium bovis
subsp.
caprae
infection have occurred in four humans, three cattle, and five red deer in western Austria since 1994. DNA-fingerprinting of the isolates suggested transmission in and between these species over several years. Contact with cattle, but not with goats, was found to be associated with three of four human cases.
Versatility of the Accessory C Proteins of Sendai Virus: Contribution to Virus Assembly as an Additional Role
Authors: Hasan Mohammad K. , Kato Atsushi , Muranaka Miki , Yamaguchi Ryoji , Sakai Yuko , Hatano Ikuyoshi , Tashiro Masato , Nagai Yoshiyuki
Published: July 2002

The P/C mRNA of
Sendai virus
(SeV) encodes a nested set of accessory proteins, C′, C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(−) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111–124, 1998). Here, we further characterized the 4C(−) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(−) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-α/β. SeV C proteins thus appear to be quite versatile.
The Long Terminal Repeat of Jaagsiekte Sheep Retrovirus Is Preferentially Active in Differentiated Epithelial Cells of the Lungs
Authors: Palmarini Massimo , Datta Shoibal , Omid Reza , Murgia Claudio , Fan Hung
Published: July 2002

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious bronchioloalveolar carcinoma of sheep known as sheep pulmonary adenomatosis (SPA; ovine pulmonary carcinoma). JSRV is unique among retroviruses because it transforms the alveolar type II cells and the nonciliated bronchiolar cells (Clara cells) of the lungs; these cells are where JSRV is specifically expressed in both naturally and experimentally SPA-affected sheep. In this study, we investigated the cell specificity of JSRV expression. By transient-transfection assays of 23 different cell lines with a reporter plasmid driven by the JSRV long terminal repeat (LTR), pJS21-luc, we found that the JSRV LTR is preferentially active in cell lines derived from type II pneumocytes and Clara cells (MLE-15 and mtCC1-2 mouse cell lines). Reporter assays using progressive 5′ deletions of pJS21-luc allowed us to establish that the JSRV enhancers are able to activate the JSRV proximal promoter in MLE-15 and mtCC1-2 cells, but they have very low activity in mouse cells of other lineages (e.g., NIH 3T3). The JSRV enhancers are able to activate heterologous promoters in both MLE-15 and 3T3 cells, although optimal activity is achieved in MLE-15 cells only with the homologous JSRV promoter. Thus, JSRV cell-specific LTR activity appears to result from an interaction between the enhancer elements and the JSRV proximal promoter elements. By mutation analysis, we established that an upstream NF-κB-like element appears to be responsible for approximately 50% of the JSRV LTR transcriptional activity in MLE-15 cells. Electrophoretic mobility shift assays showed evidence of a factor(s) that binds to this sequence. Antibody supershift experiments indicated that the factor(s) is not related to NF-κB component p50 or p52. This factor also appeared to be present in cells that do not support a high level of JSRV expression. Finally the JSRV21LTR contains putative enhancer binding motifs for transcription factors such as hepatocyte nuclear factor 3 (HNF-3) that are involved in lung-specific gene expression. Cotransfection experiments demonstrated that exogenous HNF-3 is able to enhance the expression of pJS21-luc in NIH 3T3 cells, which normally show minimal enhancer activity for the JSRV LTR.
Binding of Hepatitis C Virus E2 Glycoprotein to CD81 Does Not Correlate with Species Permissiveness to Infection
Authors: Meola Annalisa , Sbardellati Andrea , Bruni Ercole Bruno , Cerretani Mauro , Pezzanera Monica , Ceccacci Alessandra , Vitelli Alessandra , Levy Shoshana , Nicosia Alfredo , Traboni Cinzia , McKeating Jane , Scarselli Elisa
Published: July 2002

Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.
Characterization of an Essential RNA Secondary Structure in the 3′ Untranslated Region of the Murine Coronavirus Genome
Authors: Hsue Bilan , Hartshorne Toinette , Masters Paul S.
Published: July 2002

We have previously identified a functionally essential bulged stem-loop in the 3′ untranslated region of the positive-stranded RNA genome of mouse hepatitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequence, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirmed most of the previously proposed structure, but they revealed that the terminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Further, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and partially overlapping, RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication.
Systemic Polyomavirus Genome Increase and Dissemination of Capsid-Defective Genomes in Mammary Gland Tumor-Bearing Mice
Authors: Wirth Julie J. , Chen Li , Fluck Michele M.
Published: July 2002

BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.
The Conserved Carboxyl-Terminal Half of Herpes Simplex Virus Type 1 Regulatory Protein ICP27 Is Dispensable for Viral Growth in the Presence of Compensatory Mutations
Authors: Bunnell Scott M. , Rice Stephen A.
Published: July 2002

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that regulates viral gene expression by poorly characterized mechanisms. Previous data suggest that its carboxyl (C)-terminal portion is absolutely required for productive viral infection. In this study, we isolated M16R, a second-site revertant of a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion, as demonstrated by the fact that its cloned ICP27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alteration in a homopolymeric run of C residues at codons 215 to 217. This results in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation assays confirmed that the truncated, frameshifted protein can partially substitute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshift mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant,
ex
Cd305, was constructed which possesses the frameshift in the context of an ICP27 gene with the C terminus deleted. We found that both M16exC and
ex
Cd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required for the viability of M16R. Consistent with this interpretation, we isolated two viable derivatives of
ex
Cd305 which grow productively in Vero cells despite being incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the truncated, frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved widely in herpesviruses and previously believed to be absolutely essential, is dispensable for HSV-1 lytic replication in the presence of compensatory genomic mutations.
Targeted Infection of Endothelial Cells by Avian Influenza Virus A/FPV/Rostock/34 (H7N1) in Chicken Embryos
Authors: Feldmann Anke , Schäfer Martin K.-H. , Garten Wolfgang , Klenk Hans-Dieter
Published: July 2002

The tissue tropism and spread of infection of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken embryos. As shown by in situ hybridization, the virus caused generalized infection that was strictly confined to endothelial cells in all organs. Studies with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49 (H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To further analyze the factors determining endotheliotropism, the HA-activating protease furin was cloned from chicken tissue. Ubiquitous expression of furin and other proprotein convertases in the chick embryo indicated that proteolytic activation of HA was not responsible for restriction of infection to the endothelium. To determine the expression of virus receptors in embryonic tissues, histochemical analysis of α2,3- and α2,6-linked neuraminic acid was carried out by lectin-binding assays. These receptors were found on endothelial cells and on several epithelial cells, but not on tissues surrounding endothelia. Finally, we analyzed the polarity of virus maturation in endothelial cells. Studies on cultured human endothelial cells employing confocal laser scanning microscopy revealed that HA is specifically targeted to the apical surface of these cells, and electron microscopy of embryonic tissues showed that virus maturation occurs also at the luminar side. Taken together, these observations indicate that endotheliotropism of FPV in the chicken embryo is determined, on one hand, by the high cleavability of HA, which mediates virus entry into the vascular system, and, on the other hand, by restricted receptor expression and polar budding, which prevent spread of infection into tissues surrounding endothelia.
Sex Differences in Seoul Virus Infection Are Not Related to Adult Sex Steroid Concentrations in Norway Rats
Authors: Klein Sabra L. , Bird Brian H. , Glass Gregory E.
Published: July 2002

Field studies of hantavirus infection in rodents report that a higher percentage of infected individuals are males than females. To determine whether males were more susceptible to hantavirus infection than females, adult male and female Long Evans rats (Rattus norvegicus) were inoculated with doses of Seoul virus ranging from 10−4to 106PFU. The 50% infective doses (ID50) were not significantly different for male and female rats (100.05and 100.8PFU, respectively). To determine whether sex differences in response to infection were related to circulating sex steroid hormones, sex steroid concentrations were manipulated and antibody responses and virus shedding were assessed following inoculation with the ID90. Regardless of hormone treatment, males had higher anti-Seoul virus immunoglobulin G (IgG) and IgG2a (i.e., Th1) responses than females and IgG1 (i.e., Th2) responses similar to those of females. Males also shed virus in saliva and feces longer than females. Manipulation of sex steroids in adulthood did not alter immune responses or virus shedding, suggesting that sex steroids may organize adult responses to hantavirus earlier during ontogeny.
Carboxy Terminus of Human Herpesvirus 8 Latency-Associated Nuclear Antigen Mediates Dimerization, Transcriptional Repression, and Targeting to Nuclear Bodies
Authors: Schwam David R. , Luciano Randy L. , Mahajan Shahana S. , Wong LaiYee , Wilson Angus C.
Published: July 2002

Human herpesvirus 8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus) is the causative agent of Kaposi's sarcoma and certain B-cell lymphomas. In most infected cells, HHV-8 establishes a latent infection characterized by the expression of latency-associated nuclear antigen (LANA) encoded by open reading frame 73. Although unrelated by sequence, there are functional similarities between LANA and the EBNA-1 protein of Epstein-Barr virus. Both accumulate as subnuclear speckles and are required for maintenance of the viral episome. EBNA-1 also regulates viral gene expression and is required for cell immortalization, suggesting that LANA performs similar functions in the context of HHV-8 infection. Here we show that LANA forms stable dimers, or possibly higher-order multimers, and that this is mediated by a conserved region in the C terminus. By expressing a series of truncations, we show that both the N- and C-terminal regions localize to the nucleus, although only the C terminus accumulates as nuclear speckles characteristic of the intact protein. Lastly, we show that LANA can function as a potent transcriptional repressor when tethered to constitutively active promoters via a heterologous DNA-binding domain. Domains in both the N and C termini mediate repression. This suggests that one function of LANA is to suppress the expression of the viral lytic genes or cellular genes involved in the antiviral response.