bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific HLA-DRB and -DQB Alleles and Haplotypes Confer Disease Susceptibility or Resistance in Bahraini Type 1 Diabetes Patients
Authors: Al-Harbi Einas M. , Abbassi Abdul-Jabbar , Tamim Hala , al-Jenaidi Fayza , Kooheji Mariam , Kamal Madeeha , al-Mahroos Salwa , al-Nasir Faisal , Motala Ayesha A. , Almawi Wassim Y.
Published: March 2004

Insofar as genetic susceptibility to type 1 diabetes is associated with HLA class II genes, with certain allelic combinations conferring disease susceptibility or resistance, this study assessed the distributions of HLA-DR and -DQ among 107 unrelated patients with type 1 diabetes and 88 healthy controls from Bahrain, all of Arab origin. The HLA
-
DRB and -DQB genotypes were determined by PCR-sequence-specific priming. The following alleles showed the strongest association with type 1 diabetes among patients versus controls according to their frequencies:
DRB1
*
030101
(0.430 versus 0.097;
P
< 0.001),
DRB1
*
040101
(0.243 versus 0.034;
P
< 0.001),
DQB1
*
0201
(0.467 versus 0.193;
P
< 0.001), and
DQB1
*
0302
(0.229 versus 0.091;
P
< 0.001). When the frequencies of alleles in controls were compared to those in patients, negative associations were seen for
DRB1
*
100101
(0.085 versus 0.014;
P
< 0.001),
DRB1
*
110101
(0.210 versus 0.060;
P
< 0.001),
DQB1
*
030101
(0.170 versus 0.075;
P
= 0.006), and
DQB1
*
050101
(0.335 versus 0.121;
P
< 0.001). In addition, the
DRB1
*
030101
-
DQB1
*
0201
(70.1 versus 22.7%;
P
< 0.001) and
DRB1
*
030101
-
DQB1
*
0302
(21.5 versus 0.0%;
P
< 0.001) genotypes were more prevalent among patients, thereby conferring disease susceptibility, whereas the
DRB1
*
100101
-
DQB1
*
050101
(20.5 versus 2.8%;
P
< 0.001),
DRB1
*
110101
-
DQB1
*
030101
(28.4 versus 8.4%;
P
< 0.001), and
DRB1
*
110101
-
DQB1
*
050101
(30.7 versus 0.9%;
P
< 0.001) genotypes were more prevalent among controls, thus assigning a protective role. These results confirm the association of specific HLA-DR and -DQ alleles and haplotypes with type 1 diabetes and may underline several characteristics that distinguish Bahraini patients from other Caucasians patients.
Innate Immune Responses of Calves during Transient Infection with a Noncytopathic Strain of Bovine Viral Diarrhea Virus
Authors: Müller-Doblies Doris , Arquint Adrian , Schaller Patrick , Heegaard Peter M. H. , Hilbe Monika , Albini Sarah , Abril Carlos , Tobler Kurt , Ehrensperger Felix , Peterhans Ernst , Ackermann Mathias , Metzler Alfred
Published: March 2004

In this study, six immunocompetent calves were experimentally infected with a noncytopathic strain of bovine viral diarrhea virus (BVDV), and the effects of the viral infection on parameters of the innate immune response of the host were analyzed. Clinical and virological data were compared with the temporal activation of the alpha/beta interferon-regulated Mx gene in white blood cells (WBC) and skin as well as the upregulation of the acute-phase serum proteins haptoglobin (Hp) and serum amyloid A (SAA). The viral strain used did provoke transient health impairment, namely, fever and leukopenia that were associated with viremia, viral shedding with nasal secretions, and antiviral seroconversion. Complete recovery was observed within 3 weeks. Elevated levels of SAA and Hp were apparent from days 4 to 13 and 8 to 11, respectively. In WBC, the levels of Mx mRNA and Mx protein were elevated from days 2 to 15. In the context of this study with BVDV, the level of Mx protein expression in WBC provided the most telling diagnostic window to monitor the host's ongoing innate immune response.
Combined Use of Enzyme-Linked Immunosorbent Assay and Flow Cytometry To Detect Antibodies toTrypanosoma cruziin Domestic Canines in Texas
Authors: Shadomy Sean V. , Waring Stephen C. , Chappell Cynthia L.
Published: March 2004

Canines may be sentinels and/or reservoirs for humanTrypanosoma cruziexposures. This study adapted a method originally designed for human diagnostics to detect serum immunoglobulin G toT. cruziin canines. The method combined an enzyme-linked immunosorbent assay (ELISA) for screening and flow cytometry detection of anti-live trypomastigote antibodies (ALTA) for confirmation. The assays were optimized by using known positive and negative control canine sera, and cutoff values were established. The ELISA and ALTA assay easily distinguished between reactive (positive controls) and nonreactive (negative controls) sera and were used to test sera collected in a cross-sectional seroprevalence survey of 356 domestic canines from Harris County, Tex., and the surrounding area. Fifty-three (14.9%) of 356 asymptomatic canines in the survey were positive by ELISA, and 5 (1.4%) were confirmed positive with the ALTA assay, with an additional 4 (1.1%) canines classified as “suspect positive.” Thus, the overall prevalence ofT. cruziantibodies in this population was 2.6%. This is the first U.S. study to use the combination of ELISA and ALTA to detect serum antibodies toT. cruziand the first report of the prevalence ofT. cruziinfection in domestic canines in the Houston, Tex. (Harris County), region. Our results demonstrate that the combination of ELISA and ALTA has been successfully adapted for use in testing canines for serological evidence ofT. cruziinfection. Seroprevalence survey results suggest thatT. cruziantibody-positive domestic canines in the peridomestic setting are present in the Houston, Tex., region and further suggest thatT. cruziis enzootic in the region.
Molecular Cloning and Characterization of Tropomyosin, a Major Allergen of
Chironomus kiiensis
, a Dominant Species of Nonbiting Midges in Korea
Authors: Jeong Kyoung Yong , Yum Hye-Yung , Lee In-Yong , Ree Han-Il , Hong Chein-Soo , Kim Dong Soo , Yong Tai-Soon
Published: March 2004

Chironomids are widely and abundantly distributed in the vicinity of standing waters. Larvae of
Chironomus
and some other genera are known to contain hemoglobins, which have been described as a major allergen, and the adults that have no hemoglobins also have been reported to contain allergens. In this study, we tried to establish the role of chironomid allergy and characterize the allergen of
Chironomus kiiensis
adults. Skin tests using
C. kiiensis
adult extracts were performed on patients with allergic symptoms. A cDNA library of
C. kiiensis
adults was screened with
C. kiiensis
immune mouse sera to identify allergens, and results were confirmed using skin test-positive human sera. Recombinant allergen was expressed in
Escherichia coli
and purified by affinity chromatography using nickel-nitrilotriacetic acid agarose to investigate its allergenic properties. Out of 275 allergic patients 14.2% showed a positive reaction to
C. kiiensis
adult crude extracts in the skin test. The tropomyosin was cloned by immunoscreening and expressed in
Escherichia coli. C. kiiensis
tropomyosin has a high homology at the amino acid level with tropomyosins which were previously known to be allergens in various arthropods (
Periplaneta americana
, 86.3%;
Panulirus stimpson
, 78.9%;
Dermatophagoides pteronyssinus
, 76.5%). Specific immunoglobulin E antibodies reacting to recombinant tropomyosin were detected in 17 (81%) of 21 patients whose skin test results were positive. Cross-reactivity against house dust mites and other insects was noticed with mouse anti-recombinant tropomyosin immune serum.
C. kiiensis
adults were shown to be an important source of inhalant allergens in Korea. Molecular cloning of
C. kiiensis
tropomyosin was performed and IgE reactivity was demonstrated using skin test-positive human sera. Recombinant tropomyosin will be useful for further studies or clinical applications.
D8/17 and CD19 Expression on Lymphocytes of Patients with Acute Rheumatic Fever and Tourette's Disorder
Authors: Weisz Julie L. , McMahon William M. , Moore Jill C. , Augustine Nancy H. , Bohnsack John F. , Bale James F. , Johnson Michael B. , Morgan Jubel F. , Jensen Jenise , Tani Lloyd Y. , Veasy L. George , Hill Harry R.
Published: March 2004

D8/17, an alloantigen found on B lymphocytes, has been reported to be elevated in patients susceptible to rheumatic fever and may be associated with autoimmune types of neuropsychiatric disorders. The pediatric-autoimmune-neuropsychiatric-disorders-associated-with-streptococci model is a putative model of pathogenesis for a group of children whose symptoms of obsessive-compulsive disorder and Tourette's disorder (TD) are abrupt and may be triggered by an infection with group A streptococci. As a test of this model, we have examined D8/17 levels on the B cells of patients with TD and acute rheumatic fever (ARF) along with those on the B cells of normal controls by flow cytometry. We have utilized several different preparations of D8/17 antibody along with a variety of secondary antibodies but have been unable to show an association with an elevated percentage of D8/17-positive, CD19-positive B cells in either ARF or TD. We did find, however, that the percentages of CD19-positive B cells in ARF and TD patients were significantly elevated compared to those in normal controls. Group A streptococcal pharyngitis patients also had an elevated percentage of CD19 B cells, however. These studies failed to confirm the utility of determining the percentage of B cells expressing the D8/17 alloantigen in ARF patients or our sample of TD patients. In contrast, the percentage of CD19-positive B cells was significantly elevated in ARF and TD patients, as well as group A streptococcal pharyngitis patients, suggesting a role for inflammation and/or autoimmunity in the pathogenesis of these disorders.
Effects of Infection with Transmissible Gastroenteritis Virus on Concomitant Immune Responses to Dietary and Injected Antigens
Authors: Bailey Michael , Haverson Karin , Miller Bevis , Jones Philip , Sola Isabel , Enjuanes Luis , Stokes Christopher R.
Published: March 2004

Normal piglets weaned onto soy- or egg-based diets generated antibody responses to fed protein. Concurrent infection with transmissible gastroenteritis virus (TGEV) did not affect the responses to dietary antigens at weaning, nor did it affect the subsequent development of tolerance. However, TGEV infection did enhance the primary immunoglobulin M (IgM) and IgG1, but not IgG2, antibody responses to injected soy in comparison to those of uninfected animals. Paradoxically, TGEV-infected animals showed an enhanced primary IgG1 antibody response to injected soy at 4 weeks of age, but they subsequently showed a reduced secondary response after an intraperitoneal challenge at 9 weeks of age in comparison to uninfected animals. The results suggest that an enteric virus, either used as a vaccine vector or present as a subclinical infection, may not have significant effects on the development of dietary allergies but may have effects both on the primary response and on the subsequent recall response to systemic antigens to which the animal is exposed concurrently with virus antigens.
Prevalence of Neutralizing Antibodies to Adenoviral Serotypes 5 and 35 in the Adult Populations of The Gambia, South Africa, and the United States
Authors: Nwanegbo Edward , Vardas Eftyhia , Gao Wentao , Whittle Hilton , Sun Huijie , Rowe David , Robbins Paul D. , Gambotto Andrea
Published: March 2004

One of the major limitations of the use of adenoviruses as gene therapy vectors is the existence of preformed immunity in various populations. Recent studies have linked failure of adenoviral gene therapy trials to the presence of antiadenoviral neutralizing antibodies (NAb). Understanding the distribution and specificity of such antibodies will assist in the design of successful recombinant adenoviral gene therapies and vaccines. To assess the prevalence of NAb to adenovirus serotypes 5 and 35 (Ad5 and Ad35), we analyzed serum samples from adult immunocompetent individuals living in The Gambia, South Africa, and the United States by using a neutralization assay. Serum samples were incubated with A549 lung carcinoma cells and adenoviruses encoding enhanced green or yellow fluorescent proteins; results were analyzed by fluorescence microscopy and flow cytometry. Using this technique, we found a high prevalence of NAb against Ad5 in Gambian, South African, and U.S. subjects at both low and high titers. Conversely, all subjects displayed a low prevalence of NAb to Ad35; when present, anti-Ad35 NAb were seen at low titers. Because of the ability of adenoviruses to elicit systemic and mucosal immune responses, Ad35 with its low NAb prevalence appears to be an attractive candidate vector for gene therapy applications.
Sustained Lipopolysaccharide-Induced Lung Inflammation in Mice Is Attenuated by Functional Deficiency of the Fas/Fas Ligand System
Authors: Matute-Bello Gustavo , Winn Robert K. , Martin Thomas R. , Liles W. Conrad
Published: March 2004

To determine whether the Fas/Fas ligand (FasL) (CD95/CD178) system contributes to the development of an inflammatory response in vivo, 2.5 μg of bacterial lipopolysaccharide (LPS; endotoxin) per g was administered intranasally to healthy mice (C57BL/6) and mutant mice deficient in either Fas (lprmice) or FasL (gldmice). Sustained LPS-induced neutrophilic inflammation in the lungs was attenuated in bothlprandgldmice. These observations provide further evidence of a proinflammatory role for the Fas/FasL system in the lungs.
Profiles of Antibody Responses against Severe AcuteRespiratory Syndrome Coronavirus Recombinant Proteins andTheir Potential Use as DiagnosticMarkers
Authors: Tan Yee-Joo , Goh Phuay-Yee , Fielding Burtram C. , Shen Shuo , Chou Chih-Fong , Fu Jian-Lin , Leong Hoe Nam , Leo Yee Sin , Ooi Eng Eong , Ling Ai Ee , Lim Seng Gee , Hong Wanjin
Published: March 2004

A
new coronavirus (severe acute respiratory syndrome
coronavirus [SARS-CoV]) has been identified to
be the etiological agent of severe acute respiratory syndrome. Given
the highly contagious and acute nature of the disease, there is an
urgent need for the development of diagnostic assays that can detect
SARS-CoV infection. For determination of which of the viral proteins
encoded by the SARS-CoV genome may be exploited as diagnostic antigens
for serological assays, the viral proteins were expressed individually
in mammalian and/or bacterial cells and tested for reactivity with sera
from SARS-CoV-infected patients by Western blot analysis. A total of 81
sera, including 67 from convalescent patients and seven pairs from two
time points of infection, were analyzed, and all showed
immunoreactivity towards the nucleocapsid protein (N). Sera from some
of the patients also showed immunoreactivity to U274 (59 of 81
[73%]), a protein that is unique to SARS-CoV. In
addition, all of the convalescent-phase sera showed immunoreactivity to
the spike (S) protein when analyzed by an immunofluorescence method
utilizing mammalian cells stably expressing S. However, samples from
the acute phase (2 to 9 days after the onset of illness) did not react
with S, suggesting that antibodies to N may appear earlier than
antibodies to S. Alternatively, this could be due to the difference in
the sensitivities of the two methods. The immunoreactivities to these
recombinant viral proteins are highly specific, as sera from 100
healthy donors did not react with any of them. These results suggest
that recombinant N, S, and U274 proteins may be used as antigens for
the development of serological assays for
SARS-CoV.
Preventive Effects ofEscherichia coliStrain Nissle 1917 on Acute and Chronic Intestinal Inflammation in Two Different Murine Models of Colitis
Authors: Schultz Michael , Strauch Ulrike G. , Linde Hans-Jörg , Watzl Sonja , Obermeier Florian , Göttl Claudia , Dunger Nadja , Grunwald Nicole , Schölmerich Jürgen , Rath Heiko C.
Published: March 2004

Escherichia colistrain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day −2 to day +7. Chronic colitis was induced by transfer of CD4+CD62L+T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-γ], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number ofE. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-γ, 32,477 ± 6,377 versus 9,734 ± 1,717 [P= 0.004]; IL-6, 231 ± 35 versus 121 ± 17 [P= 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 ± 0.2 versus 1.9 ± 0.3 [P= 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and residentE. coliinto MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and residentE. coliinto MLN.
Immunoglobulin M Antibody Responses toMycobacterium ulceransAllow Discrimination between Cases of Active Buruli Ulcer Disease and Matched Family Controls in Areas Where the Disease Is Endemic
Authors: Okenu Daniel M. N. , Ofielu Lazarus O. , Easley Kirk A. , Guarner Jeannette , Spotts Whitney Ellen A. , Raghunathan Pratima L. , Stienstra Ymkje , Asamoa Kwame , van der Werf Tjip S. , van der Graaf Winette T. A. , Tappero Jordan W. , Ashford David A. , King C. Harold
Published: March 2004

Buruli ulcer disease (BUD) is an emerging disease caused byMycobacterium ulcerans. In the present study we have characterized the serological reactivities of sera from volunteer case patients with laboratory-confirmed BUD and controls living in three different regions of Ghana where the disease is endemic to determine if serology may be useful for disease confirmation. Our results showed highly reactive immunoglobulin G (IgG) responses among patients with laboratory-confirmed disease, healthy control family members of the case patients, and sera from patients with tuberculosis from areas where BUD is not endemic. These responses were represented by reactivities to multiple protein bands found in theM. ulceransculture filtrate (CF). In contrast, patient IgM antibody responses to theM. ulceransCF (MUCF) proteins were more distinct than those of healthy family members living in the same village. A total of 84.8% (56 of 66) of the BUD patients exhibited strong IgM antibody responses against MUCF proteins (30, 43 and 70 to 80 kDa), whereas only 4.5% (3 of 66) of the family controls exhibited such responses. The sensitivity of the total IgM response for the patients was 84.8% (95% confidence interval [CI], 74.3 to 91.6%), and the specificity determined with sera from family controls was 95.5% (95% CI, 87.5 to 98.4%). These studies suggest that the IgM responses of patients with BUD will be helpful in the identification and production of theM. ulceransrecombinant antigens required for the development of a sensitive and specific serological assay for the confirmation of active BUD.
High Prevalence of Antibodies to Hepatitis A and E Viruses and Viremia of Hepatitis B, C, and D Viruses among Apparently Healthy Populations in Mongolia
Authors: Takahashi Masaharu , Nishizawa Tsutomu , Gotanda Yuhko , Tsuda Fumio , Komatsu Fumio , Kawabata Terue , Hasegawa Kyoko , Altankhuu Murdorjyn , Chimedregzen Ulziiburen , Narantuya Luvsanbasaryn , Hoshino Hiromi , Hino Kunihiko , Kagawa Yasuo , Okamoto Hiroaki
Published: March 2004

The prevalence of infection with hepatitis A virus (HAV), HBV, HCV, HDV, and HEV was evaluated in 249 apparently healthy individuals, including 122 inhabitants in Ulaanbaatar, the capital city of Mongolia, and 127 age- and sex-matched members of nomadic tribes who lived around the capital city. Overall, hepatitis B surface antigen (HBsAg) was detected in 24 subjects (10%), of whom 22 (92%) had detectable HBV DNA. Surprisingly, HDV RNA was detectable in 20 (83%) of the 24 HBsAg-positive subjects. HCV-associated antibodies were detected in 41 (16%) and HCV RNA was detected in 36 (14%) subjects, none of whom was coinfected with HBV, indicating that HBV/HCV carriers account for one-fourth of this population. Antibodies to HAV and HEV were detected in 249 (100%) and 28 (11%) subjects, respectively. Of 22 HBV DNA-positive subjects, genotype D was detected in 21 subjects and genotype F was detected in 1 subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype I, but these differed by 2.1 to 11.4% from each other in the 522- to 526-nucleotide sequence. Of 36 HCV RNA-positive samples, 35 (97%) were genotype 1b and 1 was genotype 2a. Reflecting an extremely high prevalence of hepatitis virus infections, there were no appreciable differences in the prevalence of hepatitis virus markers between the two studied populations with distinct living place and lifestyle. A nationwide epidemiological survey of hepatitis viruses should be conducted in an effort to prevent de novo infection with hepatitis viruses in Mongolia.
Comparison of Ophthalmic Sponges for Measurements of Immune Markers from Cervical Secretions
Authors: Castle Philip E. , Rodriguez Ana-Cecilia , Bowman Frederick P. , Herrero Rolando , Schiffman Mark , Bratti M. Concepcion , Morera Lidia Ana , Schust Danny , Crowley-Nowick Peggy , Hildesheim Allan
Published: March 2004

Measurements of cervical immunity are important for evaluating immune responses to infections of the cervix and to vaccines for preventing those infections. Three ophthalmic sponges, Weck-Cel, Ultracell, and Merocel, were loaded in vitro with interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, gamma interferon (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), immunoglobulin A (IgA), or IgG, and sponges were extracted and evaluated for total recovery by enzyme-linked immunosorbent assay (ELISA). There was excellent (>75%) recovery for all immune markers from all three devices except for IL-6, which was poorly recovered (<60%) for all sponge types, IFN-γ, which was poorly recovered from both Weck-Cel and Ultracell sponges but was completely recovered from Merocel sponges, and IL-4, which was poorly recovered from Weck-Cel sponges but was completely recovered from Ultracell or Merocel sponges. We then compared the absolute recovery of selected markers (IL-10, IL-12, IgG, and IgA) from cervical secretion specimens collected from women using each type of sponge. There were no significant differences in the recoveries of IL-10, IL-12, and IgG from cervical specimens collected by any type of ophthalmic sponge, but there was reduced IgA recovery from Merocel sponges. However, the variability in these measurements attributable to sponge types (1 to 3%) was much less than was attributable to individuals (45 to 72%), suggesting that differences in sponge type contribute only in a minor way to these measurements. We infer from our data that the three collection devices are adequate for the measurements of IL-1β, IL-2, IL-5, IL-12, IL-15, IL-18, and IgG. Merocel may be a better ophthalmic sponge for the collection of cervical secretions and measurements of IL-4, IL-8, IL-10, GM-CSF, and IFN-γ, but our data from clinical specimens, not in vitro-loaded sponges, suggested the possibility of reduced recovery of IgA. These findings require confirmation.
Neutralization Assay Using a Modified Vaccinia Virus Ankara Vector Expressing the Green Fluorescent Protein Is a High-Throughput Method To Monitor the Humoral Immune Response against Vaccinia Virus
Authors: Cosma Antonio , Bühler Silja , Nagaraj Rashmi , Staib Caroline , Hammarin Anna-Lena , Wahren Britta , Goebel Frank D. , Erfle Volker , Sutter Gerd
Published: March 2004

Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.
Prevalence of
Bartonella clarridgeiae
and
Bartonella henselae
in Domestic Cats from France and Detection of the Organisms in Erythrocytes by Immunofluorescence
Authors: Rolain Jean-Marc , Locatelli Caroline , Chabanne Luc , Davoust Bernard , Raoult Didier
Published: March 2004

The prevalence of
Bartonella
infection in a pet cat population from France was found to be 8.1% (8 of 99 cats). The intraerythrocytic location of
Bartonella clarridgeiae
is shown for the first time, and we show that immunofluorescence detection of the organism in erythrocytes correlates with the number of bacteria in blood.
Performance Characteristics of Updated INNO-LiPA Assays for Molecular Typing of Human Leukocyte Antigen A (HLA-A), HLA-B, and HLA-DQB1 Alleles
Authors: De Vreese Karen , Barylski Rachel , Pughe Fiona , Bläser Miriam , Evans Colin , Norton John , Semana Gilbert , Holman Richard , Loiseau Pascale , Masson Dominique , Gielis Mieke , De Brauwer Annelies , De Canck Ilse , Verpooten Gonda , Hulstaert Frank
Published: March 2004

We carried out a multicenter performance evaluation of three new DNA-based human leukocyte antigen (HLA) typing assays: INNO-LiPA HLA-A Update, INNO-LiPA HLA-B Update, and INNO-LiPA HLA-DQB1 Update. After optimization, the accuracy rates were all 100%, and the final observed resolutions were 99.4, 92.4, and 85.6%, respectively. These rapid and easy-to-perform assays yielded results fully concordant with other DNA-based tissue typing tests.
Comparison of Hepatitis C Viral Loads in Patients with or without Coinfection with Different Genotypes
Authors: Schijman Alejandro , Colina Rodney , Mukomolov Sergey , Kalinina Olga , García Laura , Broor Shobha , Bhupatiraju Ajoy Varma , Karayiannis Peter , Khan Baldip , Mogdasy Cristina , Cristina Juan
Published: March 2004

Hepatitis C virus genotyping was assessed for 257 chronic hepatitis C patients with viral loads above 1,000 IU/ml. Twelve patients were coinfected with more than one genotype. Their median viral loads did not differ significantly from those observed for monoinfected patients, which in turn did not vary significantly among different genotypes.
Dimerization of Recombinant Tobacco Mosaic Virus Movement Protein
Authors: Brill Laurence M. , Dechongkit Songpon , DeLaBarre Byron , Stroebel Jonathon , Beachy Roger N. , Yeager Mark
Published: March 2004

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent
n
-octyl-β-
d
-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant α-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane α-helical coiled coils.
Monitoring RNA Release from Human Rhinovirus by Dynamic Force Microscopy
Authors: Kienberger Ferry , Zhu Rong , Moser Rosita , Blaas Dieter , Hinterdorfer Peter
Published: March 2004

Human rhinoviruses were imaged under physiological conditions by dynamic force microscopy. Topographical images revealed various polygonal areas on the surfaces of the 30-nm viral particles. RNA release was initiated by exposure to a low-pH buffer. The lengths of the RNAs that were released but still connected to the virus capsid varied between 40 and 330 nm, whereas RNA molecules that were completely released from the virus were observed with lengths up to 1 μm. Fork-like structure elements with 30-nm extensions were sometimes resolved at one end of the RNA molecules. They possibly correspond to the characteristic multi-stem-loop conformation, the internal ribosomal entry site, located at the 5′ region of the genome. This study demonstrates that dynamic force microscopy can be used to study viral RNA release in situ under physiological conditions.
Amino Acid 324 in the Simian Immunodeficiency Virus SIVmac V3 Loop Can Confer CD4 Independence and Modulate the Interaction with CCR5 and Alternative Coreceptors
Authors: Pöhlmann Stefan , Davis Carl , Meister Silke , Leslie George J. , Otto Claas , Reeves Jacqueline D. , Puffer Bridget A. , Papkalla Armin , Krumbiegel Mandy , Marzi Andrea , Lorenz Steffen , Münch Jan , Doms Robert W. , Kirchhoff Frank
Published: March 2004

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.
Hierarchical Targeting of Subtype C Human Immunodeficiency Virus Type 1 Proteins by CD8+T Cells: Correlation with Viral Load
Authors: Masemola Agatha , Mashishi Tumelo , Khoury Greg , Mohube Phineas , Mokgotho Pauline , Vardas Efthyia , Colvin Mark , Zijenah Lynn , Katzenstein David , Musonda Rosemary , Allen Susan , Kumwenda Newton , Taha Taha , Gray Glenda , McIntyre James , Karim Salim Abdool , Sheppard Haynes W. , Gray Clive M.
Published: March 2004

An understanding of the relationship between the breadth and magnitude of T-cell epitope responses and viral loads is important for the design of effective vaccines. For this study, we screened a cohort of 46 subtype C human immunodeficiency virus type 1 (HIV-1)-infected individuals for T-cell responses against a panel of peptides corresponding to the complete subtype C genome. We used a gamma interferon ELISPOT assay to explore the hypothesis that patterns of T-cell responses across the expressed HIV-1 genome correlate with viral control. The estimated median time from seroconversion to response for the cohort was 13 months, and the order of cumulative T-cell responses against HIV proteins was as follows: Nef > Gag > Pol > Env > Vif > Rev > Vpr > Tat > Vpu. Nef was the most intensely targeted protein, with 97.5% of the epitopes being clustered within 119 amino acids, constituting almost one-third of the responses across the expressed genome. The second most targeted region was p24, comprising 17% of the responses. There was no correlation between viral load and the breadth of responses, but there was a weak positive correlation (r= 0.297;P= 0.034) between viral load and the total magnitude of responses, implying that the magnitude of T-cell recognition did not contribute to viral control. When hierarchical patterns of recognition were correlated with the viral load, preferential targeting of Gag was significantly (r= 0.445;P= 0.0025) associated with viral control. These data suggest that preferential targeting of Gag epitopes, rather than the breadth or magnitude of the response across the genome, may be an important marker of immune efficacy. These data have significance for the design of vaccines and for interpretation of vaccine-induced responses.
Ancient Coevolution of Baculoviruses and Their Insect Hosts
Authors: Herniou Elisabeth A. , Olszewski Julie A. , O'Reilly David R. , Cory Jenny S.
Published: March 2004

If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the
Baculoviridae
, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes
lef
-
8
and
ac22
. The tree showed a clear division of the baculoviruses according to the order of their hosts. This division highlighted the need to reconsider the classification of the baculoviruses to include one or possibly two new genera. Furthermore, the specialization of distinct virus lineages to particular insect orders suggests ancient coevolutionary interactions between baculoviruses and their hosts.
Inferring the Evolutionary History of
Rice Yellow Mottle Virus
from Genomic, Phylogenetic, and Phylogeographic Studies
Authors: Fargette Denis , Pinel Agnès , Abubakar Zakia , Traoré Oumar , Brugidou Christophe , Fatogoma Sorho , Hébrard Eugénie , Choisy Marc , Séré Yacouba , Fauquet Claude , Konaté Gnissa
Published: March 2004

Fourteen isolates of
Rice yellow mottle virus
(RYMV) were selected as representative of the genetic variability of the virus in Africa from a total set of 320 isolates serologically typed or partially sequenced. The 14 isolates were fully sequenced and analyzed together with two previously reported sequences. RYMV had a genomic organization similar to that of
Cocksfoot mottle sobemovirus
. The average nucleotide diversity among the 16 isolates of RYMV was 7%, and the maximum diversity between any two isolates was 10%. A strong conservative selection was apparent on both synonymous and nonsynonymous substitutions, through the amino acid replacement pattern, on the genome size, and through the limited number of indel events. Furthermore, there was a lack of positive selection on single amino acid sites and no evidence of recombination events. RYMV diversity had a pronounced and characteristic geographic structure. The branching order of the clades correlated with the geographic origin of the isolates along an east-to-west transect across Africa, and there was a marked decrease in nucleotide diversity moving westward across the continent. The insertion-deletion polymorphism was related to virus phylogeny. There was a partial phylogenetic incongruence between the coat protein gene and the rest of the genome. Overall, our results support the hypothesis that RYMV originated in East Africa and then dispersed and differentiated gradually from the east to the west of the continent.
Modulation of Hepatitis B Virus Secretion by Naturally Occurring Mutations in the S Gene
Authors: Khan Nasser , Guarnieri Michael , Ahn Sang Hoon , Li Jisu , Zhou Yonghong , Bang Genie , Kim Kyun-Hwan , Wands Jack R. , Tong Shuping
Published: March 2004

Alteration in hepatitis B virus (HBV) secretion efficiency may have pathological consequences. Naturally occurring mutations that regulate virion secretion have not been defined. We recently identified HBV genomes displaying high (4B), substantially reduced (3.4), or negative (4C) virion secretion. In the present study, the underlying mutations were mapped. A T552C point mutation in the 4B genome was responsible for its enhanced virion secretion, whereas a G510A mutation in 3.4 and G660C in 4C impaired virus secretion. The three point mutations generate M133T, G119E, and R169P substitutions in the S domains of viral envelope proteins, respectively, without modifying the coding capacity of the overlapping polymerase gene. The mutated residues are predicted to lie in the luminal side of the endoplasmic reticulum (ER) or to be embedded in the ER membrane and thus are not involved in contact with core particles during envelopment. Of the two mutations inhibitory of virion secretion, G510A greatly reduced small envelope protein (hepatitis B surface antigen [HBsAg]) levels both inside cells and in culture medium, whereas G660C specifically abolished HBsAg secretion. Surprisingly, a T484G mutation in the 4B genome, generating an I110M substitution in the S domain, could also reduce HBsAg secretion and block virion secretion. However, its inhibitory effect was suppressed in the 4B genome by the T552C mutation, the enhancer of virion secretion. T552C can also override the inhibitory G510A mutation, but not the G660C mutation. These findings suggest a hierarchy in the regulation of virion secretion and a close link between defective virion secretion and impaired HBsAg formation or secretion.
N-Linked Glycosylation of the V3 Loop and the Immunologically Silent Face of gp120 Protects Human Immunodeficiency Virus Type 1 SF162 from Neutralization by Anti-gp120 and Anti-gp41 Antibodies
Authors: McCaffrey Ruth A. , Saunders Cheryl , Hensel Mike , Stamatatos Leonidas
Published: March 2004

We examined how asparagine-linked glycans within and adjacent to the V3 loop (C2 and C3 regions) and within the immunologically silent face (V4, C4, and V5 regions) of the human immunodeficiency virus (HIV) SF612 envelope affect the viral phenotype. Five of seven potential glycosylation sites are utilized when the virus is grown in human peripheral blood mononuclear cells, with the nonutilized sites lying within the V4 loop. Elimination of glycans within and adjacent to the V3 loop renders SF162 more susceptible to neutralization by polyclonal HIV+-positive and simian/human immunodeficiency virus-positive sera and by monoclonal antibodies (MAbs) recognizing the V3 loop, the CD4- and CCR5-binding sites, and the extracellular region of gp41. Importantly, our studies also indicate that glycans located within the immunologically silent face of gp120, specifically the C4 and V5 regions, also conferred on SF162 resistance to neutralization by anti-V3 loop, anti-CD4 binding site, and anti-gp41 MAbs but not by antibodies targeting the coreceptor binding site. We also observed that the amino acid composition of the V4 region contributes to the neutralization phenotype of SF162 by anti-V3 loop and anti-CD4 binding site MAbs. Collectively, our data support the proposal that the glycosylation and structure of the immunologically silent face of the HIV envelope plays an important role in defining the neutralization phenotype of HIV type 1.
The Last C-Terminal Residue of VP3, Glutamic Acid 257, Controls Capsid Assembly of Infectious Bursal Disease Virus
Authors: Chevalier Christophe , Lepault Jean , Da Costa Bruno , Delmas Bernard
Published: March 2004

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH
2
-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.
Multistep Regulation of Membrane Insertion of the Fusion Peptide of Semliki Forest Virus
Authors: Gibbons Don L. , Ahn Anna , Liao Maofu , Hammar Lena , Cheng R. Holland , Kielian Margaret
Published: March 2004

A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1
*
) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1
*
interaction with target membranes and trimerization. E1
*
was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.
Isolation of Human Monoclonal Antibodies That Neutralize Human Rotavirus
Authors: Higo-Moriguchi Kyoko , Akahori Yasushi , Iba Yoshitaka , Kurosawa Yoshikazu , Taniguchi Koki
Published: March 2004

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones—1-2H, 2-3E, and 2-11G—were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 μg/ml.
Qualitative T-Helper Responses to Multiple Viral Antigens Correlate with Vaccine-Induced Immunity to Simian/Human Immunodeficiency Virus Infection
Authors: Mooij Petra , Nieuwenhuis Ivonne G. , Knoop Christiaan J. , Doms Robert W. , Bogers Willy M. J. M. , ten Haaft Peter J. F. , Niphuis Henk , Koornstra Wim , Bieler Kurt , Köstler Josef , Morein Brør , Cafaro Aurelio , Ensoli Barbara , Wagner Ralf , Heeney Jonathan L.
Published: March 2004

Evidence is accumulating that CD4+T-helper (Th) responses play a critical role in facilitating effector responses which are capable of controlling and even preventing human immunodeficiency virus (HIV) infection. The present work was undertaken to determine whether immunization with multiple antigens influenced individual Th responses and increased protection relative to a single antigen. Rhesus macaques were primed with DNA and boosted (immune-stimulating complex-formulated protein) with a combination of regulatory and structural antigens (Tat-Env-Gag) or with Tat alone. Immunization with combined antigens reduced the magnitude of the responses to Tat compared to the single-antigen immunization. Interestingly, the Th immune responses to the individual antigens were noticeably different. To determine whether the qualitative differences in vaccine-induced Th responses correlated with vaccine efficacy, animals were challenged intravenously with simian/human immunodeficiency virus (strain SHIV89.6p) 2 months following the final immunization. Animals that developed combined Th1- and Th2-like responses to Gag and Th2 dominant Env-specific responses were protected from disease progression. Interestingly, one animal that was completely protected from infection had the strongest IFN-γ and interleukin-2 (IL-2) responses prior to challenge, in addition to very strong IL-4 responses to Gag and Env. In contrast, animals with only a marked vaccine-induced Tat-specific Th2 response (no IFN-γ) were not protected from infection or disease. These data support the rationale that effective HIV vaccine-induced immunity requires a combination of potent Th1- and Th2-like responses best directed to multiple antigens.