bluuebunny/crossref_metadata_2025 huggingface dataset:
Virus–host interactions in environments with a wide range of ionic strengths
Authors: Kukkaro Petra , Bamford Dennis H.
Published: February 2009

Although high‐salt environments are rich in viruses, virus–host interactions have not been much explored. Here we study the virus–host interactions occurring in a variety of salt environments, up to saturated salinity. We chose viruses from different environments with diverse morphologies that included both bacterial and archaeal viruses. To extend the test virus set five new haloviruses were isolated and initially characterized in this study. We observed adsorption rates that varied over four orders of magnitude among the virus–host cell systems used in this study. Changes in ionic strength affected the adsorption of these viruses to their host cells in a variety of ways. All the studied viruses, regardless from which environment they were isolated, were more resistant to variations in ionic strength conditions than their host cells. Our study provides a glimpse of the early events in virus life cycles for a number of viruses from different environments. We also gained information on viral responses to changing environments, a valuable piece of information in extending our understanding of the viruses in the environment.
Online program ‘vipcal’ for calculating lytic viral production and lysogenic cells based on a viral reduction approach
Authors: Luef Birgit , Luef Franz , Peduzzi Peter
Published: February 2009

Assessing viral production (VP) requires robust methodological settings combined with precise mathematical calculations. This contribution improves and standardizes mathematical calculations of VP and the assessment of the proportion of lysogenic cells in a sample. We present an online tool ‘Viral Production Calculator’ (vipcal, http://www.univie.ac.at/nuhag‐php/vipcal) that calculates lytic production and the percentage of lysogenic cells based on data obtained from a viral reduction approach (VRA). The main advantage of our method lies in its universal applicability, even to different piecewise‐linear curves. We demonstrate the application of our tool for calculating lytic VP and the proportion of lysogenic bacteria in an environmental sample. The program can also be used to calculate different parameters for estimating virus‐induced mortality, including the percentage of lytically infected cells, lysis rate of bacteria, percentage of bacterial production lysed, proportion of bacterial loss per day, viral turnover time as well as dissolved organic carbon and nitrogen release. vipcal helps avoid differences in the calculation of VP and diverse viral parameters between studies and laboratories, which facilities interpretation of results. This tool represents a methodological step forward that can help improve our understanding of the role of viral activity in aquatic systems.
New phylotypes of mesophilic filamentous anoxygenic phototrophic bacteria enriched from sulfide‐containing environments
Authors: Bañeras L. , Gich F. , Martinez‐Medina M. , Miller M. , Abella C. A. , Borrego C. M.
Published: February 2009

Agar‐based solid media with increasing concentrations of organic matter were used to isolate new members of the Chloroflexaceae (phylum Chloroflexi) from mesophilic environments containing sulfide. Inorganic media yielded less than 10% positive enrichments, which were not able to be maintained after repetitive inoculations in fresh medium. The use of casaminoacids and complex organic acid mixtures increased the number of positive enrichments (up to 45%) from both water and sediment samples. Two different green filamentous bacteria, SisoF2 and SalF, could be stably maintained as co‐cultures for long periods and their phylogeny inferred from the analysis of complete sequences of the 16S rRNA gene. Ribotype SalF showed a high homology (95–98%) to previously isolated Oscillochloris trichoides strains. The 16S rRNA gene sequence retrieved from culture SisoF2 was largely divergent (< 92% similarity) from any sequence derived from either cultured representatives or environmental samples, suggesting that ribotype SisoF2 may constitute a new genus within the phylum. The presence of the new morphotypes in the environment from where they were enriched was analysed by high‐resolution phylogenetic fingerprinting.
Evaluation of the Combined Application of Ethanol-Fixed and Formaldehyde-Fixed Neutrophil Substrates for Identifying Atypical Perinuclear Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease
Authors: Papp Maria , Altorjay Istvan , Lakos Gabriella , Tumpek Judit , Sipka Sandor , Dinya Tamas , Palatka Karoly , Veres Gabor , Udvardy Miklos , Lakatos Peter Laszlo
Published: February 2009

No clear guidelines for indirect immunofluorescence (IIF) detection and interpretation of antineutrophil cytoplasmic antibodies (ANCA) have been proposed for inflammatory bowel diseases (IBD). We evaluated the reliability of the combined use of ethanol- and formalin-fixed neutrophil substrates to identify atypical perinuclear ANCA (P-ANCA) by IIF under routine laboratory circumstances. A total of 204 IBD patients were assessed with four different fluorescent substrates in two distinct laboratories. Antibodies against myeloperoxidase, proteinase-3, and other specific granule proteins (elastase, lactoferrin, cathepsin G, lysozyme, and bactericidal permeability-increasing protein) were measured by an enzyme-linked immunosorbent assay. The combined application of ethanol- and formalin-fixed slides to detect atypical P-ANCA resulted in a lack of agreement between assays (κ, ≤0.39) in the interassay study and moderate agreement in the interobserver study (κ, 0.42). After atypical and typical P-ANCA patterns were combined, the consensus improved greatly. A total of 26.9% of patients were P-ANCA positive by at least two tests (44.3% of ulcerative colitis [UC] and 13.1% of Crohn's disease [CD] patients;
P
< 0.0001), while overall ANCA positivity was 22.5% to 34.8%. The combined application of ethanol-fixed and formaldehyde-fixed neutrophil substrates did not facilitate differentiation between P-ANCA and atypical P-ANCA, and the results were not consistent when substrates from different sources were used. Combining all P-ANCA ensures the highest sensitivity and specificity in differentiating UC from CD.
Whole-Cell Pertussis Vaccine Induces Low Antibody Levels in Human Immunodeficiency Virus-Infected Children Living in Sub-Saharan Africa
Authors: Tejiokem Mathurin C. , Njamkepo Elisabeth , Gouandjika Ionela , Rousset Dominique , Béniguel Lydie , Bilong Catherine , Tene Gilbert , Penda Ida , Ngongueu Carine , Gody Jean C. , Guiso Nicole , Baril Laurence
Published: February 2009

The WHO recommendations for the immunization of children infected with human immunodeficiency virus (HIV) differ slightly from the guidelines for uninfected children. The introduction of antiretroviral therapy for HIV-infected infants should considerably prolong their life expectancy. The question of the response to the whole-cell pertussis (wP) vaccine should now be addressed, particularly in countries in which pertussis remains endemic. To evaluate the persistence of antibodies to the wP vaccine in HIV-infected and uninfected children who had previously received this vaccine in routine clinical practice, we conducted a cross-sectional study of children aged 18 to 36 months, born to HIV-infected mothers and living in Cameroon or the Central African Republic. We tested blood samples for antibodies to the wP vaccine and for antibodies to diphtheria and tetanus toxoids (D and T, respectively) in the context of the use of a combined DTwP vaccine. We enrolled 50 HIV-infected children and 78 uninfected, HIV-exposed children in the study. A lower proportion of HIV-infected children than uninfected children had antibodies against the antigens tested for all valences of the DTwP vaccine. Agglutinin levels were substantially lower in HIV-infected than in HIV-exposed but uninfected children (30.0% versus 55.1%, respectively;
P
= 0.005). We also observed a high risk of low antibody levels in response to the DTwP vaccine in HIV-infected children with severe immunodeficiency (CD4 T-cell level, <25%). The concentrations of antibodies induced by the DTwP vaccine were lower in HIV-infected children than in uninfected children. This study supports the need for a booster dose of the DTwP vaccine in order to maintain high antibody levels in HIV-infected children.
Detection of Antibodies Secreted from CirculatingMycobacterium tuberculosis-Specific Plasma Cells in the Diagnosis of Pediatric Tuberculosis
Authors: Raqib Rubhana , Mondal Dinesh , Karim M. Anwarul , Chowdhury Fahima , Ahmed Sultan , Luby Stephen , Cravioto Alejandro , Andersson Jan , Sack David
Published: February 2009

Diagnosis of tuberculosis (TB) in children is difficult because symptoms are often nonspecific or absent in infected children, diagnostic specimens are difficult to obtain from younger children, and >50% have negative TB cultures. Thus, there is an urgent need for improved diagnosis of pediatric TB. This study aimed to evaluate the diagnostic value of a new serological method, the ALS (antibodies in lymphocyte supernatant) assay, for the diagnosis of active TB in children with clinically identified TB. The ALS test is based on the concept that antigen-specific plasma cells are present in the circulation only at times of acute infection and not in latency. A cross-sectional study of pediatric patients (age range, 11 to 167 months) who were clinically identified as TB (n= 58) or non-TB (n= 16) patients was conducted, and they were monitored for 6 months. Healthy children (n= 58) were enrolled as controls. Spontaneous release of TB antigen-specific antibodies by in vitro-cultured, unstimulated peripheral blood mononuclear cells was assessed by an enzyme-linked immunosorbent assay usingMycobacterium bovisbacillus Calmette-Guérin (BCG) as the detecting antigen. Of the patients clinically diagnosed with TB, 15% had culture-confirmed TB, 64% were positive for TB by clinically established scoring charts (K. Edwards, P. N. G. Med. J. 30: 169-178, 1987; G. Stegen, K. Jones, and P. Kaplan, Pediatrics 43: 260-263, 1969; and stop TB Partnership, Childhood TB subgroup, World Health Organization, Int. J. Tuberc. Lung Dis. 10: 1091-1097, 2006), and 91% were TB positive by the ALS method. All TB patients had significantly higher BCG-specific ALS titers at enrollment (optical density [OD], 1.06 ± 0.32) than healthy-control children (OD, 0.18 ± 0.06) and non-TB children (OD, 0.21 ± 0.10) (P= 0.001). The ALS titers declined in children with active disease from enrollment through 6 months following anti-TB therapy (P= 0.001). The ALS assay is a novel diagnostic method with potential applications in the diagnosis of pediatric TB and in subsequent monitoring of treatment effectiveness.
Novel Method for Determination of Substance P Levels in Unextracted Human Plasma by Using Acidification
Authors: Campbell Donald E. , Bruckner Paula , Tustin Nancy B. , Tustin Richard , Douglas Steven D.
Published: February 2009

Substance P (SP) is a member of the tachykinin family and has an important role in immune responses. SP is detectable in plasma in a free and bound state. Simple modification of a commercially available SP enzyme-linked immunosorbent assay allows the dissociation and capture of plasma SP without solid-phase extraction.
Application of the Dengue Virus NS1 Antigen Rapid Test for On-Site Detection of Imported Dengue Cases at Airports
Authors: Shu Pei-Yun , Yang Cheng-Fen , Kao Jeng-Fong , Su Chien-Ling , Chang Shu-Fen , Lin Chien-Chou , Yang Wen-Chih , Shih Hsiu , Yang Shih-Yan , Wu Ping-Fuai , Wu Ho-Sheng , Huang Jyh-Hsiung
Published: February 2009

We used the dengue virus NS1 antigen (Ag) rapid test for on-site detection of imported dengue cases at airports. Among 22 positive cases of dengue identified from 850 patients with a fever suspected to have dengue, 17 were NS1 Ag test positive. These findings demonstrate the usefulness of the NS1 Ag rapid test in screening imported dengue cases at airports.
Use of Spoligotyping and Large Sequence Polymorphisms To Study the Population Structure of the
Mycobacterium tuberculosis
Complex in a Cohort Study of Consecutive Smear-Positive Tuberculosis Cases in The Gambia
Authors: de Jong Bouke C. , Antonio Martin , Awine Timothy , Ogungbemi Kunle , de Jong Ype P. , Gagneux Sebastien , DeRiemer Kathryn , Zozio Thierry , Rastogi Nalin , Borgdorff Martien , Hill Philip C. , Adegbola Richard A.
Published: February 2009

Mycobacterium africanum
, first described in Senegal in 1968, causes up to half of the smear-positive pulmonary tuberculosis cases in West Africa, but it has not been found in other geographical areas except among recent West African migrants. The reasons for the geographic restriction of
M. africanum
are unknown. We used molecular tools to determine the population structure of the
Mycobacterium tuberculosis
complex in a cohort study of consecutive smear-positive tuberculosis cases in The Gambia. We collected and genotyped 386 clinical isolates using spoligotype analysis and PCRs for large sequence polymorphisms (LSPs) and compared the genotype patterns to the patterns in an international database. The results of spoligotyping and LSP analysis for the study population were also compared to determine the correlation between them. The main lineages within the
Mycobacterium tuberculosis
complex identified in The Gambia included
M. africanum
type I (38.4%), characterized by an LSP in region of difference 702 (RD702; West African type 2). Among the
M. tuberculosis
sensu stricto isolates, lineages characterized by RD182 and by RD174 were the most common. We also detected a gradient in the prevalence of
M. africanum
that extended from neighboring Guinea-Bissau. The genotypic diversity of the spoligotype patterns was greater among the isolates of
M. africanum
than among the isolates of
M. tuberculosis
. We postulate that
M. africanum
became endemic in West Africa first, before the introduction of different lineages within
M. tuberculosis
sensu stricto.
Predominance and Circulation of Enteric Viruses in the Region of Greater Cairo, Egypt
Authors: Kamel Aziza H. , Ali Mohamed A. , El-Nady Hala G. , de Rougemont Alexis , Pothier Pierre , Belliot Gaël
Published: February 2009

The circulation of enteric viruses among the population of Cairo, Egypt, between March 2006 and February 2007 was studied. At least one virus was detected in 50% of fecal samples, 57.4% of which were positive for rotavirus, 26% for norovirus, 10.4% for adenovirus, and 1.7% for astrovirus. Over 10% of infections were mixed infections. Rotavirus typing showed that G1P[8] and G2P[4] were predominant but that the unusual G12P[4] and G12P[6] reassortants were also present. Among the noroviruses, half belonged to the predominant GGII.4 cluster. The phylogenetic analysis of the capsid gene suggested that GGII.4 strains from Cairo were similar to those circulating elsewhere. It also showed the emergence of new GGII.4 variants that were not associated with any previously known GGII.4 isolate. Further studies are required to assess the disease burden of enteric viruses in Egypt and the impact of atypical strains.
Nucleic Acid Amplification Tests for Diagnosis of
Neisseria gonorrhoeae
Oropharyngeal Infections
Authors: Bachmann Laura H. , Johnson Robert E. , Cheng Hong , Markowitz Lauri E. , Papp John R. , Hook Edward W.
Published: February 2009

The optimal methods for the diagnosis of pharyngeal
Neisseria gonorrhoeae
infection are uncertain. The objective of this study was to define the performance of culture and nucleic acid amplification tests (NAATs) for the diagnosis of pharyngeal
N. gonorrhoeae
. In this cross-sectional study, males and females >15 years old who acknowledged performing fellatio or cunnilingus (in the previous 2 months) were recruited from three clinics (two human immunodeficiency virus clinics and one sexually transmitted diseases clinic) located in Birmingham, AL. The test performance of culture for
N. gonorrhoeae
, the Gen-Probe Aptima Combo 2 transcription-mediated amplification assay (TMA), the BD ProbeTec ET amplified DNA strand displacement assay (SDA), and the Roche Cobas Amplicor PCR was defined by using a rotating “gold standard” of any positive results by two or three of the three tests that excluded the test being evaluated. A total of 961 evaluable test sets were collected. On the basis of a rotating gold standard of positive results by two of three comparator tests, the sensitivity and the specificity were as follows: culture for
N. gonorrhoeae
, 50.0% and 99.4%, respectively; PCR, 80.3% and 73.0%, respectively; TMA, 83.6% and 98.6%, respectively; and SDA, 93.2% and 96.3%, respectively. On the basis of a rotating gold standard of positive results by three of three comparator tests, the sensitivity and specificity were as follows: culture for
N. gonorrhoeae
, 65.4% and 99.0%, respectively; PCR, 91.9% and 71.8%, respectively; TMA, 100% and 96.2%, respectively; and SDA, 97.1% and 94.2%, respectively. In conclusion, currently available NAATs are more sensitive than culture for the detection of pharyngeal gonorrhea in at-risk patients. PCR is substantially less specific than culture, TMA, or SDA and should not be used for the detection of pharyngeal gonorrhea.
Human Bocavirus in an Immunocompromised Child Presenting with Severe Diarrhea
Authors: de Vries Jutte J. C. , Bredius Robbert G. M. , van Rheenen Patrick F. , Smiers Frans J. W. , Schölvinck Elisabeth H. , Vossen Ann C. T. M. , Claas Eric C. J. , Niesters Hubert G. M.
Published: February 2009

Human bocavirus (HBoV) is frequently detected in young children with respiratory symptoms. However, the prevalence and pathogenicity of HBoV in immunocompromised patients are largely unknown. This report describes a case of life-threatening hypovolemic shock due to diarrhea associated with disseminated HBoV infection in an immunocompromised child.
Eight-Plex PCR and Liquid-Array Detection of Bacterial and Viral Pathogens in Cerebrospinal Fluid from Patients with Suspected Meningitis
Authors: Bøving Mette Kusk , Pedersen Lisbeth Nørum , Møller Jens Kjølseth
Published: February 2009

We here report on the development of a novel multiplex PCR with product detection in a Luminex 100 suspension array system. The assay covers the nine most important bacterial and viral pathogens found in Danish meningitis patients. The microorganisms include
Neisseria meningitidis
,
Streptococcus pneumoniae
,
Escherichia coli
,
Staphylococcus aureus
,
Listeria monocytogenes
,
Streptococcus agalactiae
, herpes simplex virus types 1 and 2, and varicella-zoster virus. The study was based on 1,187 samples, of which 55 were found to be positive by PCR. The assay was found to have an excellent sensitivity and an excellent specificity compared to the results of a “gold standard,” defined by routine laboratory tests, for the two most important pathogens,
S. pneumoniae
(95 and 99.1%, respectively) and
N. meningitidis
(100 and 99.7%, respectively). The method provides a valuable supplement to the traditional microscopy and culture of cerebrospinal fluid (CSF) samples in a routine diagnostic setting, and results can be available within 1 workday. The method is suitable for use for the initial screening and identification of nine important microorganisms in CSF samples from patients with suspected meningitis. Compared to microscopy and culture of CSF, this rapid and sensitive method will support physicians with the selection of the appropriate antimicrobial agents and the initiation of timely treatment in the absence of live microorganisms in the CSF.
Correlation between Human Immunodeficiency Virus Type 1 (HIV-1) RNA Measurements Obtained with Dried Blood Spots and Those Obtained with Plasma by Use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV Load Tests
Authors: Garrido Carolina , Zahonero Natalia , Corral Angélica , Arredondo Miguel , Soriano Vincent , de Mendoza Carmen
Published: February 2009

The plasma human immunodeficiency virus (HIV) RNA load is used in the clinical routine for the monitoring of HIV infection and the patient's response to antiretroviral therapy. Other body fluids or dried blood spots (DBS) can be used, however, to assess the level of viremia. The use of DBS may be especially helpful for the monitoring of HIV-infected patients in resource-poor settings, where access to adequate laboratory facilities is often difficult. However, the correlation between the HIV RNA levels in plasma and those in DBSs has not been well established. Paired plasma and DBS samples obtained from HIV type 1 (HIV-1)-infected patients were tested for HIV RNA copy numbers by using two different commercial assays, the Nuclisens EasyQ HIV-1 (version 1.1) test (the Nuclisens test; Biomerieux) and the m2000rt RealTime HIV test (the m2000rt test; Abbott). Nucleic acid extraction was performed manually by using either the Nuclisens isolation kit (which uses the Boom methodology) or the m2000rt sample preparation kit (an iron particle-based method). A total of 103 paired plasma and DBS samples were tested. Viral load results were obtained for 97 (94.2%) samples with the Nuclisens isolation kit and 81 (78.6%) samples with the m2000rt kit. The overall correlation between the RNA loads in plasma and DBS was good, although better results were obtained by the Nuclisens test (R2= 0.87,P< 0.001) than by the m2000rt test (R2= 0.70,P< 0.001). While the specificities were excellent and similar for both the Nuclisens and the m2000rt tests (97.1% and 100%, respectively), the sensitivity was greater by the Nuclisens test than by the m2000rt test (75.8% and 56.6%, respectively). Overall, the viral loads in DBS tended to be lower than those in plasma, with mean differences of 0.3 log unit (standard deviation, 0.5 log unit) and 0.76 log unit (standard deviation, 0.8 log unit) for the Nuclisens and the m2000rt tests, respectively. The levels of agreement between the measurements in plasma and DBS were assessed by using the Bland-Altman plot for each assay. The Nuclisens test gave results within its defined limits (−0.65 to 1.26) for 95.9% of the samples, while the m2000rt test gave results within its limits (−0.83 to 2.33) for 100% of the samples. In summary, the HIV-1 load can accurately be quantified by testing DBS by either the Nuclisens or the m2000rt test, although the Nuclisens test may outperform the m2000rt test when nucleic acids are extracted manually.
Single-Point Mutations Causing More than 100-Fold Underestimation of Human Immunodeficiency Virus Type 1 (HIV-1) Load with the Cobas TaqMan HIV-1 Real-Time PCR Assay
Authors: Korn Klaus , Weissbrich Benedikt , Henke-Gendo Cornelia , Heim Albert , Jauer Christin Mariel , Taylor Ninon , Eberle Josef
Published: February 2009

Systematic sequence analysis of human immunodeficiency virus type 1 (HIV-1) variants with RNA levels underestimated by the Cobas TaqMan HIV-1 assay demonstrated that mutations at a single position of the downstream primer can lead to the underestimation of HIV-1 RNA levels by more than 2 log and to false-negative results in minipool screening of blood donors. Mutations at this position are found in about 2% of all HIV-1 M
gag
sequences.
Comparison of the Abbott RealTime Human Immunodeficiency Virus Type 1 (HIV-1) Assay to the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test: Workflow, Reliability, and Direct Costs
Authors: Sloma Cari R. , Germer Jeffrey J. , Gerads Tara M. , Mandrekar Jayawant N. , Mitchell P. Shawn , Yao Joseph D. C.
Published: February 2009

The Abbott RealTime human immunodeficiency virus type 1 (HIV-1) assay (ART) and the Cobas AmpliPrep/Cobas TaqMan HIV-1 test (CTM) are commercially available assays for quantification of HIV-1 RNA in plasma. We evaluated performance characteristics, workflow, throughput, reliability, and direct costs of these assays. Both assays yielded good correlation of quantitative results (
r
= 0.95) among clinical specimens, with a mean difference of −0.34 log
10
copies/ml. Testing of healthy donor plasma specimens yielded “target not detected” results by ART, with “HIV-1 RNA detected, <40 copies/ml” results for 3.3% (3 of 90 samples) of these specimens by CTM. Both the m2000sp/m2000rt (ART) and docked CAP/CTM96 (CTM) instrument systems were capable of operating with continuous, uninterrupted workflow. When daily maintenance and cleaning were included, ART and CTM run durations (5 h 52 min and 6 h 4 min, respectively) and hands-on times (53 min and 46 min, respectively) were similar for a run batch size of 24. While ART was more flexible in terms of run batch size, CTM required fewer user interventions and consistently produced higher specimen throughput rates at 8, 16, and 24 h. Assay run failure rates were 6.3% (1 of 16 runs) and 4.2% (1 of 24 runs) for ART and CTM, respectively (
P
= 1.000), with invalid specimen result rates of 1.0% (5 of 495 specimens) and 2.8% (11 of 399 specimens), respectively (
P
= 0.073). Direct reagent and consumable costs for each assay were comparable (difference of <10%). In selecting an assay for implementation, laboratories should consider how various assay and instrument features might impact laboratory operation and patient care.
Distribution of Genes Encoding MSCRAMMs and Pili in Clinical and Natural Populations of
Enterococcus faecium
Authors: Sillanpää Jouko , Prakash Vittal P. , Nallapareddy Sreedhar R. , Murray Barbara E.
Published: February 2009

Enterococcus faecium
has recently emerged as an important cause of nosocomial infections. We previously identified 15 predicted surface proteins with characteristics of MSCRAMMs and/or pili and demonstrated that their genes were frequently present in 30 clinical
E. faecium
isolates studied; one of these,
acm
, has been studied in further detail. To determine the prevalence of the other 14 genes among various
E. faecium
populations, we have now assessed 433
E. faecium
isolates, including 264 isolates from human clinical infections, 69 isolates from stools of hospitalized patients, 70 isolates from stools of community volunteers, and 30 isolates from animal-related sources. A variable distribution of the 14 genes was detected, with their presence ranging from 51% to 98% of isolates. While 81% of clinical isolates carried 13 or 14 of the 14 genes tested, none of the community group isolates and only 13% of animal isolates carried 13 or 14 genes. The presence of these genes was most frequent in endocarditis isolates, with 11 genes present in all isolates, followed by isolates from other clinical sources. The number of genes significantly associated with clinical versus fecal or animal origin (
P
= 0.04 to <0.0001) varied from 10 to 13, depending on whether comparisons were made against individual clinical subgroups (endocarditis, blood, and other clinical isolates) or against all clinical isolates combined as one group. The strong association of these genes with clinical isolates raises the possibility that their preservation/acquisition has favored the adaptation of
E. faecium
to nosocomial environments and/or patients.
Identification of
Candida nivariensis
and
Candida bracarensis
in a Large Global Collection of
Candida glabrata
Isolates: Comparison to the Literature
Authors: Lockhart Shawn R. , Messer Shawn A. , Gherna Michael , Bishop Justin A. , Merz William G. , Pfaller Michael A. , Diekema Daniel J.
Published: February 2009

We analyzed 1,598
Candida glabrata
isolates for the presence of the cryptic species
Candida nivariensis
and
Candida bracarensis
. Both species were very rare in this collection (0.2% prevalence), despite the number of isolates analyzed and the global distribution of the isolates. We saw no associated antifungal resistance in
C. nivariensis
.
O-Linked
N
-Acetylglucosaminylation of Sp1 Inhibits the Human Immunodeficiency Virus Type 1 Promoter
Authors: Jochmann Ramona , Thurau Mathias , Jung Susan , Hofmann Christian , Naschberger Elisabeth , Kremmer Elisabeth , Harrer Thomas , Miller Matthew , Schaft Niels , Stürzl Michael
Published: February 2009

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5′ long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5′ LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked
N
-acetyl-
d
-glucosamine (
O
-GlcNAc), we evaluated whether increased
O
-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the
O
-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of
O
-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of
O
-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4
+
T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection.
O
-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that
O
-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1
O
-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS.
Human Rhinovirus Type 2 Uncoating at the Plasma Membrane Is Not Affected by a pH Gradient but Is Affected by the Membrane Potential
Authors: Berka Ursula , Khan Abdul , Blaas Dieter , Fuchs Renate
Published: February 2009

The minor receptor group human rhinovirus type 2 enters host cells by endocytosis via members of the low-density-lipoprotein receptor family. In late endosomes, it undergoes a conformational change solely induced by a pH of ≤5.6, resulting in RNA transfer across the endosomal membrane into the cytoplasm. To determine potential driving forces of this process, we investigated whether RNA penetration might depend on the pH gradient and/or the membrane potential between the acidic endosome lumen and the neutral cytoplasm. Since these parameters are difficult to assess in endosomes, we took advantage of the possibility of inducing structural changes, RNA release, and consequently infection from the plasma membrane. To manipulate the pH gradient, cell-bound virus was exposed to membrane-permeant or -impermeant acidic buffers at 4°C, and this was followed by a shift to 34°C in medium containing bafilomycin to prevent RNA release from endosomes. To manipulate the plasma membrane potential, similar experiments were carried out, but these included K
+
diffusion potentials in the presence of the K
+
ionophore valinomycin. We demonstrated that infection does not depend on a pH gradient but is enhanced by plasma membrane hyperpolarization compared to plasma membrane depolarization.
Characterization of a Human Immunodeficiency Virus Type 1 V3 Deletion Mutation That Confers Resistance to CCR5 Inhibitors and the Ability To Use Aplaviroc-Bound Receptor
Authors: Nolan Katrina M. , Del Prete Gregory Q. , Jordan Andrea P. O. , Haggarty Beth , Romano Josephine , Leslie George J. , Hoxie James A.
Published: February 2009

The human immunodeficiency virus type 1 (HIV-1) V3 loop is essential for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. Using the dual-tropic virus HIV-1
R3A
, we previously made an extensive panel of V3 deletions and identified subdomains within V3 that could differentially mediate R5 and X4 tropism. A deletion of residues 9 to 12 on the N-terminal side of the V3 stem ablated X4 tropism while leaving R5 tropism intact. This mutation also resulted in complete resistance to several small-molecule CCR5 inhibitors. Here, we extend these studies to further characterize a variant of this mutant, Δ9-12a, adapted for growth in CCR5
+
SupT1 cells. Studies using coreceptor chimeras, monoclonal antibodies directed against the CCR5 amino terminus (NT) and extracellular loops, and CCR5 point mutants revealed that, relative to parental R3A, R5-tropic Δ9-12a was more dependent on the CCR5 NT, a region that contacts the gp120 bridging sheet and V3 base. Neutralization sensitivity assays showed that, compared to parental R3A, Δ9-12a was more sensitive to monoclonal antibodies b12, 4E10, and 2G12. Finally, cross-antagonism assays showed that Δ9-12a could use aplaviroc-bound CCR5 for entry. These studies indicate that increased dependence on the CCR5 NT represents a mechanism by which HIV envelopes acquire resistance to CCR5 antagonists and may have more general implications for mechanisms of drug resistance that arise in vivo. In addition, envelopes such as Δ9-12a may be useful for developing new entry inhibitors that target the interaction of gp120 and the CCR5 NT.
Lambda Interferon Inhibits Human Immunodeficiency Virus Type 1 Infection of Macrophages
Authors: Hou Wei , Wang Xu , Ye Li , Zhou Lin , Yang Zhan-Qiu , Riedel Eric , Ho Wen-Zhe
Published: February 2009

The newly identified type III interferon (IFN-λ) has antiviral activity against a broad spectrum of viruses. We thus examined whether IFN-λ has the ability to inhibit human immunodeficiency virus type 1 (HIV-1) infection of blood monocyte-derived macrophages that expressed IFN-λ receptors. Both IFN-λ1 and IFN-λ2, when added to macrophage cultures, inhibited HIV-1 infection and replication. This IFN-λ-mediated anti-HIV-1 activity is broad, as IFN-λ could inhibit infection by both laboratory-adapted and clinical strains of HIV-1. Investigations of the mechanism(s) responsible for the IFN-λ action showed that although IFN-λ had little effect on HIV-1 entry coreceptor CCR5 expression, IFN-λ induced the expression of CC chemokines, the ligands for CCR5. In addition, IFN-λ upregulated intracellular expression of type I IFNs and APOBEC3G/3F, the newly identified anti-HIV-1 cellular factors. These data provide direct and compelling evidence that IFN-λ, through both extracellular and intracellular antiviral mechanisms, inhibits HIV-1 replication in macrophages. These findings indicate that IFN-λ may have therapeutic value in the treatment of HIV-1 infection.
The Human Immunodeficiency Virus Type 1 Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation I132M Confers Hypersensitivity to Nucleoside Analogs
Authors: Ambrose Zandrea , Herman Brian D. , Sheen Chih-Wei , Zelina Shannon , Moore Katie L. , Tachedjian Gilda , Nissley Dwight V. , Sluis-Cremer Nicolas
Published: February 2009

We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.
Cardioviruses Are Genetically Diverse and Cause Common Enteric Infections in South Asian Children
Authors: Blinkova Olga , Kapoor Amit , Victoria Joseph , Jones Morris , Wolfe Nathan , Naeem Asif , Shaukat Shahzad , Sharif Salmaan , Alam Muhammad Masroor , Angez Mehar , Zaidi Sohail , Delwart Eric L.
Published: February 2009

Cardioviruses cause enteric infections in mice and rats which when disseminated have been associated with myocarditis, type 1 diabetes, encephalitis, and multiple sclerosis-like symptoms. Cardioviruses have also been detected at lower frequencies in other mammals. The
Cardiovirus
genus within the
Picornaviridae
family is currently made up of two viral species,
Theilovirus
and
Encephalomyocarditis virus
. Until recently, only a single strain of cardioviruses (Vilyuisk virus within the
Theilovirus
species) associated with a geographically restricted and prevalent encephalitis-like condition had been reported to occur in humans. A second theilovirus-related cardiovirus (
Saffold virus
[SAFV]) was reported in 2007 and subsequently found in respiratory secretions from children with respiratory problems and in stools of both healthy and diarrheic children. Using viral metagenomics, we identified RNA fragments related to SAFV in the stools of Pakistani and Afghani children with nonpolio acute flaccid paralysis (AFP). We sequenced three near-full-length genomes, showing the presence of divergent strains of SAFV and preliminary evidence of a distant recombination event between the ancestors of the Theiler-like viruses of rats and those of human SAFV. Further VP1 sequencing showed the presence of five new SAFV genotypes, doubling the reported genetic diversity of human and animal theiloviruses combined. Both AFP patients and healthy children in Pakistan were found to be excreting SAFV at high frequencies of 9 and 12%, respectively. Further studies are needed to examine the roles of these highly common and diverse SAFV genotypes in nonpolio AFP and other human diseases.
Quantitating the Multiplicity of Infection with Human Immunodeficiency Virus Type 1 Subtype C Reveals a Non-Poisson Distribution of Transmitted Variants
Authors: Abrahams M.-R. , Anderson J. A. , Giorgi E. E. , Seoighe C. , Mlisana K. , Ping L.-H. , Athreya G. S. , Treurnicht F. K. , Keele B. F. , Wood N. , Salazar-Gonzalez J. F. , Bhattacharya T. , Chu H. , Hoffman I. , Galvin S. , Mapanje C. , Kazembe P. , Thebus R. , Fiscus S. , Hide W. , Cohen M. S. , Karim S. Abdool , Haynes B. F. , Shaw G. M. , Hahn B. H. , Korber B. T. , Swanstrom R. , Williamson C.
Published: February 2009

Identifying the specific genetic characteristics of successfully transmitted variants may prove central to the development of effective vaccine and microbicide interventions. Although human immunodeficiency virus transmission is associated with a population bottleneck, the extent to which different factors influence the diversity of transmitted viruses is unclear. We estimate here the number of transmitted variants in 69 heterosexual men and women with primary subtype C infections. From 1,505 env sequences obtained using a single genome amplification approach we show that 78% of infections involved single variant transmission and 22% involved multiple variant transmissions (median of 3). We found evidence for mutations selected for cytotoxic-T-lymphocyte or antibody escape and a high prevalence of recombination in individuals infected with multiple variants representing another potential escape pathway in these individuals. In a combined analysis of 171 subtype B and C transmission events, we found that infection with more than one variant does not follow a Poisson distribution, indicating that transmission of individual virions cannot be seen as independent events, each occurring with low probability. While most transmissions resulted from a single infectious unit, multiple variant transmissions represent a significant fraction of transmission events, suggesting that there may be important mechanistic differences between these groups that are not yet understood.
The Replication Cycle of Varicella-Zoster Virus: Analysis of the Kinetics of Viral Protein Expression, Genome Synthesis, and Virion Assembly at the Single-Cell Level
Authors: Reichelt Mike , Brady Jennifer , Arvin Ann M.
Published: February 2009

Varicella-zoster virus (VZV) is a human alphaherpesvirus that is highly cell associated in cell culture. Because cell-free virus yields are too low to permit the synchronous infections needed for time-resolved analyses, information is lacking about the sequence of events during the VZV replication cycle. To address this challenge, we differentially labeled VZV-infected inoculum cells (input) and uninfected (output) cells with fluorescent cell dyes or endocytosed nanogold particles and evaluated newly infected cells by confocal immunofluorescence or electron microscopy (EM) at the single-cell level at defined intervals. We demonstrated the spatiotemporal expression of six major VZV proteins, ORF61, IE62, IE63, ORF29, ORF23, and gE, representing all putative kinetic classes, for the first time. Newly synthesized ORF61, as well as IE62, the major VZV transactivator, appeared within 1 h, and they were targeted to different subnuclear compartments. The formation of VZV DNA replication compartments started between 4 and 6 h, involved recruitment of ORF29 to putative IE62 prereplication sites, and resulted in large globular nuclear compartments where newly synthesized viral DNA accumulated. Although considered a late protein, gE accumulated in the Golgi compartment at as early as 4 h. ORF23 capsid protein was present at 9 h. The assembly of viral nucleocapsids and mature enveloped VZ virions was detected by 9 to 12 h by time-resolved EM. Although syncytium formation is a hallmark of VZV infection, infection of neighboring cells did not require cell-cell fusion; its occurrence from 9 h is likely to amplify VZV replication. Our results define the productive cycle of VZV infection in a single cell as occurring in 9 to 12 h.
Amino Acid Residues in the Fusion Peptide Pocket Regulate the pH of Activation of the H5N1 Influenza Virus Hemagglutinin Protein
Authors: Reed Mark L. , Yen Hui-Ling , DuBois Rebecca M. , Bridges Olga A. , Salomon Rachelle , Webster Robert G. , Russell Charles J.
Published: February 2009

The receptor specificity and cleavability of the hemagglutinin (HA) protein have been shown to regulate influenza A virus transmissibility and pathogenicity, but little is known about how its pH of activation contributes to these important biological properties. To identify amino acid residues that regulate the acid stability of the HA protein of H5N1 influenza viruses, we performed a mutational analysis of the HA protein of the moderately pathogenic A/chicken/Vietnam/C58/04 (H5N1) virus. Nineteen HA proteins containing point mutations in the HA2 coiled-coil domain or in an HA1 histidine or basic patch were generated. Wild-type and mutant HA plasmids were transiently transfected in cell culture and analyzed for total protein expression, surface expression, cleavage efficiency, pH of fusion, and pH of conformational change. Four mutations to residues in the fusion peptide pocket, Y23H and H24Q in the HA1 subunit and E105K and N114K in the HA2 subunit, and a K58I mutation in the HA2 coiled-coil domain significantly altered the pH of activation of the H5 HA protein. In some cases, the magnitude and direction of changes of individual mutations in the H5 HA protein differed considerably from similar mutations in other influenza A virus HA subtypes. Introduction of Y23H, H24Q, K58I, and N114K mutations into recombinant viruses resulted in virus-expressed HA proteins with similar shifts in the pH of fusion. Overall, the data show that residues comprising the fusion peptide pocket are important in triggering pH-dependent activation of the H5 HA protein.
Detection and Characterization of Influenza A Virus PA-PB2 Interaction through a Bimolecular Fluorescence Complementation Assay
Authors: Hemerka Joseph N. , Wang Dan , Weng Yuejin , Lu Wuxun , Kaushik Radhey S. , Jin Jing , Harmon Aaron F. , Li Feng
Published: February 2009

The influenza virus polymerase complex, consisting of the PA, PB1, and PB2 subunits, is required for the transcription and replication of the influenza A viral genome. Previous studies have shown that PB1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In the current study, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB1 interactions in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies reveal a previously unknown PA-PB2 interaction and provide a framework for further investigation of the biological relevance of the PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus.
Kinetic Analysis of Human T-Cell Leukemia Virus Type 1 Gene Expression in Cell Culture and Infected Animals
Authors: Li Min , Kesic Matthew , Yin Han , Yu Lianbo , Green Patrick L.
Published: February 2009

Human T-cell leukemia virus type 1 (HTLV-1) infection causes adult T-cell leukemia and is associated with a variety of lymphocyte-mediated disorders. It has been hypothesized that a highly regulated pattern of HTLV-1 gene expression is critical for virus survival and disease pathogenesis. In this study, real-time reverse transcriptase PCR was used to determine the kinetics of viral gene expression in cells transiently transfected with an HTLV-1 proviral plasmid, in newly infected human peripheral blood mononuclear cells (PBMCs), and in PBMCs from newly infected rabbits. The HTLV-1 gene expression profiles in transiently transfected and infected cells were similar; over time, all transcripts increased and then maintained stable levels.
gag/pol
,
tax/rex
, and
env
mRNA were detected first and at the highest levels, whereas the expression levels of the accessory genes, including the antisense
Hbz
, were significantly lower than the
tax/rex
levels (ranging from 1 to 4 logs depending on the specific mRNA). In infected rabbits,
tax/rex
and
gag/pol
mRNA levels peaked early after inoculation and progressively decreased, which correlated inversely with the proviral load and host antibody response against viral proteins. Interestingly,
Hbz
mRNA was detectable at 1 week postinfection and increased and stabilized. The expression levels of all other HTLV-1 genes in infected rabbit PBMCs were at or below our limit of detection. This analysis provides insight into viral gene expression under various in vitro and in vivo experimental conditions. Our in vivo data indicate that in infected rabbits,
Hbz
mRNA expression over time directly correlates with the proviral load, which provides the first evidence linking
Hbz
expression to proviral load and the survival of the virus-infected cell in the host.