bluuebunny/crossref_metadata_2025 huggingface dataset:
M.SpyI, a DNA Methyltransferase Encoded on a
mefA
Chimeric Element, Modifies the Genome of
Streptococcus pyogenes
Authors: Euler Chad W. , Ryan Patricia A. , Martin Judith M. , Fischetti Vincent A.
Published: January 2007

While screening the clonality of
Streptococcus pyogenes
isolates from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh, PA, we found a correlation between the presence of the chimeric element Φ10394.4 (carrying the macrolide efflux gene,
mefA
) and genomic DNA being resistant to cleavage by SmaI restriction endonuclease. A search of the open reading frames in Φ10394.4 identified a putative type II restriction-modification (R-M) cassette containing a cytosine methyltransferase gene (
spyIM
). Heterologous expression of the cloned
spyIM
gene, as well as allelic-replacement experiments, showed that the action of this methyltransferase (M.SpyI) was responsible for the inhibition of SmaI digestion of genomic DNA in the Φ10394.4-containing isolates. Analysis of the methylation patterns of streptococcal genomic DNA from
spyIM
-positive strains, a
spyIM
deletion mutant, and a
spyIM
-negative strain determined that M.SpyI specifically recognized and methylated the DNA sequence to generate 5′-C
m
CNGG. To our knowledge, this is the first methyltransferase gene from
S. pyogenes
to be cloned and to have its activity characterized. These results reveal why pulsed field gel electrophoresis analysis of SmaI-digested genomic DNA cannot be used to analyze the clonality of some streptococci containing Φ10394.4 and may explain the inability of previous epidemiological studies to use SmaI to analyze DNAs from macrolide-resistant streptococci. The presence of the SpyI R-M cassette in Φ10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in macrolide-resistant streptococci.
COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma
Authors: Allice Tiziano , Cerutti Francesco , Pittaluga Fabrizia , Varetto Silvia , Gabella Silvia , Marzano Alfredo , Franchello Alessandro , Colucci Giuseppe , Ghisetti Valeria
Published: January 2007

Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (
r
= 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (
r
= 0.966, mean difference in quantitation = 0.36 log
10
IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection.
Intimin Types Determined by Heteroduplex Mobility Assay of Intimin Gene (
eae
)-Positive
Escherichia coli
Strains
Authors: Ito Kenitiro , Iida Mariko , Yamazaki Mitsugu , Moriya Kazuo , Moroishi Sanae , Yatsuyanagi Jun , Kurazono Takayuki , Hiruta Noriaki , Ratchtrachenchai Orn-Anong
Published: January 2007

We developed a quick genetic approach to screen variants of the intimin gene (
eae
) by using a heteroduplex mobility assay (HMA) that targets the 5′ conserved region of
eae
. The
eae
variants were categorized into 4 major HMA types and 10 minor subtypes.
Real-Time PCR Diagnostics Failure Caused by Nucleotide Variability within Exon 4 of the Human Cytomegalovirus Major Immediate-Early Gene
Authors: Lengerova Martina , Racil Zdenek , Volfova Pavlina , Lochmanova Jana , Berkovcova Jitka , Dvorakova Dana , Vorlicek Jiri , Mayer Jiri
Published: January 2007

Here we report how variability in the human cytomegalovirus genome sequence may seriously affect the outcome of its molecular diagnosis. A real-time quantitative PCR assay targeting the major immediate-early gene failed to detect the viral load in some, but not all, clinical samples from hematooncological patients. By amplification and sequencing the DNA across the regions targeted by this assay we found a number of nucleotide substitutions which accounted for decreased primer/probe binding. This decreased the sensitivity of the assay up to 1,000-fold.
Quantitative Detection and Rapid Identification of Human Adenoviruses
Authors: Miura-Ochiai Rika , Shimada Yasushi , Konno Tsunetada , Yamazaki Shudo , Aoki Koki , Ohno Shigeaki , Suzuki Eitaro , Ishiko Hiroaki
Published: January 2007

We have established a method of quantitative detection and rapid identification of human adenoviruses (hAdVs). Using LightCycler PCR with a primer set, we were able to amplify 554 bp of the hexon gene from each of 51 prototype strains of hAdVs. The sensitivity of LightCycler PCR was 10 copies of hAdV DNA/reaction. When LightCycler PCR was performed using a set of primers, hAdV was positive for 74.4% (99 of 133) of conjunctivitis patients and for 27.3% (81 of 297) of respiratory infection patients. We also attempted to measure hAdV in the potentially contaminated eye drops used by patients, detecting 5.4 × 10
2
to 1.6 × 10
6
copies/ml of hAdV. We determined the 350-bp nucleotide sequence of the amplified hexon gene and compared it with the sequences of the 51 prototype strains. Phylogenetic analysis based on 350 bp of the hexon gene identified 99 positive conjunctival swabs as 24 cases of AdV type 3 (AdV-3), 14 cases of AdV-4, 1 case of AdV-8, 19 cases of AdV-19a, and 41 cases of AdV-37. The 81 sequences from pharyngeal or nasal mucus swabs were identified as 29 cases of AdV-2, 18 cases of AdV-1, 18 cases of AdV-5, 12 cases of AdV-4, 2 cases of AdV-37, 1 case of AdV-3, and 1 case of AdV-6. LightCycler PCR followed by phylogenetic analysis provides an effective tool for the rapid identification of hAdVs and for studying molecular epidemiology.
Mutated G4P[8] Rotavirus Associated with a Nationwide Outbreak of Gastroenteritis in Nicaragua in 2005
Authors: Bucardo Filemon , Karlsson Beatrice , Nordgren Johan , Paniagua Margarita , González Alcides , Amador Juan Jose , Espinoza Felix , Svensson Lennart
Published: January 2007

During February and March 2005, one of the largest national recorded outbreaks of severe acute gastroenteritis occurred in Nicaragua, affecting ≥64,000 individuals and causing ≥56 deaths, predominantly in children under 5 years of age. Through a nationwide laboratory-based study, stool samples were collected and investigated for rotavirus. Of 108 stool samples examined, 72 (67%) were positive for rotavirus. While 69% (50/72) of the positive samples were found in children less than 2 years of age, 50% (6/12) of the adult samples were positive. A mutated G4P[8] strain was the most commonly recognized strain (85%), followed by mixed G strains (8%) and G9P[8] (7%) strains. Phylogenetic analysis of the VP7 gene revealed that the G4 strains belonged to the emerging lineage Ic and was distantly related to the ST3 and VA70 G4 strains. Secondary structure predictions of the VP7 G4 protein revealed an insert of an asparagine residue in position 76, which, combined with additional mutations, surprisingly modified two downstream β-sheets at amino acid positions 80 to 85 and 115 to 119. The 2005 G4P[8] strain compared to a G4P[8] strain from 2002 had a substitution of an asparagine residue for threonine (Asn→Thr) at position 96 within antigenic region A, thus eliminating a potential glycosylation site. The mutated G4 virus was introduced in Nicaragua after 2002 and probably emerged from Brazil, Argentina, or Uruguay.
Human Papillomavirus Quantification in Urine and Cervical Samples by Using the Mx4000 and LightCycler General Real-Time PCR Systems
Authors: Payan Christopher , Ducancelle Alexandra , Aboubaker Mohamed H. , Caer Julien , Tapia Malena , Chauvin Amelie , Peyronnet Damien , Le Hen Elodie , Arab Zohra , Legrand Marie-Christine , Tran Adissa , Postec Edith , Tourmen Françoise , Avenel Martine , Malbois Chantal , De Brux Marie-Anne , Descamps Philippe , Lunel Francoise
Published: January 2007

During the last decade, growing efforts have focused on human papillomavirus (HPV) detection using liquid hybridization, conventional PCR, and real-time PCR-based methods to increase the overall proportion of patients participating in cervical cancer screening procedures. We proposed a new general HPV DNA real-time PCR on the Mx4000 (Stratagene) and LightCycler (Roche Diagnostics) systems usable for both cervical scrape specimens and urine samples. A linear range was obtained from 5 DNA copies to 8 log
10
DNA copies/ml, and intra- and interassay variations were between 1.8 and 4%. Cervical carcinoma and HPV DNA screening was performed in 333 individual women referred for gynecological examination at the university hospitals of Angers and Brest and enrolled in the PapU study. Among cervical specimens (
n
= 333), 45% were positive for HPV DNA, with a mean viral load at 5.00 log/ml (± 1.73). Among urine samples (
n
= 177), 37% were positive with a significant 50-fold-lower mean viral load (3.77 ± 1.32 log/ml;
P
< 0.0001). Kappa agreement for HPV DNA between cervical and urine specimens was excellent (93%). Thus, we developed a highly sensitive and quantitative general HPV DNA real-time PCR method that allows mass screening of patients with HPV infection. The ongoing longitudinal and prospective multicenter PapU study should give us the opportunity to validate this method adapted to HPV DNA screening in urine samples in a larger population.
Use of Capillary Blood Samples as a New Approach for Diagnosis of Dengue Virus Infection
Authors: Matheus Séverine , Meynard Jean-Baptiste , Lacoste Vincent , Morvan Jacques , Deparis Xavier
Published: January 2007

We evaluated the use of capillary blood samples stored on filter papers for diagnosis of dengue virus infection. Venous and capillary blood samples were collected from 130 patients suspected of having dengue fever. We compared the performances of standard reference methods using capillary blood samples absorbed onto filter papers versus venous blood samples. The resulting sensitivity, specificity, and positive predictive value of tests performed on filter paper compared to those performed on venous blood samples were 81.6% (62/76; 95% confidence interval [CI], 74.9% to 88.3%), 90.7% (49/54; 95% CI, 85.7% to 95.7%), and 92.5% (62/67; 95% CI, 86.2% to 98.8%), respectively. During the acute phase of dengue virus infection (day 1 to day 4), the tests performed on capillary blood samples had a sensitivity of 88.5% (95% CI, 82.0% to 95.0%) and a specificity of 93.8% (95% CI, 88.9% to 98.7%). During the convalescent phase of infection, this method allowed the viral serotype to be determined for 4 of 15 (27%) dengue virus-infected patients for whom virological diagnosis using venous samples was negative. Capillary blood samples could therefore be a good alternative for the diagnosis of dengue virus infection in tropical areas. Indeed, these samples are convenient for storage and transport without the need for a cold chain and simplify the collection of samples from children. Moreover, our results suggest that viral particles persist longer in capillary blood than in peripheral blood. Analysis of the viability of viral particles under these conditions may give new insights into the physiopathology of dengue virus infection and the transmission of dengue virus during outbreaks.
Evaluation of Combined General Primer-Mediated PCR Sequencing and Type-Specific PCR Strategies for Determination of Human Papillomavirus Genotypes in Cervical Cell Specimens
Authors: Fontaine Véronique , Mascaux Corinne , Weyn Christine , Bernis Aurore , Celio Nathalie , Lefèvre Philippe , Kaufman Leonard , Garbar Christian
Published: January 2007

A strategy combining human papillomavirus general primer (mainly the PGMY primers)-directed PCR sequencing and type-specific PCR is presented. DNA samples were first tested in general primer-mediated PCR. The amplified fragments of positive samples after ethidium bromide-stained DNA gel analysis were further sequenced, and corresponding DNA samples were further analyzed by PCR using type-specific primers for human papillomavirus (HPV) types 16, 18, 31, and 52. The comparison of the results of 157 samples analyzed by this strategy in parallel with the Hybrid Capture 2 tests and with the HPV INNO-LiPA (Innogenetics line probe assay) shows that this method is suitable for HPV detection and genotyping in cervical cell samples. Although the PCR sequencing method is as sensitive as the HPV INNO-LiPA for HPV detection, our method allows the identification of a broader range of HPV types. In contrast, the HPV INNO-LiPA was less time-consuming and better identified coinfections.
Human Herpesvirus 6 DNA Levels in Cerebrospinal Fluid Due to Primary Infection Differ from Those Due to Chromosomal Viral Integration and Have Implications for Diagnosis of Encephalitis
Authors: Ward Katherine N. , Leong Hoe Nam , Thiruchelvam Anton D. , Atkinson Claire E. , Clark Duncan A.
Published: January 2007

The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log
10
copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log
10
copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (≥3.5 log
10
copies/ml) or whole blood (≥6.0 log
10
copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log
10
copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log
10
copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood.
Generic Detection of Coronaviruses and Differentiation at the Prototype Strain Level by Reverse Transcription-PCR and Nonfluorescent Low-Density Microarray
Authors: de Souza Luna Luciano Kleber , Heiser Volker , Regamey Nicolas , Panning Marcus , Drexler Jan Felix , Mulangu Sabue , Poon Leo , Baumgarte Sigrid , Haijema Bert Jan , Kaiser Laurent , Drosten Christian
Published: January 2007

A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.
Expression of Rotavirus NSP4 Alters the Actin Network Organization through the Actin Remodeling Protein Cofilin
Authors: Berkova Zuzana , Crawford Sue E. , Blutt Sarah E. , Morris Andrew P. , Estes Mary K.
Published: January 2007

Rotavirus is a major cause of infantile gastroenteritis with a multifactorial pathogenesis. As with many other pathogens, rotavirus infection and replication leads to rearrangement of the cytoskeleton with disorganization of cytoskeletal elements such as actin and cytokeratin through a calcium-dependent process that has not been fully characterized. The rotavirus enterotoxin NSP4, shown previously to elevate intracellular calcium levels when added exogenously as well as when expressed intracellularly, is a key player in intracellular calcium regulation during rotavirus infection. Here, we investigated the role NSP4 may play in actin rearrangement. Expression of NSP4 fused to enhanced green fluorescent protein (NSP4-EGFP), but not expression of EGFP alone, caused stabilization of long cellular projections in fully confluent HEK 293 cells. Cells expressing NSP4-EGFP for 24 h were also resistant to cell rounding induced by cytochalasin D. Quantification of filamentous actin (F-actin) content by using rhodamine-conjugated phalloidin and flow cytometry showed an elevated F-actin content in NSP4-EGFP-expressing and rotavirus-infected cells in comparison with that in nonexpressing and noninfected cells. Normalization of intracellular calcium levels prevented alterations of F-actin content. Observed changes in F-actin amounts correlated with the increased activation of the actin-remodeling protein cofilin. These calcium-dependent actin rearrangements induced by intracellular NSP4 expression may contribute to rotavirus pathogenesis by interfering with cellular processes dependent on subcortical actin remodeling, including ion transport and viral release.
The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis
Authors: Mach Michael , Osinski Karolina , Kropff Barbara , Schloetzer-Schrehardt Ursula , Krzyzaniak Magdalena , Britt William
Published: January 2007

Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.
Role of the Coronavirus E Viroporin Protein Transmembrane Domain in Virus Assembly
Authors: Ye Ye , Hogue Brenda G.
Published: January 2007

Coronavirus envelope (E) proteins are small (∼75- to 110-amino-acid) membrane proteins that have a short hydrophilic amino terminus, a relatively long hydrophobic membrane domain, and a long hydrophilic carboxy-terminal domain. The protein is a minor virion structural component that plays an important, not fully understood role in virus production. It was recently demonstrated that the protein forms ion channels. We investigated the importance of the hydrophobic domain of the mouse hepatitis coronavirus (MHV) A59 E protein. Alanine scanning insertion mutagenesis was used to examine the effect of disruption of the domain on virus production in the context of the virus genome by using a MHV A59 infectious clone. Mutant viruses exhibited smaller plaque phenotypes, and virus production was significantly crippled. Analysis of recovered viruses suggested that the structure of the presumed α-helical structure and positioning of polar hydrophilic residues within the predicted transmembrane domain are important for virus production. Generation of viruses with restored wild-type helical pitch resulted in increased virus production, but some exhibited decreased virus release. Viruses with the restored helical pitch were more sensitive to treatment with the ion channel inhibitor hexamethylene amiloride than were the more crippled parental viruses with the single alanine insertions, suggesting that disruption of the transmembrane domain affects the functional activity of the protein. Overall the results indicate that the transmembrane domain plays a crucial role during biogenesis of virions.
Contrasting Effects of Human, Canine, and Hybrid Adenovirus Vectors on the Phenotypical and Functional Maturation of Human Dendritic Cells: Implications for Clinical Efficacy
Authors: Perreau Matthieu , Mennechet Franck , Serratrice Nicolas , Glasgow Joel N. , Curiel David T. , Wodrich Harald , Kremer Eric J.
Published: January 2007

Antipathogen immune responses create a balance between immunity, tolerance, and immune evasion. However, during gene therapy most viral vectors are delivered in substantial doses and are incapable of expressing gene products that reduce the host's ability to detect transduced cells. Gene transfer efficacy is also modified by the in vivo transduction of dendritic cells (DC), which notably increases the immunogenicity of virions and vector-encoded genes. In this study, we evaluated parameters that are relevant to the use of canine adenovirus serotype 2 (CAV-2) vectors in the clinical setting by assaying their effect on human monocyte-derived DC (hMoDC). We compared CAV-2 to human adenovirus (HAd) vectors containing the wild-type virion, functional deletions in the penton base RGD motif, and the CAV-2 fiber knob. In contrast to the HAd type 5 (HAd5)-based vectors, CAV-2 poorly transduced hMoDC, provoked minimal upregulation of major histocompatibility complex class I/II and costimulatory molecules (CD40, CD80, and CD86), and induced negligible morphological changes indicative of DC maturation. Functional maturation assay results (e.g., reduced antigen uptake; tumor necrosis factor alpha, interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-10, IL-12, and IFN-α/β secretion; and stimulation of heterologous T-cell proliferation) were also significantly lower for CAV-2. Our data suggested that this was due, in part, to the use of an alternative receptor and a block in vesicular escape. Additionally, HAd5 vector-induced hMoDC maturation was independent of the aforementioned cytokines. Paradoxically, an HAd5/CAV-2 hybrid vector induced the greatest phenotypical and functional maturation of hMoDC. Our data suggest that CAV-2 and the HAd5/CAV-2 vector may be the antithesis ofAdenoviridaeimmunogenicity and that each may have specific clinical advantages.
Immunoglobulin A (IgA) Is a Natural Ligand of Hepatitis A Virus Cellular Receptor 1 (HAVCR1), and the Association of IgA with HAVCR1 Enhances Virus-Receptor Interactions
Authors: Tami Cecilia , Silberstein Erica , Manangeeswaran Mohanraj , Freeman Gordon J. , Umetsu Sarah E. , DeKruyff Rosemarie H. , Umetsu Dale T. , Kaplan Gerardo G.
Published: January 2007

The hepatitis A virus cellular receptor 1 (HAVCR1/TIM1), a member of the T-cell immunoglobulin mucin (TIM) family, is an important atopy susceptibility gene in humans. The exact natural function of HAVCR1/TIM1 and the inverse association between HAV infection and prevention of atopy are not well understood. To identify natural ligands of human HAVCR1/TIM1, we used an expression cloning strategy based on the binding of dog cells transfected with a human lymph node cDNA library to a HAVCR1/TIM1 Fc fusion protein. The transfected cells that bound to the human HAVCR1/TIM1 Fc contained cDNA of human immunoglobulin alpha 1 heavy (Igα1) and lambda light (Igλ) chain and secreted human IgA1λ antibody that bound to the cell surface. Cotransfection of the isolated Igα1 and Igλ cDNAs to naïve dog cells resulted in the secretion of IgA1λ that bound to HAVCR1/TIM1 Fc but not to a poliovirus receptor Fc fusion protein in a capture enzyme-linked immunosorbent assay. The interaction of HAVCR1/TIM1 with IgA was inhibited by monoclonal antibodies (MAbs) against Igα1 and Igλ, excess IgA1λ, or anti-HAVCR1/TIM1 MAb. IgA did not inhibit HAV infection of African green monkey cells, suggesting that the IgA and the virus binding sites are in different epitopes on HAVCR1/TIM1. IgA enhanced significantly the neutralization of HAV by HAVCR1/TIM1 Fc. Our results indicate that IgA1λ is a specific ligand of HAVCR1/TIM1 and that their association has a synergistic effect in virus-receptor interactions.
Frequency and Phenotype of JC Virus-Specific CD8+T Lymphocytes in the Peripheral Blood of Patients with Progressive Multifocal Leukoencephalopathy
Authors: Lima Marco A. , Marzocchetti Angela , Autissier Patrick , Tompkins Troy , Chen Yiping , Gordon Jennifer , Clifford David B. , Gandhi Rajesh T. , Venna Nagagopal , Berger Joseph R. , Koralnik Igor J.
Published: January 2007

JC virus (JCV)-specific CD8+cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+T cells specific for two A*0201-restricted JCV epitopes, VP1p36and VP1p100, and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+patients with PML (HIV+PML patients) classified as survivors. After magnetic preenrichment of CD8+T cells, epitope-specific cells ranged from 0.001% to 0.22% by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+T cells in healthy individuals, HIV+subjects, or HIV+PML patients. However, in one HIV+PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+and two HIV+PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1p100CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+individuals with PML.
5′-Proximal Hot Spot for an Inducible Positive-to-Negative-Strand Template Switch byCoronavirus RNA-Dependent RNA Polymerase
Authors: Wu Hung-Yi , Brian David A.
Published: January 2007

Coronaviruses have a positive-strand RNA genome and replicate through the use of a
3′ nested set of subgenomic mRNAs each possessing a leader (65
to 90 nucleotides [nt] in length, depending on the viral species)
identical to and derived from the genomic leader. One widely supported
model for leader acquisition states that a template switch takes place
during the generation of negative-strand antileader-containing
templates used subsequently for subgenomic mRNA synthesis. In this
process, the switch is largely driven by canonical heptameric donor
sequences at intergenic sites on the genome that match an acceptor
sequence at the 3′ end of the genomic leader. With
experimentally placed 22-nt-long donor sequences within a bovine
coronavirus defective interfering (DI) RNA we have shown that matching
sites occurring anywhere within a 65-nt-wide 5′-proximal
genomic acceptor hot spot (nt 33 through 97) can be used for production
of templates for subgenomic mRNA synthesis from the DI RNA. Here we
report that with the same experimental approach, template switches can
be induced in
trans
from an internal site in the DI RNA to the
negative-strand antigenome of the helper virus. For these, a
3′-proximal 89-nt acceptor hot spot on the viral antigenome (nt
35 through 123), largely complementary to that described above, was
found. Molecules resulting from these switches were not templates for
subgenomic mRNA synthesis but, rather, ambisense chimeras potentially
exceeding the viral genome in length. The results suggest the existence
of a coronavirus 5′-proximal partially double-stranded template
switch-facilitating structure of discrete width that contains both the
viral genome and
antigenome.
Poxvirus-Encoded Gamma Interferon Binding Protein Dampens the Host Immune Response to Infection
Authors: Sakala Isaac G. , Chaudhri Geeta , Buller R. Mark , Nuara Anthony A. , Bai Hongdong , Chen Nanhai , Karupiah Gunasegaran
Published: January 2007

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbpΔ, produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects.
An Extended Stem-Loop 1 Is Necessary for Human Immunodeficiency Virus Type 2 Replication and Affects Genomic RNA Encapsidation
Authors: Lanchy Jean-Marc , Lodmell J. Stephen
Published: January 2007

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3′ end of the ψ encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3′ end of the pal within ψ with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.
Dysregulation of the Ubiquitin-Proteasome System by Curcumin Suppresses Coxsackievirus B3 Replication
Authors: Si Xiaoning , Wang Yahong , Wong Jerry , Zhang Jingchun , McManus Bruce M. , Luo Honglin
Published: January 2007

Curcumin (diferuloylmethane), a natural polyphenolic compound extracted from the spice turmeric, has been reported to have anti-inflammatory, antioxidant, and antiproliferative properties by modulating multiple cellular machineries. It inhibits several intracellular signaling pathways, including the mitogen-activated protein kinases (MAPKs), casein kinase II (CKII), and the COP9 signalosome (CSN), in various cell types. It has also been recently demonstrated that exposure to curcumin leads to the dysregulation of the ubiquitin-proteasome system (UPS). Coxsackievirus infection is associated with various diseases, including myocarditis and dilated cardiomyopathy. In searching for new antiviral agents against coxsackievirus, we found that treatment with curcumin significantly reduced viral RNA expression, protein synthesis, and virus titer and protected cells from virus-induced cytopathic effect and apoptosis. We further demonstrated that reduction of viral infection by curcumin was unlikely due to inhibition of CVB3 binding to its receptors or CVB3-induced activation of MAPKs. Moreover, gene silencing of CKII and Jab1, a component of CSN, by small interfering RNAs did not inhibit the replication of coxsackievirus, suggesting that the antiviral action of curcumin is independent of these pathways. Finally, we showed that curcumin treatment reduced both the 20S proteasome proteolytic activities and the cellular deubiquitinating activities, leading to increased accumulation of ubiquitinated proteins and decreased protein levels of free ubiquitin. We have recently demonstrated that the UPS-mediated protein degradation and/or modification plays a critical role in the regulation of coxsackievirus replication. Thus, our results suggest an important antiviral effect of curcumin wherein it potently inhibits coxsackievirus replication through dysregulation of the UPS.
Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses
Authors: Ehrhardt Christina , Wolff Thorsten , Pleschka Stephan , Planz Oliver , Beermann Wiebke , Bode Johannes G. , Schmolke Mirco , Ludwig Stephan
Published: January 2007

Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3β and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.
Murine Norovirus 1 Infection Is Associated with Histopathological Changes in Immunocompetent Hosts, but Clinical Disease Is Prevented by STAT1-Dependent Interferon Responses
Authors: Mumphrey Shannon M. , Changotra Harish , Moore Tara N. , Heimann-Nichols Ellen R. , Wobus Christiane E. , Reilly Michael J. , Moghadamfalahi Mana , Shukla Deepti , Karst Stephanie M.
Published: January 2007

Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.
New Way of Regulating Alternative Splicing in Retroviruses: the Promoter Makes a Difference
Authors: Bohne Jens , Schambach Axel , Zychlinski Daniela
Published: January 2007

Alternative splicing has been recognized as a major mechanism for creating proteomic diversity from a limited number of genes. However, not all determinants regulating this process have been characterized. Using subviral human immunodeficiency virus (HIV) env constructs we observed an enhanced splicing of the RNA when expression was under control of the cytomegalovirus (CMV) promoter instead of the HIV long terminal repeat (LTR). We extended these observations to LTR- or CMV-driven murine leukemia proviruses, suggesting that retroviral LTRs are adapted to inefficient alternative splicing at most sites in order to maintain balanced gene expression.
Ability of Hyaluronidase 2 To Degrade Extracellular Hyaluronan Is Not Required for Its Function as a Receptor for Jaagsiekte Sheep Retrovirus
Authors: Vigdorovich Vladimir , Miller A. Dusty , Strong Roland K.
Published: January 2007

Jaagsiekte sheep retrovirus (JSRV) uses hyaluronidase 2 (Hyal2) as a cell entry receptor. By making inactivating mutations to the catalytic residues of human Hyal2, we found that hyaluronidase activity was dispensable for its receptor function. The affinities of the JSRV envelope glycoprotein for Hyal2 and the Hyal2 mutant were similar, and hyaluronan did not block either high-affinity interaction or virus infection. While generating the Hyal2 mutant, we discovered that our previous analysis of the hyaluronidase activity of Hyal2 was affected by a contaminating hyaluronan lyase, which we have identified as the occlusion-derived baculovirus E66 protein of the recombinant baculovirus used to produce Hyal2. We now report that purified human Hyal2 is a weak acid-active hyaluronidase.
Maintenance of Endoplasmic Reticulum (ER) Homeostasis in Herpes Simplex Virus Type 1-Infected Cells through the Association of a Viral Glycoprotein with PERK, a Cellular ER Stress Sensor
Authors: Mulvey Matthew , Arias Carolina , Mohr Ian
Published: January 2007

In
the efforts of viruses to dominate and control critical cellular
pathways, viruses generate considerable intracellular stress within
their hosts. In particular, the capacity of resident
endoplasmic reticulum (ER) chaperones to properly process the acute
increase in client protein load is significantly challenged. Such
alterations typically induce the unfolded protein response, one
component of which acts through IRE1 to restore ER homeostasis by
expanding the folding capabilities, whereas the other arm activates the
eIF-2α (α subunit of eukaryotic initiation factor 2)
kinase PERK to transiently arrest production of new polypeptide
clientele. Viruses, such as herpes simplex virus type 1 (HSV-1),
however, go to great lengths to prevent the inhibition of translation
resulting from eIF-2α phosphorylation. Here, we establish that
PERK, but not IRE1, resists activation by acute ER stress in
HSV-1-infected cells. This requires the ER luminal domain of PERK,
which associates with the viral glycoprotein gB. Strikingly, gB
regulates viral protein accumulation in a PERK-dependent manner. This
is the first description of a virus-encoded PERK-specific effector and
defines a new strategy by which viruses are able to maintain ER
homeostasis.
Innate Immune Response to Adenoviral Vectors Is Mediated by both Toll-Like Receptor-Dependent and -Independent Pathways
Authors: Zhu Jiangao , Huang Xiaopei , Yang Yiping
Published: January 2007

Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.
Ribonucleocapsid Formation of Severe Acute Respiratory Syndrome Coronavirus through Molecular Action of the N-Terminal Domain of N Protein
Authors: Saikatendu Kumar Singh , Joseph Jeremiah S. , Subramanian Vanitha , Neuman Benjamin W. , Buchmeier Michael J. , Stevens Raymond C. , Kuhn Peter
Published: January 2007

Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 Å (monoclinic) and at 1.85 Å (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the β-sheet core. The functionally important positively charged β-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.
Upregulation of Indoleamine 2,3-Dioxygenase in Hepatitis C Virus Infection
Authors: Larrea Esther , Riezu-Boj José I. , Gil-Guerrero Lucía , Casares Noelia , Aldabe Rafael , Sarobe Pablo , Civeira María P. , Heeney Jonathan L. , Rollier Christine , Verstrepen Babs , Wakita Takaji , Borrás-Cuesta Francisco , Lasarte Juan J. , Prieto Jesús
Published: January 2007

Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.