bluuebunny/crossref_metadata_2025 huggingface dataset:
The deviant citizen: Measuring potential positive relations between counterproductive work behaviour and organizational citizenship behaviour
Authors: Fox Suzy , Spector Paul E. , Goh Angeline , Bruursema Kari , Kessler Stacey R.
Published: May 2011

Studies have shown a strong negative correlation between counterproductive work behaviour (CWB) and organizational citizenship behaviour (OCB), and opposite correlations with hypothesized antecedents. Such observed correlations may have been erroneously caused by three measurement artefacts: items measuring absence of CWBs, rather than behaviours that exceed requirements or expectations in OCB scales; supervisory halo; and agreement rather than frequency response format. A new OCB scale, the OCB‐checklist (OCB‐C) was used that did not have these artefacts. Contrary to prior expectations from the literature, positive relations were found between CWB and OCB, and stressors and OCB. Theoretical explanations for positive CWB/OCB relations (demand‐elicited OCB, social loafing, work process problems, rater perceptions and attributions, and aggravated job stress processes) are discussed.
Serological Responses to Experimental Norwalk Virus Infection Measured Using a Quantitative Duplex Time-Resolved Fluorescence Immunoassay
Authors: Kavanagh Owen , Estes Mary K. , Reeck Amanda , Raju Ravikiran M. , Opekun Antone R. , Gilger Mark A. , Graham David Y. , Atmar Robert L.
Published: May 2011

A quantitative duplex time-resolved fluorescence assay, dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), was developed to measure Norwalk virus (NV)-specific IgA and IgG antibodies simultaneously. The duplex assay showed superior performance by detecting seroconversion following experimental NV infection at an earlier time point than a reference total immunoglobulin enzyme-linked immunosorbent assay (ELISA).
Distinct Effects of Anti-Tumor Necrosis Factor Combined Therapy on TH1/TH2 Balance in Rheumatoid Arthritis Patients
Authors: Herman S. , Zurgil N. , Machlav S. , Shinberg A. , Langevitz P. , Ehrenfeld M. , Deutsch M.
Published: May 2011

The immune balance in patients with rheumatoid arthritis (RA), a disease characterized by TH1 dominance, treated by the preferred combined anti-tumor necrosis factor (anti-TNF) and methotrexate (MTX) therapy was evaluated by assessing the chemokine and cytokine receptors as well as apoptosis induction. A meta-analysis of combined therapy by TNF blockers and MTX in 15 RA patients, MTX monotherapy in 20 RA patients, and 11 diagnosed but untreated RA patients was performed by assessing several immune markers in the whole lymphocyte population, as well as in specific CD4 cells, by both flow cytometry and image analysis. A significant downregulation of CXCR3 and IL-12 receptors (both TH1 markers) and a significant increase in the chemokine receptor CCR4 and, to a lesser extent, IL-4R (both TH2 markers) were found; a particularly marked increase was found in patients treated by combined therapy. This phenomenon was pronounced in CD4 cells and was accompanied by a high proportion of apoptotic cells. The therapeutic effect of MTX and TNF blockers may be due to apoptosis induction in lymphocytes infiltrating from the inflammation site and restoring the TH1/TH2 balance.
Serological Response of Shiga Toxin-Producing Escherichia coli Type III Secreted Proteins in Sera from Vaccinated Rabbits, Naturally Infected Cattle, and Humans
Authors: Asper David J. , Karmali Mohamed A. , Townsend Hugh , Rogan Dragan , Potter Andrew A.
Published: May 2011

Escherichia coliO157:H7 is an important zoonotic pathogen, causing hemolytic uremic syndrome (HUS). The colonization of cattle and human hosts is mediated through the action of effectors secreted via a type III secretion system (T3SS). The structural genes for the T3SS and many of the secreted effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE). We cloned and expressed the genes coding for 66 effectors and purified each to measure the cross-reactivity of type III secreted proteins from Shiga toxin-producingEscherichia coli(STEC) serotypes. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with type III secreted proteins (T3SPs) from four STEC serotypes, experimentally infected cattle, and human sera from six HUS patients. Twenty proteins were recognized by at least one of the STEC T3SP-vaccinated rabbits by Western blotting. Several structural proteins (EspA, EspB, and EspD) and a number of effectors (Tir, NleA, and TccP) were recognized by O26-, O103-, O111-, and O157-specific sera. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA, and NleA were recognized by the majority of the samples tested. A number of other proteins also were recognized by individual serum samples. Overall, proteins such as Tir, EspB, EspD, NleA, and EspA were highly immunogenic in vaccinated and naturally infected subjects and could be candidates for a cross-protective STEC vaccine.
Measurement of Functional Anti-Meningococcal Serogroup A Activity Using Strain 3125 as the Target Strain for Serum Bactericidal Assay
Authors: Poolman Jan T. , De Vleeschauwer Isabel , Durant Nathalie , Devos Nathalie , Feron Christiane , Lestrate Pascal , Weynants Vincent , Boutriau Dominique
Published: May 2011

Functional anti-N. meningitidisserogroup A (MenA) activity in human serum is detected by serum bactericidal assay (SBA), using either rabbit (rSBA) or human (hSBA) complement, with F8238 as the recommended MenA SBA target strain. However, the F8238 strain may not be optimal for this purpose because, as we show here, it expresses the L11 immunotype, whereas most MenA invasive strains express the L(3,7)9 or L10 immunotype. Moreover, SBA results may be strain dependent, because immunotypes differ in their sensitivity to complement, emphasizing the need to choose the most appropriate strain. Sera from random subsets of infants, toddlers, children, and adolescents in clinical trials of MenA conjugate vaccines were tested by rSBA using strains 3125 (L10) and F8238 (L11). In unvaccinated subjects from all age groups, the percentages of seropositive samples (rSBA-MenA titer, ≥1:8) was lower using strain 3125 than using strain F8238. However, in toddlers and adolescents immunized with a conjugate MenA vaccine, the percentages of seropositive samples generally were similar using either strain in the rSBA. In two studies, sera also were tested with hSBA. Using hSBA, the differences in the percentages of seroprotective samples (hSBA-MenA titer, ≥1:4) between strains 3125 and F8238 was less apparent, and in contrast with rSBA, the percentage of seroprotective samples from unvaccinated subjects was slightly higher using strain 3125 than using strain F8238. In adults vaccinated with plain MenA polysaccharide, the percentage of seroprotective samples was higher using strain 3125 than with strain F8238, and the vaccine response rates using strain 3125 were better aligned with the demonstrated efficacy of MenA vaccination. In conclusion, SBA results obtained using the MenA L10 3125 strain better reflected vaccine-induced immunity.
Most Multidrug-Resistant Pseudomonas aeruginosa Isolates from Hospitals in Eastern France Belong to a Few Clonal Types
Authors: Cholley Pascal , Thouverez Michelle , Hocquet Didier , van der Mee-Marquet Nathalie , Talon Daniel , Bertrand Xavier
Published: May 2011

This study aimed to determine the genetic diversity of clinical multidrug-resistant
Pseudomonas aeruginosa
. We used pulsed-field gel electrophoresis and multilocus sequence typing to analyze 187 strains isolated in different French hospitals. To illustrate the diversity of resistance mechanisms to antibiotics in a given clone, we identified β-lactamases with an extended spectrum by using phenotypic and genotypic methods. Typing results showed that the majority of our multidrug-resistant isolates belong to a few clonal types (ST235, ST111, and ST175) that are already spreading worldwide. These successful international clones sporadically produced extended-spectrum β-lactamase-encoding genes but mostly became extensively resistant to β-lactams after derepression of intrinsic resistance mechanisms (i.e., AmpC cephalosporinase). Our results indicate that cross-transmission plays a major role in the spread of multidrug-resistant
P. aeruginosa
in hospital settings.
Rapid and Simultaneous Detection of Mycobacterium tuberculosis Complex and Beijing/W Genotype in Sputum by an Optimized DNA Extraction Protocol and a Novel Multiplex Real-Time PCR
Authors: Leung Eric T. Y. , Zheng L. , Wong Rity Y. K. , Chan Edward W. C. , Au T. K. , Chan Raphael C. Y. , Lui Grace , Lee Nelson , Ip Margaret
Published: May 2011

Rapid diagnosis and genotyping of
Mycobacterium tuberculosis
by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of
M. tuberculosis
complex and the Beijing/W genotype (a hypervirulent sublineage of
M. tuberculosis
) by using multiple fluorogenic probes targeting both the
M. tuberculosis
IS
6110
and the
Rv0927c-pstS3
intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W
M. tuberculosis
strains from 17 different species of nontuberculosis
Mycobacterium
(NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74
M. tuberculosis
culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (
n
= 31). The detection limit of the assay is 10
M. tuberculosis
bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of
M. tuberculosis
, including Beijing/W
M. tuberculosis
, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.
Streptomycin Resistance and Lineage-Specific Polymorphisms in Mycobacterium tuberculosis
gidB
Gene
Authors: Spies Fernanda S. , Ribeiro Andrezza W. , Ramos Daniela F. , Ribeiro Marta O. , Martin Anandi , Palomino Juan Carlos , Rossetti Maria Lucia R. , da Silva Pedro Eduardo A. , Zaha Arnaldo
Published: May 2011

Mutations related to streptomycin resistance in the
rpsL
and
rrs
genes are well known and can explain about 70% of this phenotypic resistance. Recently, the
gidB
gene was found to be associated with low-level streptomycin resistance in
Mycobacterium tuberculosis
. Mutations in
gidB
have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycin-susceptible and streptomycin-resistant strains. In this study, mutations in
gidB
appeared in 27% of streptomycin-resistant strains that contained no mutations in the
rpsL
or
rrs
genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in
gidB
with streptomycin resistance needs to be further investigated, as we also found mutations in
gidB
in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the
gidB
16
polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the
gidB
92
polymorphism (92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.
Genotypic Prediction of Human Immunodeficiency Virus Type 1 Tropism by Use of Plasma and Peripheral Blood Mononuclear Cells in the Routine Clinical Laboratory
Authors: Paar Christian , Geit Maria , Stekel Herbert , Berg Jörg
Published: May 2011

We developed a sequencing assay for genotypic HIV-1 tropism determination. The assay allows examination of HIV RNA from plasma and HIV DNA from peripheral blood mononuclear cells (PBMC), including PBMC samples from patients with undetectable viral loads. Assessment of 100 pairs of plasma and PBMC samples showed a high concordance of 90%. With the limitations of population-based sequencing, the assay was found to be robust and suitable for the routine clinical laboratory.
Genotype Diversity and Distribution of Orientia tsutsugamushi Causing Scrub Typhus in Thailand
Authors: Ruang-areerate Toon , Jeamwattanalert Pimmada , Rodkvamtook Wuttikorn , Richards Allen L. , Sunyakumthorn Piyanate , Gaywee Jariyanart
Published: May 2011

Scrub typhus, caused by antigenically disparate isolates of
Orientia tsutsugamushi
, is a widely distributed mite-borne human disease in the Asia Pacific region. Information regarding the heterogeneity of the immunodominant 56-kDa type-specific antigen (TSA) gene is crucial for the design and evaluation of scrub typhus-specific diagnostic assays and vaccines. Using indirect immunofluorescence assays (IFA) and PCR assays,
O. tsutsugamushi
was detected samples from rodents and patients with fever of unknown origin obtained from six provinces of Thailand during 2004 to 2007. Sequences were determined for a fragment of the 56-kDa TSA gene, and the relationship between these sequences and those previously determined were assessed. The phylogenetic analyses of partial 56-kDa TSA gene sequences demonstrated wide diversity and distribution of
O. tsutsugamushi
genotypes in Thailand. Furthermore, the genetic diversity grouped the scrub typhus agents into two commonly and five infrequently found genotypes within six provinces of Thailand. The two most commonly found genotypes of
O. tsutsugamushi
described in this study do not associate with the prototype strains that are widely used for the design and evaluation of diagnostic assays and vaccine candidates. Thus, these new genotypes should be considered for future scrub typhus assay and vaccine development.
Rapid Detection and Molecular Differentiation of Toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans Strains by LightCycler PCR
Authors: Sing Andreas , Berger Anja , Schneider-Brachert Wulf , Holzmann Thomas , Reischl Udo
Published: May 2011

The systemic symptoms of diphtheria are caused by thetox-encoded diphtheria toxin (DT) which is produced by toxigenicCorynebacteriumspp. Besides the classical agentC. diphtheriae, the zoonotic pathogenC. ulceranshas increasingly been reported as an emerging pathogen for diphtheria. The reliable detection of toxigenicCorynebacteriumspp. is of substantial importance for both diphtheria surveillance in the public health sector and the clinical workup of a patient with diphtherialike symptoms. Since the respectivetoxgenes ofC. diphtheriaeandC. ulceransdiffer from each other in both DNA and amino acid sequence, bothtoxgenes should be covered by novel real-time PCR methods. We describe the development and validation of a LightCycler PCR assay which reliably recognizestoxgenes from bothC. diphtheriaeandC. ulceransand differentiates the respective target genes by fluorescence resonance energy transfer (FRET) hybridization probe melting curve analysis.
Evaluation of a Single-Tube Multiplex Real-Time PCR for Differentiation of Members of the Mycobacterium tuberculosis Complex in Clinical Specimens
Authors: Halse Tanya A. , Escuyer Vincent E. , Musser Kimberlee A.
Published: May 2011

Members of the
Mycobacterium tuberculosis
complex (MTBC) differ in virulence attributes, drug resistance patterns, and host preferences. The rapid differentiation of these species to determine zoonotic or human sources of tuberculosis disease or to direct treatment can benefit both public health and patient management. Commercially available assays cannot differentiate these species, and published assays have not been evaluated directly on clinical specimens. A real-time PCR assay for the differentiation of
M. tuberculosis
,
M. bovis
,
M. bovis
BCG,
M. africanum
,
M. microti
, and
M. canettii
was developed. The presence or absence of regions of difference (RD) between the genomes of members of the MTBC allowed for the design of a single-tube five-plex real-time PCR assay to differentiate these species. This assay assesses the presence of RD1, RD4, RD9, RD12, and a region exterior to RD9 which is present in all MTBC members. To evaluate the performance of this assay, 192 clinical specimens positive for MTBC by real-time PCR were tested, resulting in a 94% correlation of the real-time PCR with the identification results obtained with cultured material. Additionally, 727 Bactec MGIT 960-positive cultures were tested, resulting in a 97% concordance between the methods. This real-time PCR is an inexpensive and rapid (2.5-h) method performed in a closed-format system and requiring minimal hands-on time that can be implemented in a clinical laboratory and used directly on clinical specimens.
Adamantane- and Oseltamivir-Resistant Seasonal A (H1N1) and Pandemic A (H1N1) 2009 Influenza Viruses in Guangdong, China, during 2008 and 2009
Authors: Zhou Jie , Zou Lirong , Zhang Xin , Liao Jiazheng , Ni Hanzhong , Hou Nianmei , Wang Yajing , Li Hui , Wu Jie , Jonges Marcel , Meijer Adam , Koopmans Marion , Ke Changwen
Published: May 2011

Adamantane and oseltamivir resistance among influenza viruses is a major concern to public health officials. To determine the prevalence of antiviral-resistant influenza viruses in Guangdong, China, 244 seasonal A (H1N1) and 222 pandemic A (H1N1) 2009 viruses were screened for oseltamivir resistance by a fluorescence-based neuraminidase (NA) inhibition assay along with NA gene sequencing. Also, 147 seasonal A (H1N1) viruses were sequenced to detect adamantane resistance markers in M2. Adamantane-resistant seasonal A (H1N1) viruses clustering to clade 2C were dominant in 2008, followed by oseltamivir-resistant seasonal A (H1N1) viruses, clustering to clade 2B during January and May 2009. In June 2009, a lineage of double-resistant seasonal A (H1N1) viruses emerged, until it was replaced by the pandemic A (H1N1) 2009 viruses. The lineage most likely resulted from reassortment under the pressure of the overuse of adamantanes. As all viruses were resistant to at least one of the two types of antiviral agents, the need for close monitoring of the prevalence of antiviral resistance is stressed.
Encephalitozoon cuniculi Genotype I as a Causative Agent of Brain Abscess in an Immunocompetent Patient
Authors: Ditrich Oleg , Chrdle Aleš , Sak Bohumil , Chmelík Václav , Kubále Jiří , Dyková Iva , Kváč Martin
Published: May 2011

A brain abscess caused by
Encephalitozoon cuniculi
genotype I together with
Streptococcus intermedius
occurred in a patient without major immunocompromise and with diabetes. The distinguishing clinical signs were hemiparesis and epilepsy. The microsporidium was observed in the abscess aspirate, and its specific DNA was also detected in stool and urine. The patient was successfully treated with albendazole and mebendazole.
Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay
Authors: Bennett Susan , Harvala Heli , Witteveldt Jeroen , McWilliam Leitch E. Carol , McLeish Nigel , Templeton Kate , Gunson Rory , Carman William F. , Simmonds Peter
Published: May 2011

Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly
in vitro
and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5′-untranslated regions (5′UTR). Amplification dynamics (threshold cycle [

C
T

]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction).

C
T

values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame.
Achromobacter xylosoxidans Infection Presenting as a Pulmonary Nodule Mimicking Cancer
Authors: Claassen Stephanie L. , Reese Jason M. , Mysliwiec Vincent , Mahlen Steven D.
Published: May 2011

Achromobacter xylosoxidansis typically isolated from pulmonary sources, presenting as pneumonia in immunosuppressed individuals. We describe a novel clinical presentation ofA. xylosoxidansinfection presenting as multiple spiculated, pulmonary nodules mimicking cancer for which the patient underwent a wedge resection of the lung for diagnosis and staging of presumptive cancer.
Human T Cell Leukemia Virus Type 1 Tax Inhibits Innate Antiviral Signaling via NF-κB-Dependent Induction of SOCS1
Authors: Charoenthongtrakul Soratree , Zhou Qinjie , Shembade Noula , Harhaj Nicole S. , Harhaj Edward W.
Published: May 2011

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19
Gag
levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.
Epithelial Cell Retention of Transcriptionally Active, P3HR-1-Derived Heterogeneous Epstein-Barr Virus DNA with Concurrent Loss of Parental Virus
Authors: Ikuta Kazufumi , Ding Mingyu , Zhang Fangfang , Sixbey John W. , Scott Rona S.
Published: May 2011

Deleted, rearranged, heterogeneous (het) Epstein-Barr virus (EBV) DNA with the distinctive capability of disrupting EBV latency has been reported in biopsy samples of EBV-associated tumors whose onset in immunocompetent hosts is characteristically preceded by an antibody response indicative of EBV reactivation. Using the EBV P3HR-1 strain, we have reproduced in long-term culture of SVK epithelial cells an unusual pattern of infection previously observed in a subset of tumor biopsy samples: the persistence of het DNA in the absence of the parental helper virus. Fluorescence
in situ
hybridization (FISH) of infected cell subclones indicated the retention of het DNA in an integrated form. Incorporation of an intact het DNA molecule was confirmed by PCR, using primers that framed junctions of the four rearranged EBV DNA segments comprising P3HR-1-derived het DNA. Structural analysis of EBV terminal repeats revealed a banding pattern consistent with the integration of het DNA as a concatemer. Linkage of concatemeric monomers was defined at a nucleotide level, and that junctional sequence was detected in cell-free P3HR-1 virion DNA, confirming that subgenomic het DNA was packaged into infectious particles in a concatemeric configuration. Stable integration into cells having lost the standard viral genome allowed the unambiguous designation of het DNA as the source for viral gene products potentially encoded by both. Continuous expression of the latency-to-lytic switch protein Zta and detection of the BALF4 gene product gB, known to expand the target cell range of standard virus when incorporated at augmented levels into infectious progeny, add to a presumption of het DNA-enhanced pathogenesis in diseases of EBV reactivation.
Viral Interleukin-10 Expressed by Human Cytomegalovirus during the Latent Phase of Infection Modulates Latently Infected Myeloid Cell Differentiation
Authors: Avdic Selmir , Cao John Z. , Cheung Allen K. L. , Abendroth Allison , Slobedman Barry
Published: May 2011

The human cytomegalovirus UL111A gene is expressed during latent and productive infections, and it codes for homologs of interleukin-10 (IL-10). We examined whether viral IL-10 expressed during latency altered differentiation of latently infected myeloid progenitors. In comparison to infection with parental virus or mock infection, latent infection with a virus in which the gene encoding viral IL-10 has been deleted upregulated cytokines associated with dendritic cell (DC) formation and increased the proportion of myeloid DCs. These data demonstrate that viral IL-10 restricts the ability of latently infected myeloid progenitors to differentiate into DCs and identifies an immunomodulatory role for viral IL-10 which may limit the host's ability to clear latent virus.
Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation
Authors: Albright Brandon S. , Nellissery Jacob , Szczepaniak Renata , Weller Sandra K.
Published: May 2011

The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol.
81:
8868–8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.
Mutation of a C-Terminal Motif Affects Kaposi's Sarcoma-Associated Herpesvirus ORF57 RNA Binding, Nuclear Trafficking, and Multimerization
Authors: Taylor Adam , Jackson Brian R. , Noerenberg Marko , Hughes David J. , Boyne James R. , Verow Mark , Harris Mark , Whitehouse Adrian
Published: May 2011

The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is essential for virus lytic replication. ORF57 regulates virus gene expression at multiple levels, enhancing transcription, stability, nuclear export, and translation of viral transcripts. To enhance the nuclear export of viral intronless transcripts, ORF57 (i) binds viral intronless mRNAs, (ii) shuttles between the nucleus, nucleolus, and the cytoplasm, and (iii) interacts with multiple cellular nuclear export proteins to access the TAP-mediated nuclear export pathway. We investigated the implications on the subcellular trafficking, cellular nuclear export factor recruitment, and ultimately nuclear mRNA export of an ORF57 protein unable to bind RNA. We observed that mutation of a carboxy-terminal RGG motif, which prevents RNA binding, affects the subcellular localization and nuclear trafficking of the ORF57 protein, suggesting that it forms subnuclear aggregates. Further analysis of the mutant shows that although it still retains the ability to interact with cellular nuclear export proteins, it is unable to export viral intronless mRNAs from the nucleus. Moreover, computational molecular modeling and biochemical studies suggest that, unlike the wild-type protein, this mutant is unable to self-associate. Therefore, these results suggest the mutation of a carboxy-terminal RGG motif affects ORF57 RNA binding, nuclear trafficking, and multimerization.
Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052
Authors: Fridell Robert A. , Qiu Dike , Valera Lourdes , Wang Chunfu , Rose Ronald E. , Gao Min
Published: May 2011

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC
50
s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a
trans
-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a
cis
-acting function that likely occurs as part of the HCV replication complex and a
trans
-acting function that may occur outside the replication complex. The
cis
-acting function is likely performed by basally phosphorylated NS5A, while the
trans
-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the
cis
-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the
trans
-acting function.
Longer V1V2 Region with Increased Number of Potential N-Linked Glycosylation Sites in the HIV-1 Envelope Glycoprotein Protects against HIV-Specific Neutralizing Antibodies
Authors: van Gils Marit J. , Bunnik Evelien M. , Boeser-Nunnink Brigitte D. , Burger Judith A. , Terlouw-Klein Marijke , Verwer Naomi , Schuitemaker Hanneke
Published: May 2011

Human immunodeficiency virus type 1 (HIV-1) has the ability to adapt to the host environment by escaping from host immune responses. We previously observed that escape from humoral immunity, both at the individual and at a population level, coincided with longer variable loops and an increased number of potential N-linked glycosylation sites (PNGS) in the viral envelope glycoprotein (Env) and, in particular, in variable regions 1 and 2 (V1V2). Here, we provide several lines of evidence for the role of V1V2 in the resistance of HIV-1 to neutralizing antibodies. First, we determined that the increasing neutralization resistance of a reference panel of tier-categorized neutralization-sensitive and -resistant HIV-1 variants coincided with a longer V1V2 loop containing more PNGS. Second, an exchange of the different variable regions of Env from a neutralization-sensitive HIV-1 variant into a neutralization-resistant escape variant from the same individual revealed that the V1V2 loop is a strong determinant for sensitivity to autologous-serum neutralization. Third, exchange of the V1V2 loop of neutralization-sensitive HIV-1 variants from historical seroconverters with the V1V2 loop of neutralization-resistant HIV-1 variants from contemporary seroconverters decreased the neutralization sensitivity to CD4-binding site-directed antibodies. Overall, we demonstrate that an increase in the length of the V1V2 loop and/or the number of PNGS in that same region of the HIV-1 envelope glycoprotein is directly involved in the protection of HIV-1 against HIV-specific neutralizing antibodies, possibly by shielding underlying epitopes in the envelope glycoprotein from antibody recognition.
The Requirement for Nucleoporin NUP153 during Human Immunodeficiency Virus Type 1 Infection Is Determined by the Viral Capsid
Authors: Matreyek Kenneth A. , Engelman Alan
Published: May 2011

Lentiviruses likely infect nondividing cells by commandeering host nuclear transport factors to facilitate the passage of their preintegration complexes (PICs) through nuclear pore complexes (NPCs) within nuclear envelopes. Genome-wide small interfering RNA screens previously identified karyopherin β transportin-3 (TNPO3) and NPC component nucleoporin 153 (NUP153) as being important for infection by human immunodeficiency virus type 1 (HIV-1). The knockdown of either protein significantly inhibited HIV-1 infectivity, while infection by the gammaretrovirus Moloney murine leukemia virus (MLV) was unaffected. Here, we establish that primate lentiviruses are particularly sensitive to NUP153 knockdown and investigate HIV-1-encoded elements that contribute to this dependency. Mutants lacking functional Vpr or the central DNA flap remained sensitive to NUP153 depletion, while MLV/HIV-1 chimera viruses carrying MLV matrix, capsid, or integrase became less sensitive when the latter two elements were substituted. Two capsid missense mutant viruses, N74D and P90A, were largely insensitive to NUP153 depletion, as was wild-type HIV-1 when cyclophilin A was depleted simultaneously or when infection was conducted in the presence of cyclosporine A. The codepletion of NUP153 and TNPO3 yielded synergistic effects that outweighed those calculated based on individual knockdowns, indicating potential interdependent roles for these factors during HIV-1 infection. Quantitative PCR revealed normal levels of late reverse transcripts, a moderate reduction of 2-long terminal repeat (2-LTR) circles, and a relatively large reduction in integrated proviruses upon NUP153 knockdown. These results suggest that capsid, likely by the qualities of its uncoating, determines whether HIV-1 requires cellular NUP153 for PIC nuclear import.
Antibody-Dependent Cell-Mediated Cytotoxicity in Simian Immunodeficiency Virus-Infected Rhesus Monkeys
Authors: Sun Yue , Asmal Mohammed , Lane Sophie , Permar Sallie R. , Schmidt Stephen D. , Mascola John R. , Letvin Norman L.
Published: May 2011

With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.
Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Promoters Drive Gene Expression in All Airway Epithelial Cells of Mice but Only Induce Tumors in the Alveolar Region of the Lungs
Authors: Yu Darrick L. , Linnerth-Petrik Nicolle M. , Halbert Christine L. , Walsh Scott R. , Miller A. Dusty , Wootton Sarah K.
Published: May 2011

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats, while the closely related enzootic nasal tumor virus type 1 (ENTV-1) and ENTV-2 induce tumors in the nasal epithelium of sheep and goats, respectively. When expressed using a strong Rous sarcoma virus promoter, the envelope proteins of these viruses induce tumors in the respiratory tract of mice, but only in the distal airway. To examine the role of the retroviral long terminal repeat (LTR) promoters in determining tissue tropism, adeno-associated virus (AAV) vectors expressing alkaline phosphatase under the control of the JSRV, ENTV-1, or ENTV-2 LTRs were generated and administered to mice. The JSRV LTR was active in all airway epithelial cells, while the ENTV LTRs were active in the nasal epithelium and alveolar type II cells but poorly active in tracheal and bronchial epithelial cells. When vectors were administered systemically, the ENTV-1 and -2 LTRs were inactive in major organs examined, whereas the JSRV showed high-level activity in the liver. When a putative transcriptional enhancer from the 3′ end of the
env
gene was inserted upstream of the JSRV and ENTV-1 LTRs in the AAV vectors, a dramatic increase in transgene expression was observed. However, intranasal administration of AAV vectors containing any combination of ENTV or JSRV LTRs and Env proteins induced tumors only in the lower airway. Our results indicate that mice do not provide an adequate model for nasal tumor induction by ENTV despite our ability to express genes in the nasal epithelium.
The Signal Peptide of a Recently Integrated Endogenous Sheep Betaretrovirus Envelope Plays a Major Role in Eluding Gag-Mediated Late Restriction
Authors: Armezzani Alessia , Arnaud Frédérick , Caporale Marco , di Meo GiuliaPia , Iannuzzi Leopoldo , Murgia Claudio , Palmarini Massimo
Published: May 2011

The exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with highly related and biologically active endogenous retroviruses (enJSRVs). The endogenous enJS56A1 locus possesses a defective Gag polyprotein which blocks the late replication steps of related exogenous and endogenous retroviruses by a mechanism known as
J
SRV
l
ate
r
estriction (JLR). Conversely, enJSRV-26, which most likely integrated into the sheep genome less than 200 years ago, is able to escape JLR. In this study, we demonstrate that the ability of enJSRV-26 to escape JLR is due to a single-amino-acid substitution in the signal peptide (SP) of its envelope glycoprotein. We show that enJSRV-26 SP does not localize to the nucleolus, unlike the functional SPs of related exogenous and endogenous sheep betaretroviruses. In addition, enJSRV-26 SP function as a posttranscriptional regulator of viral gene expression is impaired. enJSRV-26 JLR escape relies on the presence of the functional enJS56A1 SP. Moreover, we show that the ratio between enJSRV-26 and enJS56A1 Gag is critical to elude JLR. Interestingly, we found that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus. These data further reinforce the notion that transdominant enJSRV proviruses have been positively selected in domestic sheep, and that the coevolution between endogenous and exogenous sheep betaretroviruses and their host is still occurring.
Viral Determinants of Polarized Assembly for the Murine Leukemia Virus
Authors: Jin Jing , Li Fei , Mothes Walther
Published: May 2011

Retrovirus transmission via direct cell-cell contact is more efficient than diffusion through the extracellular milieu. This is believed to be due to the ability of viruses to efficiently coordinate several steps of the retroviral life cycle at cell-cell contact sites (D. C. Johnson et al., J. Virol. 76:1–8, 2002; D. M. Phillips, AIDS
8:
719–731, 1994; Q. Sattenau, Nat. Rev. Microbiol.
6:
815–826, 2008). Using the murine leukemia virus (MLV) as a model retrovirus, we have previously shown that interaction between viral envelope (Env) and receptor directs viral assembly to cell-cell contact sites to promote efficient viral spreading (J. Jin et al., PLoS Biol.
7:
e1000163, 2009). In addressing the underlying mechanism, we observed that Env cytoplasmic tail directs this contact-induced polarized assembly. We present here the viral determinants in the Env cytoplasmic tail and Gag that are important in this process. A tyrosine residue within the cytoplasmic tail of Env was identified, which directs polarized assembly. MLV matrix-mediated membrane targeting is required for Gag recruitment to sites of cell-cell contact. Our results suggest that MLV polarized assembly is mediated by a direct or indirect interaction between both domains, thereby coupling Gag recruitment and virus assembly to Env accumulation at the cell-cell interface. In contrast, HIV Gag that assembles outside of cell-cell interfaces can subsequently be drawn into contact zones mediated by MLV Env and receptor, a finding that is consistent with the previously observed lateral movement of HIV into the virological synapse (W. Hubner et al., Science
323:
1743–1747, 2009; D. Rudnicka et al., J. Virol.
83:
6234–6246, 2009). As such, we observed two distinct modes of virus cell-to-cell transmission that involve either polarized or nonpolarized assembly, but both result in virus transmission.
Comparative Pathology in Ferrets Infected with H1N1 Influenza A Viruses Isolated from Different Hosts
Authors: Smith Jennifer Humberd , Nagy Tamas , Driskell Elizabeth , Brooks Paula , Tompkins S. Mark , Tripp Ralph A.
Published: May 2011

Virus replication and pulmonary disease pathogenesis in ferrets following intranasal infection with a pandemic influenza virus strain (A/California/4/09 [CA09]), a human seasonal influenza H1N1 virus isolate (A/New Caledonia/20/99 [Ncal99]), a classical swine influenza H1N1 virus isolate (A/Swine/Iowa/15/30 [Sw30]), or an avian H1N1 virus isolate (A/Mallard/MN/A108-2355/08 [Mal08]) were compared. Nasal wash virus titers were similar for Ncal99 and Sw30, with peak virus titers of 10
5.1
50% tissue culture infectious doses (TCID
50
)/ml and 10
5.5
TCID
50
/ml occurring at day 3 postinfection (p.i.), respectively. The mean peak titer for CA09 also occurred at day 3 p.i. but was higher (10
7
TCID
50
/ml). In contrast, the peak virus titers (10
3.6
to 10
4.3
TCID
50
/ml) for Mal08 were delayed, occurring between days 5 and 7 p.i. Disease pathogenesis was characterized by microscopic lesions in the nasal turbinates and lungs of all ferrets; however, Sw30 infection was associated with severe bronchointerstitial pneumonia. The results demonstrate that although CA09 is highly transmissible in the human population and replicates well in the ferret model, it causes modest disease compared to other H1N1 viruses, particularly Sw30 infection.
Wild-Type Measles Virus Interferes with Short-Term Engraftment of Human CD34
+
Hematopoietic Progenitor Cells
Authors: Boussaad Ibrahim , Varagnolo Linda , Hornich Veronika , Rieger Lorenz , Krockenberger Matthias , Stuehmer Thorsten , Kranzfelder Dietmar , Mueller Albrecht M. , Schneider-Schaulies Sibylle
Published: May 2011

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected, we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells
in vitro
. Infection of human CD34
+
HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs, where infection is established in CD34
+
CD150
−
and CD34
+
CD150
+
(in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection, as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability, expansion, and colony-forming activity of either CD150
+
or CD150
−
HS/PCs
in vitro
, it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether, these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression.