bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Three Different Methods for Detection of Shiga Toxin-Producing Escherichia coli in a Tertiary Pediatric Care Center
Authors: Vallières Emilie , Saint-Jean Maude , Rallu Fabien
Published: November 2012

Shiga toxin-producing
Escherichia coli
(STEC) is a well-known cause of sporadic and epidemic food-borne gastroenteritis. A low infectious dose, approximately 10 microorganisms, is sufficient to cause disease that may lead to hemolytic-uremic syndrome. The objective of this study was to compare the performances of an in-house real-time PCR, a commercial enzyme immunoassay (EIA) (Premier EHEC; Meridian Bioscience), and culture on sorbitol MacConkey agar for the detection of STEC in a tertiary care pediatric hospital. Of 632 stool samples tested, 21 were positive for STEC. All were detected by PCR, 6 were detected by EIA, and only 5 O157 STEC isolates were identified by culture. Among the 15 specimens falsely negative by EIA, there were 9 Stx1, 2 Stx2, and 4 Stx1 and Stx2 STEC isolates. The latter group included 2 O157 STEC isolates that would have been missed if only EIA had been performed. To our knowledge, this is the first prospective study performed in a pediatric hospital which demonstrates the superiority of PCR over EIA for the detection of STEC. We conclude that PCR is specific and more sensitive than EIA. PCR should be considered for routine use in clinical settings where molecular detection facilities are available. Its lower limit of detection, equivalent to the infectious dose, is an obvious advantage for patient care and public health surveillance.
Gamma Interferon Release Assay for Monitoring of Treatment Response for Active Tuberculosis: an Explosion in the Spaghetti Factory
Authors: Denkinger Claudia M. , Pai Madhukar , Patel Meena , Menzies Dick
Published: November 2012

Few studies have correlated the results of interferon (gamma interferon) release assays (IGRAs) with known markers of tuberculosis (TB) treatment response. We report the results of serial QuantiFERON-TB gold in-tube assay (QFT) testing on 149 patients with active tuberculosis and correlate the results with smear and culture conversion. We show that QFT results do not offer much value for treatment monitoring of TB disease.
Rapid Detection of Panton-Valentine Leukocidin in Staphylococcus aureus Cultures by Use of a Lateral Flow Assay Based on Monoclonal Antibodies
Authors: Monecke Stefan , Müller Elke , Buechler Joseph , Rejman John , Stieber Bettina , Akpaka Patrick Eberechi , Bandt Dirk , Burris Rob , Coombs Geoffrey , Hidalgo-Arroyo G. Aida , Hughes Peter , Kearns Angela , Abós Sonia Molinos , Pichon Bruno , Skakni Leila , Söderquist Bo , Ehricht Ralf
Published: November 2012

Panton-Valentine leukocidin (PVL) is a virulence factor of
Staphylococcus aureus
, which is associated with skin and soft-tissue infections and necrotizing pneumonia. To develop a rapid phenotypic assay, recombinant PVL F component was used to generate monoclonal antibodies by phage display. These antibodies were spotted on protein microarrays and screened using different
lukF-PV
preparations and detection antibodies. This led to the identification of the optimal antibody combination that was then used to establish a lateral flow assay. This test was used to detect PVL in
S. aureus
cultures. The detection limit of the assay with purified native and recombinant antigens was determined to be around 1 ng/ml. Overnight cultures from various solid and liquid media proved suitable for PVL detection. Six hundred strains and clinical isolates from patients from America, Europe, Australia, Africa, and the Middle East were tested. Isolates were genotyped in parallel by DNA microarray hybridization for confirmation of PVL status and assignment to clonal complexes. The sensitivity, specificity, and positive and negative predictive values of the assay in this trial were 99.7, 98.3, 98.4, and 99.7%, respectively. A total of 302 clinical isolates and reference strains were PVL positive and were assigned to 21 different clonal complexes. In summary, the lateral flow test allows rapid and economical detection of PVL in a routine bacteriology laboratory. As the test utilizes cultures from standard media and does not require sophisticated equipment, it can be easily integrated into a laboratory's workflow and might contribute to timely therapy of PVL-associated infections.
Severe Hepatitis Associated with an Echovirus 18 Infection in an Immune-Compromised Adult
Authors: Lefterova Martina I. , Rivetta Claudia , George Tracy I. , Pinsky Benjamin A.
Published: November 2012

Enteroviruses are recognized as important pathogens in pediatric patients; however, they are often overlooked as etiologic agents of disease in adults. Here, we report a case of echovirus 18-associated severe systemic infection and acute liver failure in an adult hematopoietic stem cell transplant recipient. Additionally, we illustrate the utility of molecular methods for the detection and typing of enteroviral infections.
Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database
Authors: Iakhiaeva Elena , McNulty Steven , Brown Elliott Barbara A. , Falkinham Joseph O. , Williams Myra D. , Vasireddy Ravikiran , Wilson Rebecca W. , Turenne Christine , Wallace Richard J.
Published: November 2012

Strain comparison is important to population genetics and to evaluate relapses in patients with
Mycobacterium avium
complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of
M. intracellulare
from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of
M. intracellulare
were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167
M. intracellulare
isolates, VNTR distinguished
M. intracellulare
into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.
First Clinical Cases of OXA-48-Producing Carbapenem-Resistant Klebsiella pneumoniae in the United States: the “Menace” Arrives in the New World
Authors: Mathers Amy J. , Hazen Kevin C. , Carroll Joanne , Yeh Anthony J. , Cox Heather L. , Bonomo Robert A. , Sifri Costi D.
Published: November 2012

OXA-48 has emerged as a major carbapenemase associated with the
Enterobacteriaceae
in Europe, North Africa, and Asia. We report the first two clinical cases of OXA-48-type carbapenemase-producing
Enterobacteriaceae
in the United States from patients recently hospitalized in Saudi Arabia and India. Each is more carbapenem resistant than nearly all previously reported OXA-48-type-producing
Enterobacteriaceae
.
Seroepidemiological Evidence of Avian Influenza A Virus Transmission to Pigs in Southern China
Authors: Su Shuo , Qi Wenbao , Chen Jidang , Zhu Wanjun , Huang Zhen , Xie Jiexiong , Zhang Guihong
Published: November 2012

Recently, three novel avian-origin swine influenza viruses (SIVs) were first isolated from pigs in Guangdong Province, southern China, yet little is known about the seroprevalence of avian influenza viruses among pigs in southern China. Here, we report for the first time the seroprevalence of avian H3, H4, and H6 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 influenza virus transmission to pigs in China.
Development and Evaluation of a Novel, Semiautomated Clostridium difficile Typing Platform
Authors: Westblade Lars F. , Chamberland Robin R. , MacCannell Duncan , Collins Rachel , Dubberke Erik R. , Dunne W. Michael , Burnham Carey-Ann D.
Published: November 2012

We describe a novel, semiautomated
Clostridium difficile
typing platform that is based on PCR-ribotyping in conjunction with a semiautomated molecular typing system. The platform is reproducible with minimal intra- or interassay variability. This method exhibited a discriminatory index of 0.954 and is therefore comparable to more arduous typing systems, such as pulsed-field gel electrophoresis.
A Laboratory-Developed TaqMan Array Card for Simultaneous Detection of 19 Enteropathogens
Authors: Liu Jie , Gratz Jean , Amour Caroline , Kibiki Gibson , Becker Stephen , Janaki Lalitha , Verweij Jaco J. , Taniuchi Mami , Sobuz Shihab U. , Haque Rashidul , Haverstick Doris M. , Houpt Eric R.
Published: November 2012

The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (
Campylobacter jejuni/C. coli
,
Clostridium difficile
,
Salmonella
,
Vibrio cholerae
, diarrheagenic
Escherichia coli
strains including enteroaggregative
E. coli
[EAEC], enterotoxigenic
E. coli
[ETEC], enteropathogenic
E. coli
[EPEC], and Shiga-toxigenic
E. coli
[STEC]),
Shigella
/enteroinvasive
E. coli
(EIEC), protozoa (
Cryptosporidium
,
Giardia lamblia
, and
Entamoeba histolytica
), and helminths (
Ascaris lumbricoides
and
Trichuris trichiura
), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.
Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery
Authors: Sèle Céleste , Gabel Frank , Gutsche Irina , Ivanov Ivan , Burmeister Wim P. , Iseni Frédéric , Tarbouriech Nicolas
Published: November 2012

Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the
Orthopoxvirus
genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.
The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89
Authors: Borst Eva Maria , Kleine-Albers Jennifer , Gabaev Ildar , Babić Marina , Wagner Karen , Binz Anne , Degenhardt Inga , Kalesse Markus , Jonjić Stipan , Bauerfeind Rudolf , Messerle Martin
Published: November 2012

Cleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.
An Increasing Proportion of Monotypic HIV-1 DNA Sequences during Antiretroviral Treatment Suggests Proliferation of HIV-Infected Cells
Authors: Wagner Thor A. , McKernan Jen L. , Tobin Nicole H. , Tapia Ken A. , Mullins James I. , Frenkel Lisa M.
Published: November 2012

Understanding how HIV-1 persists during effective antiretroviral therapy (ART) should inform strategies to cure HIV-1 infection. We hypothesize that proliferation of HIV-1-infected cells contributes to persistence of HIV-1 infection during suppressive ART. This predicts that identical or monotypic HIV-1 DNA sequences will increase over time during ART. We analyzed 1,656
env
and
pol
sequences generated following single-genome amplification from the blood and sputum of six individuals during long-term suppressive ART. The median proportion of monotypic sequences increased from 25.0% prior to ART to 43.2% after a median of 9.8 years of suppressive ART. The proportion of monotypic sequences was estimated to increase 3.3% per year (95% confidence interval, 2.3 to 4.4%;
P
< 0.001). Drug resistance mutations were not more common in the monotypic sequences, arguing against viral replication during times with lower antiretroviral concentrations. Bioinformatic analysis found equivalent genetic distances of monotypic and nonmonotypic sequences from the predicted founder virus sequence, suggesting that the relative increase in monotypic variants over time is not due to selective survival of cells with viruses from the time of acute infection or from just prior to ART initiation. Furthermore, while the total HIV-1 DNA load decreased during ART, the calculated concentration of monotypic sequences was stable in children, despite growth over nearly a decade of observation, consistent with proliferation of infected CD4
+
T cells and slower decay of monotypic sequences. Our findings suggest that proliferation of cells with proviruses is a likely mechanism of HIV-1 DNA persistence, which should be considered when designing strategies to eradicate HIV-1 infection.
Comparative Analysis of Simian Immunodeficiency Virus Gag-Specific Effector and Memory CD8
+
T Cells Induced by Different Adenovirus Vectors
Authors: Tan Wendy G. , Jin Hyun-Tak , West Erin E. , Penaloza-MacMaster Pablo , Wieland Andreas , Zilliox Michael J. , McElrath M. Juliana , Barouch Dan H. , Ahmed Rafi
Published: November 2012

Adenovirus (Ad) vectors are widely used as experimental vaccines against several infectious diseases, but the magnitude, phenotype, and functionality of CD8
+
T cell responses induced by different adenovirus serotypes have not been compared. To address this question, we have analyzed simian immunodeficiency virus Gag-specific CD8
+
T cell responses in mice following vaccination with Ad5, Ad26, and Ad35. Our results show that although Ad5 is more immunogenic than Ad26 and Ad35, the phenotype, function, and recall potential of memory CD8
+
T cells elicited by these vectors are substantially different. Ad26 and Ad35 vectors generated CD8
+
T cells that display the phenotype and function of long-lived memory T cells, whereas Ad5 vector-elicited CD8
+
T cells are of a more terminally differentiated phenotype. In addition, hepatic memory CD8
+
T cells elicited by Ad26 and Ad35 mounted more robust recall proliferation following secondary challenge than those induced by Ad5. Furthermore, the boosting potential was higher following priming with alternative-serotype Ad vectors than with Ad5 vectors in heterologous prime-boost regimens. Anamnestic CD8
+
T cell responses were further enhanced when the duration between priming and boosting was extended from 30 to 60 days. Our results demonstrate that heterologous prime-boost vaccine regimens with alternative-serotype Ad vectors elicited more functional memory CD8
+
T cells than any of the regimens containing Ad5. In summary, these results suggest that alternative-serotype Ad vectors will prove useful as candidates for vaccine development against human immunodeficiency virus type 1 and other pathogens and also emphasize the importance of a longer rest period between prime and boost for generating optimal CD8
+
T cell immunity.
The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells
Authors: Díaz-Salinas Marco A. , Romero Pedro , Espinosa Rafaela , Hoshino Yasutaka , López Susana , Arias Carlos F.
Published: November 2012

Rotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface protein VP7 and the spike protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway.
Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture
Authors: Sadeghipour Sara , Bek Emily J. , McMinn Peter C.
Published: November 2012

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 μM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 μM ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 μM ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.
Holoendemic Malaria Exposure Is Associated with Altered Epstein-Barr Virus-Specific CD8
+
T-Cell Differentiation
Authors: Chattopadhyay Pratip K. , Chelimo Kiprotich , Embury Paula B. , Mulama David H. , Sumba Peter Odada , Gostick Emma , Ladell Kristin , Brodie Tess M. , Vulule John , Roederer Mario , Moormann Ann M. , Price David A.
Published: November 2012

Coinfection with
Plasmodium falciparum
malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8
+
T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8
+
T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8
+
T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8
+
T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8
+
T cells or the global CD8
+
memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8
+
T-cell compartment that illuminates the etiology of eBL.
Differential Requirements for HIV-1 Vif-Mediated APOBEC3G Degradation and RUNX1-Mediated Transcription by Core Binding Factor Beta
Authors: Du Juan , Zhao Ke , Rui Yajuan , Li Peng , Zhou Xiaohong , Zhang Wenyan , Yu Xiao-Fang
Published: November 2012

Core binding factor beta (CBFβ), a transcription regulator through RUNX binding, was recently reported critical for Vif function. Here, we mapped the primary functional domain important for Vif function to amino acids 15 to 126 of CBFβ. We also revealed that different lengths and regions are required for CBFβ to assist Vif or RUNX. The important interaction domains that are uniquely required for Vif but not RUNX function represent novel targets for the development of HIV inhibitors.
Screening and Rational Design of Hepatitis C Virus Entry Inhibitory Peptides Derived from GB Virus A NS5A
Authors: Liu Xiuying , Huang Yibing , Cheng Min , Pan Ling , Si Youhui , Li Guirong , Niu Yuqiang , Zhao Lianjing , Zhao Jin , Li Xiang , Chen Yuxin , Yang Wei
Published: November 2012

Chronic infection by hepatitis C virus (HCV) is a cause of the global burden of liver diseases. HCV entry into hepatocytes is a complicated and multistep process that represents a promising target for antiviral intervention. The recently reported amphipathic α-helical virucidal peptide (C5A) from the HCV NS5A protein suggests a new category of antiviral drug candidates. In this study, to identify C5A-like HCV inhibitors, synthetic peptides derived from the C5A-corresponding NS5 protein region of selected
Flaviviridae
viruses were evaluated for their anti-HCV activities. A peptide from GB virus A (GBV-A), but not other flaviviruses, demonstrated an inhibitory effect on HCV infection. Through a series of sequence optimizations and modifications of the peptide helicity and hydrophobicity, we obtained a peptide designated GBVA10-9 with highly potent anti-HCV activity. GBVA10-9 suppressed infection with both cell culture-derived and pseudotyped HCV
in vitro
, and the 50% cell culture inhibitory concentration ranged from 20 nM to 160 nM, depending on the genotypic origin of the envelope proteins. GBVA10-9 had no detectable effects on either HCV attachment to Huh7.5.1 cells or viral RNA replication. No virucidal activity was found with GBVA10-9, suggesting an action mechanism distinct from that of C5A. The inhibitory effect of GBVA10-9 appeared to occur at the postbinding step during viral entry. Taken together, the results with GBVA10-9 demonstrated a potent activity for blocking HCV entry that might be used in combination with other antivirals directly targeting virus-encoded enzymes. Furthermore, GBVA10-9 also provides a novel tool to dissect the detailed mechanisms of HCV entry.
A New Rabies Vaccine Based on a Recombinant Orf Virus (Parapoxvirus) Expressing the Rabies Virus Glycoprotein
Authors: Amann Ralf , Rohde Jörg , Wulle Ulrich , Conlee Douglas , Raue Rudiger , Martinon Olivier , Rziha Hanns-Joachim
Published: November 2012

The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (107PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally,in vivoantibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals.
Antibodies with High Avidity to the gp120 Envelope Protein in Protection from Simian Immunodeficiency Virus SIV
mac251
Acquisition in an Immunization Regimen That Mimics the RV-144 Thai Trial
Authors: Pegu Poonam , Vaccari Monica , Gordon Shari , Keele Brandon F. , Doster Melvin , Guan Yongjun , Ferrari Guido , Pal Ranajit , Ferrari Maria Grazia , Whitney Stephen , Hudacik Lauren , Billings Erik , Rao Mangala , Montefiori David , Tomaras Georgia , Alam S. Munir , Fenizia Claudio , Lifson Jeffrey D. , Stablein Donald , Tartaglia Jim , Michael Nelson , Kim Jerome , Venzon David , Franchini Genoveffa
Published: November 2012

The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8
+
T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIV
mac251
that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4
+
and CD8
+
T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIV
mac251
acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIV
mac251
-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIV
mac251
infectivity in cells that express high levels of α
4
β
7
integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines.
Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein
Authors: Wahid Ahmed , Helle François , Descamps Véronique , Duverlie Gilles , Penin François , Dubuisson Jean
Published: November 2012

Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.
Aerosol Transmission of Chronic Wasting Disease in White-Tailed Deer
Authors: Denkers Nathaniel D. , Hayes-Klug Jeanette , Anderson Kelly R. , Seelig Davis M. , Haley Nicholas J. , Dahmes Sallie J. , Osborn David A. , Miller Karl V. , Warren Robert J. , Mathiason Candace K. , Hoover Edward A.
Published: November 2012

While the facile transmission of chronic wasting disease (CWD) remains incompletely elucidated, studies in rodents suggest that exposure of the respiratory mucosa may be an efficient pathway. The present study was designed to address this question in the native cervid host. Here, we demonstrate aerosol transmission of CWD to deer with a prion dose >20-fold lower than that used in previous oral inoculations. Inhalation of prions may facilitate transmission of CWD and, perhaps, other prion infections.
Reactive Oxygen Species Are Induced by Kaposi's Sarcoma-Associated Herpesvirus Early during Primary Infection of Endothelial Cells To Promote Virus Entry
Authors: Bottero Virginie , Chakraborty Sayan , Chandran Bala
Published: November 2012

The entry of Kaposi's sarcoma-associated herpesvirus (KSHV) into human dermal microvascular endothelial cells (HMVEC-d), natural
in vivo
target cells, via macropinocytosis is initiated through a multistep process involving the binding of KSHV envelope glycoproteins with cell surface α3β1, αVβ3, and αVβ5 integrin molecules and tyrosine kinase ephrin-A2 receptor, followed by the activation of preexisting integrin-associated signaling molecules such as focal adhesion kinase (FAK), Src, c-Cbl, phosphoinositide 3-kinase (PI-3K), and Rho-GTPases. Many viruses, including KSHV, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells; however, the role of ROS in early events of viral entry and the induction of signaling has not been elucidated. Here we show that KSHV induced ROS production very early during the infection of HMVEC-d cells and that ROS production was sustained over the observation period (24 h postinfection). ROS induction was dependent on the binding of KSHV to the target cells, since pretreatment of the virus with heparin abolished ROS induction. Pretreatment of HMVEC-d cells with the antioxidant
N
-acetylcysteine (NAC) significantly inhibited KSHV entry, and consequently gene expression, without affecting virus binding. In contrast, H
2
O
2
treatment increased the levels of KSHV entry and infection. In addition, NAC inhibited KSHV infection-induced translocation of αVβ3 integrin into lipid rafts, actin-dependent membrane perturbations, such as blebs, observed during macropinocytosis, and activation of the signal molecules ephrin-A2 receptor, FAK, Src, and Rac1. In contrast, H
2
O
2
treatment increased the activation of ephrin-A2, FAK, Src, and Rac1. These studies demonstrate that KSHV infection induces ROS very early during infection to amplify the signaling pathways necessary for its efficient entry into HMVEC-d cells via macropinocytosis.
Descriptive analysis of the respiratory health status of persons exposed to Libby amphibole asbestos
Authors: Winters Charlene A , Hill Wade G , Rowse Kimberly , Black Brad , Kuntz Sandra W , Weinert Clarann
Published: November 2012

Objective

Describe respiratory health and quality of life in persons exposed to Libby amphibole asbestos (LAA) contaminated vermiculite.

Design

Cross-sectional descriptive.

Setting

Asbestos-related disease clinic in Libby, Montana USA.

Participants

329 individuals exposed to LAA; mostly men, married, between 50 and 69 years; two-thirds lived in the surrounding county; one-third lived elsewhere in the state and USA.

Primary outcome measures

Chest radiograph (CXR), pulmonary function data and the St George Respiratory Questionnaire (SGRQ).

Results

Exposure categories included vermiculite workers=7.6%; family/household contact of vermiculite worker=32%; and environmental exposure only=60%. Of the participants, 55% had only pleural abnormalities; 5.4% had only interstitial abnormalities; nearly 21% had both abnormalities and 18% had no lung abnormality on chest x-ray. Mean forced vital capacity (FVC) 95.3% (SD=18.7); forced expiratory volume (FEV1) mean 87% (SD=20.2); ratio of FEV11/FVC 95.5% (SD=12.0); and diffusing capacity (DLCO) of 83% (SD=21.7) of the percent predicted. The mean total SGRQ (38.5; SD=22.1) indicated a lower quality of life than healthy persons and persons with other chronic conditions. SGRQ subscale means were Symptoms 52.1 (SD=24.9), activity 49.4 (SD=26.9) and impacts 27.5 (SD=21.9). Participants with normal CXR differed significantly from those with both interstitial and pleural abnormalities on total, activity and impacts scores. For activity alone, subjects with normal CXR differed significantly from those with pleural disease; no differences were found for those with interstitial disease. Significant findings were found for smoking history across all pulmonary measures, and for exposure status, radiographic findings, age and gender for select pulmonary parameters. Subjects with any smoking history had significantly worse average total and subscale scores on the SGRQ.

Conclusions

Of 329 persons exposed to LAA, the majority (182) had pleural abnormalities identified on CXR. SGRQ scores for persons with abnormalities (pleural, interstitial or both) (269) differed significantly from those with a normal CXR.
A randomised clinical trial of comprehensive cardiac rehabilitation versus usual care for patients treated for infective endocarditis—the CopenHeartIEtrial protocol
Authors: Rasmussen Trine Bernholdt , Zwisler Ann-Dorthe , Sibilitz Kirstine Lærum , Risom Signe Stelling , Bundgaard Henning , Gluud Christian , Moons Philip , Winkel Per , Thygesen Lau Caspar , Hansen Jane Lindschou , Norekvål Tone Merete , Berg Selina Kikkenborg ,   the CopenHeartIE Group
Published: November 2012

Introduction

Infective endocarditis (IE) is among the most serious infectious diseases in the western world. Treatment requires lengthy hospitalisation, high-dosage antibiotic therapy and possible valve replacement surgery. Despite advances in treatment, the 1-year mortality remains at 20–40%. Studies indicate that patients experience persisting physical symptoms, diminished quality of life and difficulties returning to work up to a year postdischarge. No studies investigating the effects of rehabilitation have been published. We present the rationale and design of the CopenHeartIEtrial, which investigates the effect of comprehensive cardiac rehabilitation versus usual care for patients treated for IE.

Methods and analysis

We will conduct a randomised clinical trial to investigate the effects of comprehensive cardiac rehabilitation versus usual care on the physical and psychosocial functioning of patients treated for IE. The trial is a multicentre, parallel design trial with 1 : 1 individual randomisation to either the intervention or control group. The intervention consists of five psychoeducational consultations provided by specialised nurses and a 12-week exercise training programme. The primary outcome is mental health (MH) measured by the standardised Short Form 36 (SF-36). The secondary outcome is peak oxygen uptake measured by the bicycle ergospirometry test. Furthermore, a number of exploratory analyses will be performed. Based on sample size calculation, 150 patients treated for left-sided (native or prosthetic valve) or cardiac device endocarditis will be included in the trial. A qualitative and a survey-based complementary study will be undertaken, to investigate postdischarge experiences of the patients. A qualitative postintervention study will explore rehabilitation participation experiences.

Ethics and dissemination

The study complies with the Declaration of Helsinki and was approved by the regional research ethics committee (no H-1-2011-129) and the Danish Data Protection Agency (no 2007-58-0015). Study findings will be disseminated widely through peer-reviewed publications and conference presentations.

Registration

Clinicaltrials.gov identifier:NCT01512615.
Coordinate My Care: a clinical service that coordinates care, giving patients choice and improving quality of life
Authors: Smith Clare , Hough Libby , Cheung Chi-Chi , Millington-Sanders Catherine , Sutton Eileen , Ross Joy R , Thick Michael , Riley Julia
Published: November 2012

Introduction

If palliative care is to lead the way towards a new model for integrated care, the ability to share information across the whole of health and social care is essential. Coordinate My Care (CMC) is a service dedicated to preserving dignity and autonomy at the end of life. Its care pathways enable health professionals from primary and secondary care to put the patient at the centre of health care delivery. This service is underpinned by an electronic solution. The CMC record can be accessed 24/7 by all health and social care professionals who have a legitimate relationship with the patient.

Patients and methods

The record displays diagnosis, prognosis, advanced care plan, resuscitation status and patients wishes for end of life care patients across London. This article is a review of CMC use and outcomes from August 2010- March 2012.

Results

1087 CMC patient records were analysed. The primary diagnosis was non-cancer in 52.3%, cancer in 46.3% and unknown in 1.4%, 42% of all patients were cared for by generalist only and had no specialist palliative care involvement. At the time of analysis 207 had died. Actual place of death was home 38.6%, care/nursing home 16.4%, hospice 12.1% and 30% in hospital. This equates to 55% of patients dying in their usual place of residence and reflects that 70% died outside of hospital.

Conclusion

The CMC service has a well defined pathway underpinned by an electronic solution. It has been shown to change culture and deliver fully integrated, personalised end of life care. A pan-London CMC roll out will take place over the next 12 months.
The impact of adding clinical assistants on patient waiting time in a crowded emergency department
Authors: Huang Edward Pei-Chuan , Liu Sot Shih-Hung , Fang Cheng-Chung , Chou Hao-Chang , Wang Chih-Hung , Yen Zui-Shen , Chen Shyr-Chyr
Published: November 2012

Background

Emergency department (ED) crowding causes prolonged waiting times.

Objective

To evaluate the potential benefit of introducing clinical assistants to a busy and crowded ED.

Methods

This was a retrospective cohort study at an urban, academic tertiary medical centre. We introduced one clinical assistant to each ED shift. The main task of clinical assistants was managing the flow of incoming ED patients. The case group consisted of all adult non-trauma emergency patients during the case period from 1 September to 30 November 2008. The first control group consisted of all adult non-trauma emergency patients between 1 June and 31 August 2008 and the second control group consisted of all patients treated between 1 September and 30 November 2007. The primary outcome was the ‘waiting time’, defined as the time from triage to the time of the first medical order entered into the computer system. The secondary outcome was the number of adult non-trauma emergency patients who left the ED without being seen.

Results

There were 12 257 cases and 25 950 controls. The mean and median waiting times were significantly shorter in the case group. The mean waiting time of the case group was 20.86 min, which was 4.51 min (17.8%) shorter than that of the first control group and 7.41 min (26.2%) shorter than that of the second control group. The median waiting time of the case group was also significantly shorter than those of the control groups. The number of the patients who left without being seen was significantly smaller in the case period.

Conclusions

In a busy and crowded ED, the introduction of clinical assistants to an existing emergency health service effectively reduces patient waiting times and decreases the number of patients leaving without being seen.
Human alpha-actinin-3 genotype association with exercise-induced muscle damage and the repeated-bout effect
Authors: Venckunas Tomas , Skurvydas Albertas , Brazaitis Marius , Kamandulis Sigitas , Snieckus Audrius , Moran Colin N.
Published: November 2012

Alpha-actinin-3 (ACTN3) is an integral part of the Z line of the sarcomere. The ACTN3 R577X (rs1815739) polymorphism determines the presence or absence of functional ACTN3, which may influence the extent of exercise-induced muscle damage. This study aimed to compare the impact of, and recovery from, muscle-damaging eccentric exercise on subjects with or without functional ACTN3. Seventeen young men (20–33 years old), homozygous for the R (n = 9) or X (n = 8) alleles, performed two bouts of stretch–shortening exercise (50 drop jumps) two weeks apart. Muscle soreness, plasma creatine kinase (CK) activity, jump height, maximal voluntary isometric torque (MVC), peak concentric isokinetic torque (IT), and electrically stimulated knee extension torques at 20 and 100 Hz were measured at baseline and at a number of time points up to 14 days after each bout. There were no significant baseline differences between the groups. However, significant time point × genotype interactions were observed for MVC (p = 0.021) and IT (p = 0.011) for the immediate effect of eccentric exercise in bout 1. The RR group showed greater voluntary force decrements (RR vs. XX: MVC, –33.3% vs. –24.5%; IT, –35.9% vs. –23.2%) and slower recovery. A repeated-bout effect was clearly observed, but there were no differences by genotype group. The ACTN3 genotype modulates the response of muscle function to plyometric jumping exercise, although the differences are modest. The ACTN3 genotype does not influence the clearly observed repeated-bout effect; however, XX homozygotes recover baseline voluntary torque values faster and thus may be able to undertake more frequent training sesssions.