bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-Protective Antigen IgG Enzyme-Linked Immunosorbent Assay for Diagnosis of Cutaneous Anthrax in India
Authors: Ghosh N. , Goel A. K.
Published: June 2012

Anthrax caused byBacillus anthracisis a public health problem in several developing countries whose main source of income is farming. Anthrax is a disease of herbivorous animals, and humans can be infected by handling infected animals or contaminated animal products. Specific diagnostic tests are unavailable in India for the detection and confirmation of cutaneous anthrax in humans. Here, we describe the development of an enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies againstBacillus anthracisprotective antigen in the Indian population. A total of 405 serum samples collected from different groups were tested by the developed ELISA. The assay provided a specificity of 99.41% (95% confidence interval [CI], 97.89 to 99.93) and a sensitivity of 100% (CI, 94.4 to 100) using a cutoff value of 0.29 ELISA unit (EU). The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 97% and 100%, respectively. The efficiency and J index for the reliability of the assay were 99.5% and 0.994, respectively. The assay can be a very useful tool for surveillance as well as for diagnosis of cutaneous anthrax cases in India.
Risk Factors Associated with Indeterminate Gamma Interferon Responses in the Assessment of Latent Tuberculosis Infection in a High-Incidence Environment
Authors: Oni Tolu , Gideon Hannah P. , Bangani Nonzwakazi , Tsekela Relebohile , Seldon Ronnett , Wood Kathryn , Wilkinson Katalin A. , Goliath Rene T. , Ottenhoff Tom H. M. , Wilkinson Robert J.
Published: June 2012

The performance of gamma interferon (IFN-γ) release assays (IGRA) in the detection of latent tuberculosis (TB) infection is limited by the higher rates of indeterminate results in HIV-infected persons, who bear the brunt of TB disease in some high-burden settings. The objective of the study was to evaluate predictors of indeterminate IGRA results in the overall study population and in HIV-infected persons. The study setting is Khayelitsha, an informal township in the Western Cape of South Africa, with a high burden of TB and HIV infection. A total of 561 asymptomatic persons were recruited from the day hospital and youth centers. A questionnaire was used to collect demographic information, and blood tests, including CD4 counting and a 7-day in-house IGRA, were performed. The overall prevalence of indeterminate IGRA results was 8.6% (48/561), and this was higher in HIV-infected than in HIV-uninfected persons (11.5% [38/330] versus 4.3% [10/231], respectively;
P
= 0.003). In the overall study population, predictors of indeterminate IGRA results were the presence of HIV infection (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.10 to 5.08) and the presence of a
Mycobacterium bovis
BCG scar (OR, 2.48; 95% CI, 1.23 to 5.01). Long-term township residents were significantly less likely to have indeterminate results than recent migrants (OR, 0.30; 95% CI, 0.11 to 0.80). Among HIV-infected persons, participants with CD4 counts of >200 cells/mm
3
and long-term residents were significantly less likely to have indeterminate IGRA results (OR of 0.21 with a 95% CI of 0.09 to 0.48 and OR of 0.22 with a 95% CI of 0.07 to 0.68, respectively). We evaluated risk factors for indeterminate IGRA results and report a higher rate of indeterminate results among HIV-infected persons, particularly those with lower CD4 counts. Of note, a recent move to the township was associated with a higher risk of indeterminate IGRA results.
Evaluation of Three Commercial Varicella-Zoster Virus IgG Enzyme-Linked Immunosorbent Assays in Comparison to the Fluorescent-Antibody-to-Membrane-Antigen Test
Authors: Sauerbrei Andreas , Schäfler Anna , Hofmann Jörg , Schacke Michael , Gruhn Bernd , Wutzler Peter
Published: June 2012

Commercial serologic assays for varicella-zoster virus (VZV), which enable reliable determination of VZV immune status and are amenable to automation, are needed. The present study compares the automated performance of the VZV whole-cell enzyme-linked immunosorbent assay (ELISA) Enzygnost anti-VZV/IgG, the Euroimmun anti-VZV ELISA (IgG) based on highly purified viral proteins, and the VZV glycoprotein (gp)-based Serion ELISA Classic VZV IgG. The fluorescent-antibody-to-membrane-antibody (FAMA) test was used as a reference. A total of 638 serum samples from VZV-negative children, blood donors, varicella vaccinees, and bone marrow transplant recipients were included. The Enzygnost anti-VZV/IgG and the Serion ELISA Classic VZV IgG showed sensitivities of 99.6% and 99.2%, respectively, and the Euroimmun anti-VZV ELISA (IgG) had a significantly lower sensitivity of 90.5%. Specificity was calculated as 100% for both the Euroimmun anti-VZV ELISA (IgG) and for the Enzygnost anti-VZV/IgG, and the Serion ELISA Classic VZV IgG had a significantly lower specificity of 89.4%. Quantitative results of all ELISAs correlated well, but there was a poor quantitative correlation between the ELISAs and FAMA. In conclusion, this study does not show any superiority of a gp- and a protein-based ELISA compared to a whole-cell ELISA for the automated detection of VZV-specific IgG. The automated performance of the Enzygnost anti-VZV/IgG assay correlated best with the FAMA reference assay.
A High Percentage of Serum Samples That Test Reactive by Enzyme Immunoassay for Anti-Brucella Antibodies Are Not Confirmed by the Standard Tube Agglutination Test
Authors: Binnicker Matthew J. , Theel Elitza S. , Larsen Staci M. , Patel Robin
Published: June 2012

We describe a retrospective analysis ofBrucellaenzyme immunoassay (EIA) IgM and IgG results compared to those of the standard tube agglutination test (SAT). Among 1,091 samples tested, 104 (9.5%) and 24 (2.2%) sera were positive by IgM and IgG EIA, respectively. Supplemental testing by SAT showed that 82.7% (86/104) of IgM EIA-reactive samples and 54.2% (13/24) of IgG EIA-reactive samples were negative by SAT. Testing all EIA screen-reactive samples by SAT is required when evaluating patients for potential brucellosis. Due to the limitations of serology, culture remains the gold standard for detectingBrucellainfection.
Duration of Immunity against Mycobacterium bovis following Neonatal Vaccination with Bacillus Calmette-Guérin Danish: Significant Protection against Infection at 12, but Not 24, Months
Authors: Thom M. L. , McAulay M. , Vordermeier H. M. , Clifford D. , Hewinson R. G. , Villarreal-Ramos B. , Hope J. C.
Published: June 2012

Vaccination of neonatal calves withMycobacterium bovisbacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulentM. bovis. In two independent experiments, we assessed the duration of the protective immunity induced in calves by neonatal vaccination with BCG Danish. Protection from disease was assessed at 12 and 24 months postvaccination in cattle challenged via the endotracheal route withM. bovis. We also assessed antigen-specific immune responses to assess their utility as correlates of protection. At 12 months postvaccination, significant reductions in lung and lymph node pathologies were observed compared to nonvaccinatedM. bovis-challenged control cattle. At 24 months post-BCG vaccination, there was a reduction in lung and lymph node pathology scores and in bacterial burden. However, when comparing vaccinated and control groups, this did not reach statistical significance. Vaccination induced long-lived antigen (purified protein derivative [PPD])-specific gamma interferon (IFN-γ) release in whole-blood cultures, which remained above baseline levels for more than 20 months (approximately 90 weeks). The number of antigen-specific IFN-γ-secreting central memory T cells present at the time ofM. bovischallenge was significantly higher in vaccinated than in control animals at 12 months postvaccination, but not at 24 months. Vaccination of neonatal calves with BCG Danish induced protective immune responses against bovine TB which were maintained for at least 12 months postvaccination. These studies provide data on the immunity induced by BCG vaccination in calves; the results could inform vaccination strategies for the control of bovine TB in United Kingdom cattle herds.
Differentiation of Streptococcus pneumoniae from Nonpneumococcal Streptococci of the Streptococcus mitis Group by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Authors: Werno Anja M. , Christner Martin , Anderson Trevor P. , Murdoch David R.
Published: June 2012

The differentiation of species within the
Streptococcus mitis
group has posed a problem in the routine diagnostic microbiology laboratory for some time. It also constitutes a major weakness of recently introduced matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) fingerprinting systems. As the phylogenetic resolution of the spectral similarity measures employed by these systems is insufficient to reliably distinguish between the most closely related members of the group, the major pathogen
Streptococcus pneumoniae
is frequently misidentified. In this study, a comparative analysis of MALDI-TOF spectra of several species from the
S. mitis
group has been performed in order to identify single peaks that could be used to improve mass spectrometry-based identification of the respective species. A characteristic peak profile could be identified that unambiguously distinguished the 14
S. pneumoniae
isolates studied from 33 nonpneumococcal isolates of the
S. mitis
group. In addition, specific peak combinations could be assigned to other members of the group. The findings of this study suggest that it is possible to distinguish different species of the
S. mitis
group by close analysis of their mass peak profiles.
Molecular Characterization Reveals Three Distinct Clonal Groups among Clinical Shiga Toxin-Producing Escherichia coli Strains of Serogroup O103
Authors: Iguchi Atsushi , Iyoda Sunao , Ohnishi Makoto
Published: June 2012

Shiga toxin-producingEscherichia coli(STEC) is one of the most important groups of food-borne pathogens, and STEC strains belonging to the serotype O103:H2 can cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. STEC O103:non-H2 strains are also sometimes isolated from human patients, but their genetic characteristics and role in significant human enteric disease are not yet understood. Here, we investigated 17 STEC O103:non-H2 strains, including O103:H11, O103:H25, O103:HUT (UT [untypeable]), and O103:H− (nonmotile) isolated in Japan, and their characteristics were compared to those of STEC O103:H2 and other serotype STEC strains. Sequence analyses offliCandeaegenes revealed that strains possessed any of the following combinations:fliC-H2/eae-epsilon,fliC-H11/eae-beta1, andfliC-H25/eae-theta, wherefliC-H2, -H11, and -H25 indicatefliCgenes encoding H2, H11, and H25 flagella antigens, respectively, andeae-epsilon, -beta1, and -theta indicateeaegenes encoding epsilon, beta1, and theta subclass intimins, respectively. Phylogenetic analysis based on the sequences of seven housekeeping genes demonstrated that the O103:H11/[fliC-H11] and O103:H25/[fliC-H25] strains formed two distinct groups, different from that of the O103:H2/[fliC-H2] strains. Interestingly, a group consisting of O103:H11 strains was closely related to STEC O26:H11, which is recognized as a most important non-O157 serotype, suggesting that the STEC O103:H11 and STEC O26:H11 clones evolved from a common ancestor. The multiplex PCR system for the rapid typing of STEC O103 strains described in the present study may aid clinical and epidemiological studies of the STEC O103:H2, O103:H11, and O103:H25 groups. In addition, our data provide further insights into the high variability of STEC stains with emerging new serotypes.
Phylogenetic Analysis of the Spirochete Borrelia microti, a Potential Agent of Relapsing Fever in Iran
Authors: Naddaf Saied Reza , Ghazinezhad Behnaz , Bahramali Golnaz , Cutler Sally Jane
Published: June 2012

We report a role for
Borrelia microti
as a cause of relapsing fever in Iran supported by robust epidemiological evidence. The molecular identity of this spirochete and its relation with other relapsing fever borreliae have, until now, been poorly delineated. We analyzed an isolate of
B. microti
, obtained from
Ornithodoros erraticus
ticks, by sequencing four loci (16S rRNA,
flaB
,
glpQ
, intragenic spacer [IGS]) and comparing these sequences with those of other relapsing fever borreliae. Phylogenetic analysis using concatenated sequences of 16S rRNA,
flaB
, and
glpQ
grouped
B. microti
alongside three members of the African group,
B. duttonii
,
B. recurrentis
, and
B. crocidurae
, which are distinct from
B. persica
, the most prevalent established cause of tick-borne relapsing fever in Iran. The similarity values for 10 concatenated sequences totaling 2,437 nucleotides ranged from 92.11% to 99.84%, with the highest homologies being between
B. duttonii
and
B. microti
and between
B. duttonii
and
B. recurrentis
. Furthermore, the more discriminatory IGS sequence analysis corroborated the close similarity (97.76% to 99.56%) between
B. microti
and
B. duttonii
. These findings raise the possibility that both species may indeed be the same and further dispel the one-species, one-vector theory that has been the basis for classification of relapsing fever
Borrelia
for the last 100 years.
High Hepatitis E Virus Seroprevalence in Forestry Workers and in Wild Boars in France
Authors: Carpentier Audrey , Chaussade Hélène , Rigaud Emma , Rodriguez Josefa , Berthault Camille , Boué Franck , Tognon Mauro , Touzé Antoine , Garcia-Bonnet Nathalie , Choutet Patrick , Coursaget Pierre
Published: June 2012

Hepatitis E virus (HEV) is a fecally and orally transmitted human pathogen of worldwide distribution. In industrial countries, HEV is observed in an increasing number of autochthonous cases and is considered to be an emerging pathogen. A growing body of evidence suggests that HEV is a zoonotic disease, and pig handlers and pig veterinarians have been reported to be high-risk groups for HEV infection. The aims of the present study were to establish the prevalence of anti-HEV in wild boars in France and to identify whether forestry workers are at a higher risk of HEV infection. Three different anti-HEV tests were used to compare their effectiveness in detecting anti-HEV in the general population. The most sensitive test was then used to investigate HEV seroprevalence in 593 forestry workers and 421 wild boars. Anti-HEV was detected in 31% of the forestry workers and 14% of the wild boars. Detection of anti-HEV in humans was correlated with age, geographical location, and occupational activity and in wild boars was correlated with geographical location. HEV infection is frequent in woodcutters in France, and it varies geographically. Further studies are needed to confirm these findings and to elucidate the transmission route and the exact virus reservoirs.
Field Monitoring of Avian Influenza Viruses: Whole-Genome Sequencing and Tracking of Neuraminidase Evolution Using 454 Pyrosequencing
Authors: Croville Guillaume , Soubies Sébastien Mathieu , Barbieri Johanna , Klopp Christophe , Mariette Jérôme , Bouchez Olivier , Camus-Bouclainville Christelle , Guérin Jean-Luc
Published: June 2012

Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than
de novo
emergence of viral mutants.
Assessment of Whole-Genome Mapping in a Well-Defined Outbreak of Salmonella enterica Serotype Saintpaul
Authors: Fey P. D. , Iwen P. C. , Zentz E. B. , Briska A. M. , Henkhaus J. K. , Bryant K. A. , Larson M. A , Noel R. K. , Hinrichs S. H.
Published: June 2012

We investigated the use of whole-genome mapping and pulsed-field gel electrophoresis (PFGE) with isolates from an outbreak of
Salmonella enterica
serotype Saintpaul. PFGE and whole-genome mapping were concordant with 22 of 23 isolates. Whole-genome mapping is a viable alternative tool for the epidemiological analysis of
Salmonella
food-borne disease investigations.
Optimization of the Preanalytical Steps of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Identification Provides a Flexible and Efficient Tool for Identification of Clinical Yeast Isolates in Medical Laboratories
Authors: Goyer Marianne , Lucchi Geraldine , Ducoroy Patrick , Vagner Odile , Bonnin Alain , Dalle Frederic
Published: June 2012

We report here that modifications of the preanalytical steps of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification of yeasts, with regard to the original protocol provided by the manufacturers, appear to be efficient for the reliable routine identification of clinical yeast isolates in medical laboratories. Indeed, when one colony was sampled instead of five and the protein extraction protocol was modified, the performance of MALDI-TOF MS was superior to that of the API ID 32C method (discrepancies were confirmed by using molecular identification), allowing the correct identification of 94% of the 335 clinical isolates prospectively tested. We then demonstrated that the time for which the primary cultures were preincubated on CHROMagar did not impact the identification of yeasts by MALDI-TOF MS, since 95.1 and 96.2% of the 183 clinical yeast isolates prospectively tested were correctly identified after 48 and 72 h of preincubation, respectively.
Critical Analysis of Rhinovirus RNA Load Quantification by Real-Time Reverse Transcription-PCR
Authors: Schibler Manuel , Yerly Sabine , Vieille Gaël , Docquier Mylène , Turin Lara , Kaiser Laurent , Tapparel Caroline
Published: June 2012

Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an
in vitro
-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5′-untranslated regions (5′UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5′UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.
Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections
Authors: Schiffer Joshua T. , Aubert Martine , Weber Nicholas D. , Mintzer Esther , Stone Daniel , Jerome Keith R.
Published: June 2012

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches.
The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing
Authors: Raupp Christina , Naumer Matthias , Müller Oliver J. , Gurda Brittney L. , Agbandje-McKenna Mavis , Kleinschmidt Jürgen A.
Published: June 2012

Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the
in vivo
gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2
cap
sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids
588
QQNTA
592
of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process.
Expansion of Polyfunctional HIV-Specific T Cells upon Stimulation with mRNA Electroporated Dendritic Cells in the Presence of Immunomodulatory Drugs
Authors: De Keersmaecker Brenda , Allard Sabine D. , Lacor Patrick , Schots Rik , Thielemans Kris , Aerts Joeri L.
Published: June 2012

Recently, it has been demonstrated that disease progression during HIV infection is not determined merely by the number of HIV-specific T cells but also by their quality (J. R. Almeida, et al., J. Exp. Med. 204:2473–2485, 2007; C. T. Berger, et al., J. Virol. 85:9334–9345, 2011; M. R. Betts, et al., Blood 107:4781–4789, 2006; V. V. Ganusov, et al., J. Virol. 85:10518–10528, 2011; P. Kiepiela, et al., Nat. Med. 13:46–53, 2007; and F. Pereyra, et al., J. Infect. Dis. 197:563–571, 2008). Therefore, strategies to specifically enhance or induce high-quality, HIV-specific T-cell responses are necessary to develop effective immune therapies. Thalidomide, lenalidomide, and pomalidomide have a strong capacity to boost immune responses and are therefore referred to as immunomodulatory drugs (IMiDs). We evaluated the effects of lenalidomide and pomalidomide on HIV-specific T cells. We found that the presence of IMiDs duringin vitroT-cell stimulation with dendritic cells electroporated with Gag- or Nef-encoding mRNA resulted in higher numbers of cytokine-secreting HIV-specific CD8+T cells, particularly inducing polyfunctional HIV-specific CD8+T cells with an enhanced lytic capacity. Furthermore, CD8+T-cell responses were detected upon stimulation with lower antigenic peptide concentrations, and a higher number of Gag epitopes was recognized upon addition of IMiDs. Finally, IMiDs reduced the proliferation of the HIV-specific CD4+T cells while increasing the number of polyfunctional CD4+T cells. These results provide new information about the effects of IMiDs on antigen-specific T cells and suggest that these drugs increase the efficacy of immune therapies for infectious diseases and cancer.
DNA Prime-Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-Cell Response against Hepadnaviral Proteins in the Mouse and Woodchuck Model
Authors: Kosinska Anna D. , Johrden Lena , Zhang Ejuan , Fiedler Melanie , Mayer Anja , Wildner Oliver , Lu Mengji , Roggendorf Michael
Published: June 2012

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8+T-cell response than with the previously used vaccines. Using multicolor flow cytometry and anin vivocytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.
Foot-and-Mouth Disease Virus 3C Protease Cleaves NEMO To Impair Innate Immune Signaling
Authors: Wang Dang , Fang Liurong , Li Kui , Zhong Huijuan , Fan Jinxiu , Ouyang Chao , Zhang Huan , Duan Erzhen , Luo Rui , Zhang Zhongming , Liu Xiangtao , Chen Huanchun , Xiao Shaobo
Published: June 2012

Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (L
pro
) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C
pro
, also inhibits IFN-α/β response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C
pro
of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-κB and interferon-regulatory factor signaling pathways. 3C
pro
specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3C
pro
abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C
pro
and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.
Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation
Authors: McNamara Lucy A. , Ganesh Janani A. , Collins Kathleen L.
Published: June 2012

The ability of HIV-1 to establish a latent infection presents a barrier to curing HIV. The best-studied reservoir of latent virusin vivois resting memory CD4+T cells, but it has recently been shown that CD34+hematopoietic progenitor cells (HPCs) can also become latently infected by HIV-1in vitroandin vivo. CD34+cells are not homogenous, however, and it is not yet known which types of CD34+cells support a latent infection. Furthermore, the mechanisms through which latency is established in this cell type are not yet known. Here we report the development of a primary cell model for latent HIV-1 infection in HPCs. We demonstrate that in this model, latent infection can be established in all subsets of HPCs examined, including HPCs with cell surface markers consistent with immature hematopoietic stem and progenitor cells. We further show that the establishment of latent infection in these cells can be reversed by tumor necrosis factor alpha (TNF-α) through an NF-κB-dependent mechanism. In contrast, we do not find evidence for a role of positive transcription elongation factor b (P-TEFb) in the establishment of latent infection in HPCs. Finally, we demonstrate that prostratin and suberoylanilide hydroxamic acid (SAHA), but not hexamethylene bisacetamide (HMBA) or 5-aza-2′-deoxycytidine (Aza-CdR), reactivate latent HIV-1 in HPCs. These findings illuminate the mechanisms through which latent infection can be established in HPCs and suggest common pathways through which latent virus could be reactivated in both HPCs and resting memory T cells to eliminate latent reservoirs of HIV-1.
STAT4 Regulates Antiviral Gamma Interferon Responses and Recurrent Disease during Herpes Simplex Virus 2 Infection
Authors: Svensson Alexandra , Tunbäck Petra , Nordström Inger , Shestakov Andrey , Padyukov Leonid , Eriksson Kristina
Published: June 2012

STAT4 is an important transcription factor that contributes to the incidence and severity of different autoimmune diseases and is implicated in the antiviral immune responses in mice. In this study, we evaluated the role of STAT4 in human and murine herpes simplex virus 2 (HSV-2) infections. We show that STAT4 regulates antiviral gamma interferon (IFN-γ) responses and disease severity during chronic HSV-2 infections in humans and vaccine-induced IFN-γ-mediated protection against HSV-2 infection in mice. In a cohort of 228 HSV-2-infected individuals, representing both patients with recurrent disease and asymptomatic HSV-2 carriers, we found that genetic variations in theSTAT4gene were associated with asymptomatic HSV-2 infection, as well as with increasedin vitrosecretion of IFN-γ in response to the virus. Mice that lacked STAT4 had impaired HSV-2-specific IFN-γ production and delayed-type hypersensitivity responses following vaccination, which led to impaired viral clearance in the genital tract of vaccinated animals after a genital HSV-2 challenge. We conclude that STAT4 plays an important role in IFN-γ-mediated HSV-2-specific immunity, affecting the severity of genital HSV-2 infection.
Large Ribosomal Protein 4 Increases Efficiency of Viral Recoding Sequences
Authors: Green Lisa , Houck-Loomis Brian , Yueh Andrew , Goff Stephen P.
Published: June 2012

Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end ofgag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release.
Reevaluation of the Coding Potential and Proteomic Analysis of the BAC-Derived Rhesus Cytomegalovirus Strain 68-1
Authors: Malouli Daniel , Nakayasu Ernesto S. , Viswanathan Kasinath , Camp David G. , Chang W. L. William , Barry Peter A. , Smith Richard D. , Früh Klaus
Published: June 2012

Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Mostin vivoexperiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown, and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several related Old World monkey (OWM) CMVs, we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs could be detected. Taken together, these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes, and OWMs than previously assumed.
The First Full-Length Endogenous Hepadnaviruses: Identification and Analysis
Authors: Liu Wei , Pan Shaokun , Yang Huijuan , Bai Weiya , Shen Zhongliang , Liu Jing , Xie Youhua
Published: June 2012

In silico
screening of metazoan genome data identified multiple endogenous hepadnaviral elements in the budgerigar (
Melopsittacus undulatus
) genome, most notably two elements comprising about 1.3× and 1.0× the full-length genome. Phylogenetic and molecular dating analyses show that endogenous budgerigar hepatitis B viruses (eBHBV) share an ancestor with extant avihepadnaviruses and infiltrated the budgerigar genome millions of years ago. Identification of full-length genomes with preserved key features like ε signals could enable resurrection of ancient BHBV.
A Nuclear Localization Signal in Herpesvirus Protein VP1-2 Is Essential for Infection via Capsid Routing to the Nuclear Pore
Authors: Abaitua F. , Hollinshead M. , Bolstad M. , Crump C. M. , O'Hare P.
Published: June 2012

To initiate infection, herpesviruses must navigate to and transport their genomes across the nuclear pore. VP1-2 is a large structural protein of the virion that is conserved in all herpesviruses and plays multiple essential roles in virus replication, including roles in early entry. VP1-2 contains an N-terminal basic motif which functions as an efficient nuclear localization signal (NLS). In this study, we constructed a mutant HSV strain, K.VP1-2ΔNLS, which contains a 7-residue deletion of the core NLS at position 475. This mutant fails to spread in normal cells but can be propagated in complementing cell lines. Electron microscopy (EM) analysis of infection in noncomplementing cells demonstrated capsid assembly, cytoplasmic envelopment, and the formation of extracellular enveloped virions. Furthermore, extracellular virions isolated from noncomplementing cells had similar profiles and abundances of structural proteins. Virions containing VP1-2ΔNLS were able to enter and be transported within cells. However, further progress of infection was prevented, with at least a 500- to 1,000-fold reduction in the efficiency of initiating gene expression compared to that in the revertant. Ultrastructural and immunofluorescence analyses revealed that the K.VP1-2ΔNLS mutant was blocked at the microtubule organizing center or immediately upstream of nuclear pore docking and prior to gene expression. These results indicate that the VP1-2 NLS is not required for the known assembly functions of the protein but is a key requirement for the early routing to the nuclear pore that is necessary for successful infection. Given its conservation, we propose that this motif may also be critical for entry of other classes of herpesviruses.
Inhibition of Human Cytomegalovirus Immediate-Early Gene Expression by Cyclin A2-Dependent Kinase Activity
Authors: Oduro Jennifer D. , Uecker Ralf , Hagemeier Christian , Wiebusch Lüder
Published: June 2012

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G
0
/G
1
phase of the cell division cycle. S/G
2
cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G
2
-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G
1
was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.
Clinicopathological significance of CADM4 expression, and its correlation with expression of E-cadherin and Ki-67 in colorectal adenocarcinomas
Authors: Jang Se Min , Han Hulin , Jun Young Jin , Jang Si-Hyong , Min Kyueng-Whan , Sim Jongmin , Ahn Hye In , Lee Kang Hong , Jang Ki-Seok , Paik Seung Sam
Published: June 2012

Aims

Cell adhesion molecule 4 (CADM4) is a novel tumour suppressor. The purpose of this study was to investigate the correlation between its expression and the expression of E-cadherin and Ki-67 in colorectal adenocarcinomas, as well as its effect on patient survival.

Methods

We evaluated CADM4 expression in tissue microarrays of 513 colorectal adenocarcinomas by immunohistochemistry.

Results

CADM4 was highly expressed in 210 of the 513 colorectal adenocarcinomas; expression was reduced in 185 cases and absent in the remaining cases. Loss of CADM4 expression was correlated with larger tumour size (6.2±2.1 cm vs 5.3±2.0 cm, p<0.001), mucinous tumour type (61.5% vs 20.9%, p<0.001), lymph node metastasis (31.4% vs 20.9%, p=0.022), higher Dukes stage (25.5% vs 19.6%, p=0.044), poorer differentiation (38.5% vs 18.8%, p<0.001), absence of E-cadherin expression (28.5% vs 16.0%, p=0.007) and presence of Ki-67 expression (27.3% vs 12.3%, p<0.001). In univariable Cox regression analysis, absence of CADM4 expression was associated with poorer overall survival (HR 0.712; 95% CI 0.512 to 0.989, p=0.042) and disease-free survival (HR 0.732; 95% CI 0.546 to 0.981, p=0.037). In multivariate analysis with the Cox proportional hazards model, CADM4 expression was not an independent prognostic factor of overall survival (HR 0.726; 95% CI 0.516 to 1.021, p=0.066) and disease-free survival (HR 0.762; 95% CI 0.563 to 1.033, p=0.080).

Conclusions

Loss of CADM4 expression is relatively frequent in colorectal adenocarcinomas and may play an important role in cancer progression and patient survival.
Pathology and prognosis in pseudomyxoma peritonei: a review of 274 cases
Authors: Carr Norman J , Finch Jenny , Ilesley Ian Charles , Chandrakumaran Kandiah , Mohamed Faheez , Mirnezami Alex , Cecil Tom , Moran Brendan
Published: June 2012

Aims

The classification of abdominal mucinous neoplasia is a controversial area. In 2010, WHO published a classification which divides pseudomyxoma peritonei (PMP) into low and high grades. The aim of the authors was to correlate this classification with the prognosis and site of primary neoplasm.

Methods

The authors reviewed 274 patients with PMP who had undergone surgery at a single institution and classified them according to WHO criteria. The findings were correlated with clinical information and survival data.

Results

PMP was low grade in 78% of patients and high grade in 22%. The appendix accounted for 94% of lesions, and the most common primary tumour was a low grade appendiceal mucinous neoplasm. Colorectal primaries were more likely to be associated with high grade PMP. There was an excellent correlation between the grade of the PMP and the primary neoplasm; only two cases showed discordant morphology: both were high grade appendiceal adenocarcinomas that were associated with low grade PMP. Nodal metastases were more likely in high grade lesions, but there was no significant difference in the rate of parenchymal organ invasion between low grade and high grade. Low grade morphology was associated with significantly longer survival than high grade (overall 5-year survival of 63% for low grade and 23% for high grade).

Conclusions

Categorisation as either low grade or high grade by WHO criteria correlates with prognosis. The grade of the PMP is generally consistent with the grade of the primary neoplasm. Colorectal primaries are more likely to be associated with high grade PMP.
A Swiss neighbourhood index of socioeconomic position: development and association with mortality
Authors: Panczak Radoslaw , Galobardes Bruna , Voorpostel Marieke , Spoerri Adrian , Zwahlen Marcel , Egger Matthias ,   for the Swiss National Cohort and the Swiss Household Panel
Published: June 2012

Background

Area-based measures of socioeconomic position (SEP) suitable for epidemiological research are lacking in Switzerland. The authors developed the Swiss neighbourhood index of SEP (Swiss-SEP).

Methods

Neighbourhoods of 50 households with overlapping boundaries were defined using Census 2000 and road network data. Median rent per square metre, proportion households headed by a person with primary education or less, proportion headed by a person in manual or unskilled occupation and the mean number of persons per room were analysed in principle component analysis. The authors compared the index with independent income data and examined associations with mortality from 2001 to 2008.

Results

1.27 million overlapping neighbourhoods were defined. Education, occupation and housing variables had loadings of 0.578, 0.570 and 0.362, respectively, and median rent had a loading of −0.459. Mean yearly equivalised income of households increased from SFr42 000 to SFr72 000 between deciles of neighbourhoods with lowest and highest SEP. Comparing deciles of neighbourhoods with lowest to highest SEP, the age- and sex-adjusted HR was 1.38 (95% CI 1.36 to 1.41) for all-cause mortality, 1.83 (95% CI 1.71 to 1.95) for lung cancer, 1.48 (95% CI 1.44 to 1.51) for cardiovascular diseases, 2.42 (95% CI 1.94 to 3.01) for traffic accidents, 0.93 (95% CI 0.85 to 1.02) for breast cancer and 0.86 (95% CI 0.78 to 0.95) for suicide.

Conclusions

Developed using a novel approach to define neighbourhoods, the Swiss-SEP index was strongly associated with household income and some causes of death. It will be useful for clinical- and population-based studies, where individual-level socioeconomic data are often missing, and to investigate the effects on health of the socioeconomic characteristics of a place.
Physician payment outlook for 2012
Authors: Manchikanti Laxmaiah , Hirsch Joshua A , Barr Robert M , Donovan William D , Nicola Greg N
Published: June 2012

Physician spending is complex and intrinsically related to national health care spending, government regulations, health care reform, private insurers, physician practice and patient utilization patterns. Consequently, since the inception of Medicare programs in 1965, several methods have been used to determine the amounts paid to physicians for each covered service. The sustainable growth rate (SGR) was enacted in 1997 to determine physician payment updates under Medicare part B with an intent to reduce Medicare physician payment updates to offset the growth and utilization of physician services that exceeds the gross domestic product growth. This is achieved by setting an overall target amount of spending for physicians' services and adjusting payment rates annually to reflect differences between actual spending and the spending target. Since 2002, the SGR has annually recommended reductions in Medicare reimbursements. Payments were cut by 4.8% in 2002. Since then, Congress has intervened on 13 separate occasions to prevent additional cuts from being imposed. This manuscript describes certain important aspects of the 2012 physician fee schedule.