bluuebunny/crossref_metadata_2025 huggingface dataset:
Multilocus Sequence Typing Subtypes of Poultry Clostridium perfringens Isolates Demonstrate Disease Niche Partitioning
Authors: Hibberd M. C. , Neumann A. P. , Rehberger T. G. , Siragusa G. R.
Published: January 2011

Clostridium perfringens
is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139
C. perfringens
isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated
C. perfringens
isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified
cpb2
throughout these isolates and correlated the presence of
netB
with NE pathology. Previous investigations into the genetic basis of
C. perfringens
pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of
C. perfringens
isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated
C. perfringens
isolates, a concept with both epidemiological and evolutionary significance.
Gordonia bronchialis Bacteremia and Pleural Infection: Case Report and Review of the Literature
Authors: Johnson Jennifer A. , Onderdonk Andrew B. , Cosimi Lisa A. , Yawetz Sigal , Lasker Brent A. , Bolcen Shanna J. , Brown June M. , Marty Francisco M.
Published: January 2011

Gordonia
species are aerobic actinomycetes recently recognized as causing human disease, often in the setting of intravascular catheter-related infections. We describe a case of
Gordonia bronchialis
bacteremia and pleural space infection in the absence of an indwelling intravascular catheter and review the breadth of reported infections with this emerging pathogen.
Salmonella enterica Serotype Gambia with CTX-M-3 and
armA
Resistance Markers: Nosocomial Infections with a Fatal Outcome
Authors: Moissenet D. , Weill F.-X. , Arlet G. , Harrois D. , Girardet J. P. , Vu-Thien H.
Published: January 2011

We report two cases of bacteremia caused by the
Salmonella enterica
serotype Gambia in our children's hospital, with one fatal outcome. The isolates showed indistinguishable genotypes and infrequent resistance markers: CTX-M-3 extended-spectrum β-lactamase and
armA
methyltransferase. This is the first report of
S
. Gambia exhibiting CTX-M-3 and
armA
markers involved in serious infections.
Diagnostic Accuracy Assessment of Sensititre and Agar Disk Diffusion for Determining Antimicrobial Resistance Profiles of Bovine Clinical Mastitis Pathogens
Authors: Saini V. , Riekerink R. G. M. Olde , McClure J. T. , Barkema H. W.
Published: January 2011

Determining the accuracy and precision of a measuring instrument is pertinent in antimicrobial susceptibility testing. This study was conducted to predict the diagnostic accuracy of the Sensititre MIC mastitis panel (Sensititre) and agar disk diffusion (ADD) method with reference to the manual broth microdilution test method for antimicrobial resistance profiling of
Escherichia coli
(
n
= 156),
Staphylococcus aureus
(
n
= 154), streptococcal (
n
= 116), and enterococcal (
n
= 31) bovine clinical mastitis isolates. The activities of ampicillin, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin, the penicillin-novobiocin combination, pirlimycin, and tetracycline were tested against the isolates. Diagnostic accuracy was determined by estimating the area under the receiver operating characteristic curve; intertest essential and categorical agreements were determined as well. Sensititre and the ADD method demonstrated moderate to highly accurate (71 to 99%) and moderate to perfect (71 to 100%) predictive accuracies for 74 and 76% of the isolate-antimicrobial MIC combinations, respectively. However, the diagnostic accuracy was low for
S. aureus
-ceftiofur/oxacillin combinations and other streptococcus-ampicillin combinations by either testing method. Essential agreement between Sensititre automatic MIC readings and MIC readings obtained by the broth microdilution test method was 87%. Essential agreement between Sensititre automatic and manual MIC reading methods was 97%. Furthermore, the ADD test method and Sensititre MIC method exhibited 92 and 91% categorical agreement (sensitive, intermediate, resistant) of results, respectively, compared with the reference method. However, both methods demonstrated lower agreement for
E. coli
-ampicillin/cephalothin combinations than for Gram-positive isolates. In conclusion, the Sensititre and ADD methods had moderate to high diagnostic accuracy and very good essential and categorical agreement for most udder pathogen-antimicrobial combinations and can be readily employed in veterinary diagnostic laboratories.
Isolation and Characterization of “Pseudomonas andersonii” from Four Cases of Pulmonary Granulomas and Emended Species Description
Authors: Simmon Keith E. , Fang Deanna C. , Tesic Vera , Khot Prasanna D. , Giangeruso Enrico , Bolesta E. Stephen , Wagner-Reiss Katherine M. , Fisher Mark A. , Petti Cathy A. , Han Xiang Y. , She Rosemary C.
Published: January 2011

“
Pseudomonas andersonii
” is a Gram-negative bacillus initially isolated from a granulomatous lung lesion. Novel species status has not been validated for this single strain. We report four additional cases of pulmonary granuloma involving
P. andersonii
and further characterize the organism. These patients had pulmonary nodules that were surgically resected and which grew
P. andersonii
on routine culture.
Mycobacterium avium
complex was concomitantly isolated in two cases, and fungal structures were identified histopathologically in two other cases. The five
P. andersonii
strains described to date were similar in growth characteristics, biochemical reactions, matrix-assisted laser desorption ionization–time of flight mass spectrometry protein profiles, and susceptibility to antimicrobial agents. Their 16S rRNA genes were 99.9 to 100% identical but less than 95.0% similar to those of all other known bacteria. The
gyrA
genes of these strains were 99.5 to 100% identical. These shared features illustrate
P. andersonii
as a unique and distinct bacterium and support the novel species status of the organism.
Comparison of BD Bactec Plus Aerobic/F Medium to VersaTREK Redox 1 Blood Culture Medium for Detection of Candida spp. in Seeded Blood Culture Specimens Containing Therapeutic Levels of Antifungal Agents
Authors: Riedel Stefan , Eisinger Stephen W. , Dam Lisa , Stamper Paul D. , Carroll Karen C.
Published: January 2011

Recovery of
Candida
spp. using the BD Bactec FX blood culture (BC) system (Bactec Plus Aerobic/F medium) and the VersaTREK system (aerobic Redox medium) was evaluated using seeded BC bottles with and without the addition of commonly used antifungal agents. BC bottles (
n
= 1,442) were each inoculated with 10 ml human whole blood and 0.1 ml of suspensions of
Candida
spp., with or without antifungal agents. BC bottles were incubated in the corresponding system for a maximum of 5 days. In the absence of antifungal agents, Bactec FX recovered 97.4% of
Candida
spp., and VersaTREK recovered 99.1% (
P
= 0.154). With regard to length of time to detection (LTD) and overall recovery, both systems had various levels of effectiveness in recovering
C. glabrata
. In bottles containing antifungal agents, Bactec FX recovered 83.1% of isolates, whereas VersaTREK recovered 50.7% of
Candida
spp. (
P
< 0.001). For BC bottles without the addition of antifungal agents, the median LTD for VersaTREK was 2.2 h faster than that of Bactec FX (
P
< 0.001). In the presence of antifungal agents, the Bactec FX recovery time was significantly faster than that of VersaTREK (median difference of 10.8 h,
P
< 0.001). We conclude that both systems have comparable abilities to recover
Candida
spp. from seeded blood cultures in the absence of antifungal agents. In the presence of therapeutic levels of commonly used antifungal agents, the Bactec FX system demonstrated a significantly greater recovery of various
Candida
spp., as well as a shorter LTD.
Development of a Selective Culture Medium for Primary Isolation of the Main Brucella Species
Authors: De Miguel M. J. , Marín C. M. , Muñoz P. M. , Dieste L. , Grilló M. J. , Blasco J. M.
Published: January 2011

Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of
Brucella ovis
and some
B. melitensis
and
B. abortus
strains. In contrast, mTM is adequate for growth of all
Brucella
species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating
B. suis
has never been established properly. We first determined the performance of both media for
B. suis
isolation, proving that FM significantly inhibits
B. suis
growth. We also determined the susceptibility of
B. suis
to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for
B. suis
isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main
Brucella
species, including
B. suis
. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all
Brucella
species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all
Brucella
species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main
Brucella
species, including
B. suis
.
Emergence of Disseminated Infections Due to Geosmithia argillacea in Patients with Chronic Granulomatous Disease Receiving Long-Term Azole Antifungal Prophylaxis
Authors: Machouart Marie , Garcia-Hermoso Dea , Rivier Alexandre , Hassouni Noura , Catherinot Emilie , Salmon Alexandra , Debourgogne Anne , Coignard Hélène , Lecuit Marc , Bougnoux Marie-Elisabeth , Blanche Stéphane , Lortholary Olivier
Published: January 2011

We report two cases of invasive infections due to
Geosmithia argillacea
, an emerging mold, in patients with chronic granulomatous disease receiving prolonged azole antifungal prophylaxis. One patient died despite receiving a combination of four antifungals, and the other developed cerebral and medullary lesions under a combination of caspofungin, posaconazole, terbinafine, and gamma interferon.
The TY3 Gag3 Spacer Controls Intracellular Condensation and Uncoating
Authors: Clemens Kristina , Larsen Liza , Zhang Min , Kuznetsov Yurii , Bilanchone Virginia , Randall Arlo , Harned Adam , DaSilva Rhonda , Nagashima Kunio , McPherson Alexander , Baldi Pierre , Sandmeyer Suzanne
Published: January 2011

Cells expressing the yeast retrotransposon Ty3 form concentrated foci of Ty3 proteins and RNA within which virus-like particle (VLP) assembly occurs. Gag3, the major structural protein of the Ty3 retrotransposon, is composed of capsid (CA), spacer (SP), and nucleocapsid (NC) domains analogous to retroviral domains. Unlike the known SP domains of retroviruses, Ty3 SP is highly acidic. The current studies investigated the role of this domain. Although deletion of Ty3 SP dramatically reduced retrotransposition, significant Gag3 processing and cDNA synthesis occurred. Mutations that interfered with cleavage at the SP-NC junction disrupted CA-SP processing, cDNA synthesis, and electron-dense core formation. Mutations that interfered with cleavage of CA-SP allowed cleavage of the SP-NC junction, production of electron-dense cores, and cDNA synthesis but blocked retrotransposition. A mutant in which acidic residues of SP were replaced with alanine failed to form both Gag3 foci and VLPs. We propose a speculative “spring” model for Gag3 during assembly. In the first phase during concentration of Gag3 into foci, intramolecular interactions between negatively charged SP and positively charged NC domains of Gag3 limit multimerization. In the second phase, the NC domain binds RNA, and the bound form is stabilized by intermolecular interactions with the SP domain. These interactions promote CA domain multimerization. In the third phase, a negatively charged SP domain destabilizes the remaining CA-SP shell for cDNA release.
Identification of a Small-Molecule Entry Inhibitor for Filoviruses
Authors: Basu Arnab , Li Bing , Mills Debra M. , Panchal Rekha G. , Cardinale Steven C. , Butler Michelle M. , Peet Norton P. , Majgier-Baranowska Helena , Williams John D. , Patel Ishan , Moir Donald T. , Bavari Sina , Ray Ranjit , Farzan Michael R. , Rong Lijun , Bowlin Terry L.
Published: January 2011

Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC50s) of 10 μM and 12 μM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.
T Cell-, Interleukin-12-, and Gamma Interferon-Driven Viral Clearance in Measles Virus-Infected Brain Tissue
Authors: Stubblefield Park Samantha R. , Widness Mi , Levine Alan D. , Patterson Catherine E.
Published: January 2011

Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successfulex vivosystem to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain-derived leukocytes or recombinant IFN-γ. These results reveal that primed T cells directly act to clear MV infection of the brain by using a noncytolytic IL-12- and IFN-γ-dependent mechanism in the CNS and that this mechanism relies upon Jak/STAT signaling.
Regulation of Nm23-H1 and Cell Invasiveness by Kaposi's Sarcoma-Associated Herpesvirus
Authors: Qin Zhiqiang , Dai Lu , Toole Bryan , Robertson Erle , Parsons Chris
Published: January 2011

The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and the induction of an invasive cellular phenotype by KSHV following
de novo
infection is an important pathogenic component mediating tumor progression. The metastasis suppressor gene known as Nm23-H1 regulates tumor cell invasiveness, but whether KSHV itself regulates Nm23-H1 expression or subcellular localization, and whether this impacts cell invasiveness, has not been established. We found that KSHV increases expression and nuclear translocation of Nm23-H1 and that nuclear translocation of Nm23-H1 is regulated by the KSHV-encoded latency-associated nuclear antigen (LANA). Moreover, activation of the Ras-BRaf-MAPK (mitogen-activated protein kinase) signal transduction pathway, secretion of promigratory factors associated with this pathway, and cell invasiveness are dependent on KSHV regulation of Nm23-H1. Finally, induction of cytoplasmic overexpression of Nm23-H1 using a pharmacologic inhibitor of DNA methylation reduced KSHV-associated Ras-BRaf-MAPK pathway activation and suppressed KSHV-induced invasiveness. These data provide the first evidence for KSHV regulation of Nm23-H1 as a mechanism for KSHV induction of an invasive cellular phenotype and support the potential utility of targeting Nm23-H1 as a therapeutic approach for the treatment of KS.
Identification of Domains on the Fusion (F) Protein Trimer That Influence the Hemagglutinin-Neuraminidase Specificity of the F Protein in Mediating Cell-Cell Fusion
Authors: Tsurudome Masato , Ito Morihiro , Nishio Machiko , Nakahashi Mito , Kawano Mitsuo , Komada Hiroshi , Nosaka Tetsuya , Ito Yasuhiko
Published: January 2011

For most paramyxoviruses, virus type-specific interaction between fusion (F) protein and attachment protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoprotein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F protein contains the site(s) that determines its specificity for the HPIV2 HN protein. In the present study, we further investigated the sites of the F protein that could be critical for determining the HN protein specificity. By analyzing the reported structure of the F protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN protein, suggesting that these domains are involved in determining the HN protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself.
Chromatin Reassembly Factors Are Involved in Transcriptional Interference Promoting HIV Latency
Authors: Gallastegui Edurne , Millán-Zambrano Gonzalo , Terme Jean-Michel , Chávez Sebastián , Jordan Albert
Published: January 2011

The establishment of a stable reservoir of latently infected cells allows HIV to persist in the host. Usually, HIV infection of T cells results in integration of the viral genome, with a preference for regions in the human genome containing active genes, viral expression, and production of new viruses. However, in rare cases T cells become latently infected, and this is presumed to be due to a combination of two factors: integrated viruses are not efficiently transcribed and infected T cells revert to a resting memory state. HIV latency has been associated with provirus integration in regions of constitutive heterochromatin, gene deserts, or very highly expressed genes. We have investigated the transcriptional consequences of latent HIV integration into cellular genes and the involvement of chromatin reassembly factors (CRFs) in the transcriptional interference that a host gene exerts on the integrated cryptic HIV promoter. Chimeric transcripts containing sequences from the host gene and HIV can be detected, having been initiated at promoters of either the cell or the virus. Reactivation of HIV downregulates host gene expression. Cryptic promoters might remain inactive due to the repressive chromatin configuration established by CRFs during transcription elongation. Depletion of CRFs such as Spt6, Chd1, and FACT, or the histone chaperones ASF1a and HIRA, promoted HIV reactivation, concomitantly with chromatin relaxation and a decrease in general RNA polymerase activity. Overall, our results indicate that CRFs play a role in maintaining HIV latency by transcriptional interference when the provirus is integrated into an intron of a highly active gene.
Analysis of Human APOBEC3H Haplotypes and Anti-Human Immunodeficiency Virus Type 1 Activity
Authors: Wang Xiaojun , Abudu Aierken , Son SungMo , Dang Ying , Venta Patrick J. , Zheng Yong-Hui
Published: January 2011

Human APOBEC3H (A3H) has one cytidine deaminase domain (CDD) and inhibits the replication of retrotransposons and human immunodeficiency virus type 1 (HIV-1) in a Vif-resistant manner. Human
A3H
has five single amino acid polymorphisms (N15Δ, R18L, G105R, K121D, and E178D), and four haplotypes (I to IV) have previously been identified in various human populations. Haplotype II was primarily found in African-derived populations, and it was the only one that could be stably expressed. Here, we identified three new haplotypes from six human population samples, which we have named V, VI, and VII. Haplotypes V and VII are stably expressed and inhibit HIV-1 replication. Notably, haplotype V was identified in samples from all African-, Asian-, and Caucasian-derived populations studied. Using haplotype VII, we investigated the A3H anti-HIV-1 mechanism. We found that A3H virion packaging is independent of its CDD but dependent on a
112
YYXW
115
motif. This motif binds HIV-1 nucleocapsid in an RNA-dependent manner, and a single Y112A mutation completely disrupts A3H virion incorporation. We further studied the mechanism of A3H resistance to Vif. Although the previously identified APOBEC3G Vif-responsive motif
128
DPDY
131
is not conserved in A3H, placement of this motif into A3H does not make it become less resistant to HIV-1 Vif. We conclude that stably expressed A3H haplotypes may be more broadly distributed in humans than previously realized, and A3H protein is resistant to Vif. These results have important implications for the role of A3H in retrotransposon and HIV-1 inhibition.
Simian Immunodeficiency Virus SIVmac239Δnef Vaccination Elicits Different Tat
28-35
SL8-Specific CD8
+
T-Cell Clonotypes Compared to a DNA Prime/Adenovirus Type 5 Boost Regimen in Rhesus Macaques
Authors: Burwitz Benjamin J. , Ende Zachary , Sudolcan Benjamin , Reynolds Matthew R. , Greene Justin M. , Bimber Benjamin N. , Almeida Jorge R. , Kurniawan Monica , Venturi Vanessa , Gostick Emma , Wiseman Roger W. , Douek Daniel C. , Price David A. , O'Connor David H.
Published: January 2011

Different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) vaccine vectors expressing the same viral antigens can elicit disparate T-cell responses. Within this spectrum, replicating variable vaccines, like SIVmac239Δnef, appear to generate particularly efficacious CD8
+
T-cell responses. Here, we sequenced T-cell receptor β-chain (
TRB
) gene rearrangements from immunodominant Mamu-A*01-restricted Tat
28-35
SL8-specific CD8
+
T-cell populations together with the corresponding viral epitope in four rhesus macaques during acute SIVmac239Δnef infection. Ultradeep pyrosequencing showed that viral variants arose with identical kinetics in SIVmac239Δnef and pathogenic SIVmac239 infection. Furthermore, distinct Tat
28-35
SL8-specific T-cell receptor (TCR) repertoires were elicited by SIVmac239Δnef compared to those observed following a DNA/Ad5 prime-boost regimen, likely reflecting differences in antigen sequence stability.
Assembly and Replication of HIV-1 in T Cells with Low Levels of Phosphatidylinositol-(4,5)-Bisphosphate
Authors: Monde Kazuaki , Chukkapalli Vineela , Ono Akira
Published: January 2011

HIV-1 Gag assembles into virus particles predominantly at the plasma membrane (PM). Previously, we observed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P
2
] is essential for Gag binding to the plasma membrane and virus release in HeLa cells. In the current study, we found that PI(4,5)P
2
also facilitates Gag binding to the PM and efficient virus release in T cells. Notably, serial passage of HIV-1 in an A3.01 clone that expresses polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P
2
, yielded an adapted mutant with a Leu-to-Arg change at matrix residue 74 (74LR). Virus replication in T cells expressing 5ptaseIV was accelerated by the 74LR mutation relative to replication of wild type HIV-1 (WT). This accelerated replication of the 74LR mutant was not due to improved virus release. In control T cells, the 74LR mutant releases virus less efficiently than does the WT, whereas in cells expressing 5ptaseIV, the WT and the 74LR mutant are similarly inefficient in virus release. Unexpectedly, we found that the 74LR mutation increased virus infectivity and compensated for the inefficient virus release. Altogether, these results indicate that PI(4,5)P
2
is essential for Gag-membrane binding, targeting of Gag to the PM, and efficient virus release in T cells, which in turn likely promotes efficient virus spread in T cell cultures. In T cells with low PI(4,5)P
2
levels, however, the reduced virus particle production can be compensated for by a mutation that enhances virus infectivity.
The Transmembrane Domain Sequence Affects the Structure and Function of the Newcastle Disease Virus Fusion Protein
Authors: Gravel Kathryn A. , McGinnes Lori W. , Reitter Julie , Morrison Trudy G.
Published: January 2011

The role of specific sequences in the transmembrane (TM) domain of Newcastle disease virus (NDV) fusion (F) protein in the structure and function of this protein was assessed by replacing this domain with the F protein TM domains from two other paramyxoviruses, Sendai virus (SV) and measles virus (MV), or the TM domain of the unrelated glycoprotein (G) of vesicular stomatitis virus (VSV). Mutant proteins with the SV or MV F protein TM domains were expressed, transported to cell surfaces, and proteolytically cleaved at levels comparable to that of the wild-type protein, while mutant proteins with the VSV G protein TM domain were less efficiently expressed on cell surfaces and proteolytically cleaved. All mutant proteins were defective in all steps of membrane fusion, including hemifusion. In contrast to the wild-type protein, the mutant proteins did not form detectable complexes with the NDV hemagglutinin-neuraminidase (HN) protein. As determined by binding of conformation-sensitive antibodies, the conformations of the ectodomains of the mutant proteins were altered. These results show that the specific sequence of the TM domain of the NDV F protein is important for the conformation of the preactivation form of the ectodomain, the interactions of the protein with HN protein, and fusion activity.
NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells
Authors: Sakakibara Shuhei , Sakakibara Kaori , Tosato Giovanna
Published: January 2011

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus linked to prostate carcinoma and chronic fatigue syndrome. Here we report that NF-κB activation can markedly increase XMRV production. The inflammatory cytokine tumor necrosis factor alpha (TNF-α), which activates NF-κB, significantly augmented viral Gag protein production in XMRV-infected cells. Reporter assays showed that TNF-α and Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), an intrinsic NF-κB activator, increased long terminal repeat (LTR)-dependent XMRV transcription. We identified two NF-κB binding sites (designated κB-1 and κB-2) in the LTR U3 region of XMRV and demonstrated that both sites bind to the NF-κB component p65/RelA. Mutation of the κB-1 site, but not the κB-2 site, impaired responsiveness to TNF-α and LMP1 in reporter assays. A mutant XMRV with a mutation at the κB-1 site replicated significantly less efficiently than the wild-type XMRV in the prostate carcinoma LNCaP, DU145, and PC-3 cell lines, HEK293 cells, the EBV-immortalized cell line IB4, and the Burkitt's lymphoma cell line BJAB. These results demonstrate that TNF-α and EBV LMP1 enhance XMRV replication in prostate carcinoma and B-lineage cells through the κB-1 site in the XMRV LTR, suggesting that inflammation, EBV infection, and other conditions leading to NF-κB activation may promote XMRV spread in humans.
Transcriptionally Abundant Major Histocompatibility Complex Class I Alleles Are Fundamental to Nonhuman Primate Simian Immunodeficiency Virus-Specific CD8+T Cell Responses
Authors: Budde Melisa L. , Lhost Jennifer J. , Burwitz Benjamin J. , Becker Ericka A. , Burns Charles M. , O'Connor Shelby L. , Karl Julie A. , Wiseman Roger W. , Bimber Benjamin N. , Zhang Guang Lan , Hildebrand William , Brusic Vladimir , O'Connor David H.
Published: January 2011

Simian immunodeficiency virus (SIV)-infected macaques are the preferred animal model for human immunodeficiency virus (HIV) vaccines that elicit CD8+T cell responses. Unlike humans, whose CD8+T cell responses are restricted by a maximum of six HLA class I alleles, macaques express up to 20 distinct major histocompatibility complex class I (MHC-I) sequences. Interestingly, only a subset of macaque MHC-I sequences are transcriptionally abundant in peripheral blood lymphocytes. We hypothesized that highly transcribed MHC-I sequences are principally responsible for restricting SIV-specific CD8+T cell responses. To examine this hypothesis, we measured SIV-specific CD8+T cell responses in MHC-I homozygous Mauritian cynomolgus macaques. Each of eight CD8+T cell responses defined by full-proteome gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay were restricted by four of the five transcripts that are transcriptionally abundant (>1% of total MHC-I transcripts in peripheral blood lymphocytes). The five transcriptionally rare transcripts shared by these animals did not restrict any detectable CD8+T cell responses. Further, seven CD8+T cell responses were defined by identifying peptide binding motifs of the three most frequent MHC-I transcripts on the M3 haplotype. Combined, these results suggest that transcriptionally abundant MHC-I transcripts are principally responsible for restricting SIV-specific CD8+T cell responses. Thus, only a subset of the thousands of known MHC-I alleles in macaques should be prioritized for CD8+T cell epitope characterization.
Global Analysis of the Transcriptional Response of Whitefly to
Tomato Yellow Leaf Curl China Virus
Reveals the Relationship of Coevolved Adaptations
Authors: Luan Jun-Bo , Li Jun-Min , Varela Nélia , Wang Yong-Liang , Li Fang-Fang , Bao Yan-Yuan , Zhang Chuan-Xi , Liu Shu-Sheng , Wang Xiao-Wei
Published: January 2011

The begomoviruses are the largest and most economically important group of plant viruses transmitted exclusively by the whitefly
Bemisia tabaci
in a circulative, persistent manner. The circulation of the viruses within the insect vectors involves complex interactions between virus and vector components; however, the molecular mechanisms of these interactions remain largely unknown. Here we investigated the transcriptional response of the invasive
B. tabaci
Middle East-Asia Minor 1 species to
Tomato yellow leaf curl China virus
(TYLCCNV) using Illumina sequencing technology. Results showed that 1,606 genes involved in 157 biochemical pathways were differentially expressed in the viruliferous whiteflies. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that TYLCCNV can perturb the cell cycle and primary metabolism in the whitefly, which explains the negative effect of this virus on the longevity and fecundity of
B. tabaci
. Our data also demonstrated that TYLCCNV can activate whitefly immune responses, such as autophagy and antimicrobial peptide production, which might lead to a gradual decrease of viral particles within the body of the viruliferous whitefly. Furthermore, PCR results showed that TYLCCNV can invade the ovary and fat body tissues of the whitefly, and Lysotracker and Western blot analyses revealed that the invasion of TYLCCNV induced autophagy in both the ovary and fat body tissues. Surprisingly, TYLCCNV also suppressed the whitefly immune responses by downregulating the expression of genes involved in Toll-like signaling and mitogen-activated protein kinase (MAPK) pathways. Taken together, these results reveal the relationship of coevolved adaptations between begomoviruses and whiteflies and will provide a road map for future investigations into the complex interactions between plant viruses and their insect vectors.
Immunization with Herpes Simplex Virus 2 (HSV-2) Genes plus Inactivated HSV-2 Is Highly Protective against Acute and Recurrent HSV-2 Disease
Authors: Morello Christopher S. , Levinson Michael S. , Kraynyak Kimberly A. , Spector Deborah H.
Published: January 2011

To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.
Differential Sensitivities of Retroviruses to Integrase Strand Transfer Inhibitors
Authors: Koh Yasuhiro , Matreyek Kenneth A. , Engelman Alan
Published: January 2011

Integrase inhibitors are emerging anti-human immunodeficiency virus (HIV) drugs, and multiple retroviruses and transposable elements were evaluated here for susceptibilities to raltegravir (RAL) and elvitegravir (EVG). All viruses, including primate and nonprimate lentiviruses, a
Betaretrovirus
, a
Gammaretrovirus
, and the
Alpharetrovirus
Rous sarcoma virus (RSV), were susceptible to inhibition by RAL. EVG potently inhibited all lentiviruses and intermediately inhibited
Betaretrovirus
and
Gammaretrovirus
infections yet was basically ineffective against RSV. Substitutions based on HIV type 1 (HIV-1) resistance changes revealed that integrase residue Ser150 contributed significantly to the resistance of RSV. The drugs intermediately inhibited intracisternal A-particle retrotransposition but were inactive against Sleeping Beauty transposition and long interspersed nucleotide element 1 (LINE-1) retrotransposition.
Reproduction
In Vitro
of a Quasispecies from a Hepatitis C Virus-Infected Patient and Determination of Factors That Influence Selection of a Dominant Species
Authors: Kawaguchi Kazunori , Faulk Kristina , Purcell Robert H. , Emerson Suzanne U.
Published: January 2011

Hepatitis C virus infections proceed to chronicity in the majority of cases. In patients, hepatitis C viruses exist as a dynamic and complex quasispecies. The dominant species at any one time arises in response to host immune pressure and other, incompletely understood factors. It is critical to understand all the mechanisms by which dominance is achieved, but this is difficult to study
in vivo
. Therefore, it would be useful to develop a cell culture system in which naturally occurring quasispecies could be studied. Hepatitis C virus glycoprotein genes E1 and E2 were PCR amplified as a cassette from the plasma of a chronically infected patient and shotgun cloned into a modified 1a/JFH1 infectious cDNA clone. Following transformation of bacteria, plasmids were batch harvested, transcribed, and transfected into Huh7.5 cells to produce a quasispecies of hypervariable region 1 (HVR1) that mimicked that circulating
in vivo
. Serial passage of the quasispecies
in vitro
resulted in replacement of the initially dominant species with a new HVR1 species coexisting with selected growth-enhancing mutations located outside HVR1. Antibody raised against one HVR1 sequence neutralized virus with the homologous HVR1 and cross-neutralized virus with a different sequence. Reciprocal swapping of the HVR1 regions between the two dominating species demonstrated that the HVR1 sequence affects the efficiency of replication and of neutralization by anti-HVR1 but that both processes are strongly influenced by regions outside HVR1.
Potent and Broad Neutralization of HIV-1 Subtype C by Plasma Antibodies Targeting a Quaternary Epitope Including Residues in the V2 Loop
Authors: Moore Penny L. , Gray Elin S. , Sheward Daniel , Madiga Maphuti , Ranchobe Nthabeleng , Lai Zhong , Honnen William J. , Nonyane Molati , Tumba Nancy , Hermanus Tandile , Sibeko Sengeziwe , Mlisana Koleka , Abdool Karim Salim S. , Williamson Carolyn , Pinter Abraham , Morris Lynn
Published: January 2011

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers. Repair of the FN/LRD-K-K motif in resistant heterologous viruses conferred sensitivity, with titers sometimes exceeding 1:10,000. Comparison of the CAP256 epitope with that of the PG9/PG16 monoclonal antibodies suggested that these epitopes overlapped, adding to the mounting evidence that this may represent a common neutralization target that should be further investigated as a potential vaccine candidate.
Cytomegalovirus Capsid Protease: Biological Substrates Are Cleaved More Efficiently by Full-Length Enzyme (pUL80a) than by the Catalytic Domain (Assemblin)
Authors: Fernandes Steve M. , Brignole Edward J. , Gibson Wade
Published: January 2011

We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na
2
SO
4
reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.
Determination of lead(II) by online novel electrolyte dropping electrode using 1,10-phenanthroline in flow injection system
Authors: Shu Feifei , Xie Shaoai , Jia Jinping
Published: January 2011

The transfer of Pb(II) facilitated by 1,10-phenanthroline (phen) presented in the methyl isobutyl ketone (MIBK) across the polarized water|MIBK interface was systematically investigated by novel electrolyte dropping electrode using cyclic voltammetry and square wave voltammetry. The protonation of 1,10-phenanthroline in polarized water|MIBK interface was explored. The dependence of the half-wave potential on PbCl2 and phen concentration was investigated and the stoichiometry of resulting metal ligand complex was formulated. Different concentrations of PbCl2 was determined online and the transfer peak current was proportional to the bulk concentration of the Pb2+ varied between 8 μM to 0.5 mM and 0.5 to 1 mM, and the linear regression coefficients were 0.9953 and 0.9995, respectively. The standard deviations (RSD) were 4.33, 5.80, 4.13, 3.88, 2.23, 3.39, 1.38, 2.42, 2.00, 2.73 and 2.37% for 8 times successive determinations of Pb2+ for the concentrations of 0.008, 0.05, 0.1, 0.2, 0.3, 0.35, 0.4, 0.5, 0.8, 0.9 and 1 mM, respectively. The long time stability of the electrolyte dropping electrode was tested and the result showed that this novel method was simple, rapid and efficient.
Cathodic stripping voltammetry of clothianidin: Application to environmental studies
Authors: Guziejewski Dariusz , Skrzypek Sławomira , Łuczak Adam , Ciesielski Witold
Published: January 2011

A new, rapid and very sensitive electrochemical method for the determination of a new pesticide clothianidin in tap and river water was developed. The electrochemical reduction and determination of clothianidin have been carried out at a hanging mercury drop electrode (HMDE) in various aqueous solutions in the pH range of 2–10 by cyclic voltammetry (CV) and cathodic stripping square wave voltammetry (SW CSV). The best results were obtained for the clothianidin determination by SW CSV method in 0.04 mol l–1 Britton Robinson buffer at pH 8.1. Various conditions of the procedure were checked. Elaborated electroanalytical procedure enable clothianidin determination in the concentration range of 2.0 × 10–8–9.9 × 10–7 mol l–1. The limit of detection (LOD) and limit of quantification (LOQ) were obtained as 2.00 × 10–9 and 2.36 × 10–8 mol l–1, respectively. Precision and accuracy of the developed method were checked by recovery studies in spiked tap and river water. The voltammetric determination has been validated using HPLC with UV detection.