bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Surveillance of HIV-1 in Madrid, Spain: a Phylogeographic Analysis
Authors: González-Alba José M. , Holguín África , Garcia Rosa , García-Bujalance Silvia , Alonso Roberto , Suárez Avelina , Delgado Rafael , Cardeñoso Laura , González Rosa , García-Bermejo Isabel , Portero Francisca , de Mendoza Carmen , González-Candelas Fernando , Galán Juan-Carlos 
Published: July 2011

ABSTRACT

The molecular epidemiology of HIV-1 is constantly changing, mainly as a result of human migratory flows and the high adaptive ability of the virus. In recent years, Spain has become one of Europe's main destinations for immigrants and one of the western European countries with the highest rates of HIV-positive patients. Using a phylogeographic approach, we have analyzed the relationship between HIV-1 variants detected in immigrant and native populations of the urban area of Madrid. Our project was based on two coincidental facts. First, resistance tests were extended to naïve and newly diagnosed patients, and second, the Spanish government legislated the provision of legal status to many immigrants. This allowed us to obtain a large data set (
n
= 2,792) from 11 Madrid hospitals of viral
pol
sequences from the two populations, and with this unique material, we explored the impact of immigration in the epidemiological trends of HIV-1 variants circulating in the largest Spanish city. The prevalence of infections by non-B HIV-1 variants in the studied cohort was 9%, rising to 25% among native Spanish patients. Multiple transmission events involving different lineages and subsubtypes were observed in all the subtypes and recombinant forms studied. Our results also revealed strong social clustering among the most recent immigrant groups, such as Russians and Romanians, but not in those groups who have lived in Madrid for many years. Additionally, we document for the first time the presence of CRF47_BF and CRF38_BF in Europe, and a new BG recombinant form found in Spaniards and Africans is tentatively proposed. These results suggest that the HIV-1 epidemic will evolve toward a more complex epidemiological landscape.
Major Histocompatibility Complex-Dependent Cytotoxic T Lymphocyte Repertoire and Functional Avidity Contribute to Strain-Specific Disease Susceptibility after Murine Respiratory Syncytial Virus Infection
Authors: Jessen Birthe , Faller Simone , Krempl Christine D. , Ehl Stephan 
Published: July 2011

ABSTRACTSusceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2dallele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2dmice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2+CTL directed against the immunodominant epitope M2-1 82, while in H-2bmice only 20% of antiviral CTL were Vβ9+CTL specific for the immunodominant epitope M187. The immunodominant H-2d-restricted CTL lysed target cells less efficiently than the immunodominant H-2bCTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2dmating (C57BL/6-H-2dxbmice) attenuated disease. Moreover, disease in H-2dmice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2+cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.
Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase
Authors: Gazina Elena V. , Smidansky Eric D. , Holien Jessica K. , Harrison David N. , Cromer Brett A. , Arnold Jamie J. , Parker Michael W. , Cameron Craig E. , Petrou Steven 
Published: July 2011

ABSTRACT

Amiloride and its derivative 5-(
N
-ethyl-
N
-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D
pol
conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol.
82:
1465–1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D
pol
in vitro
, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D
pol
by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg
2+
. Docking analysis suggested a binding site for amiloride and EIPA in 3D
pol
, located in close proximity to one of the Mg
2+
ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.
Analysis of a Clonal Lineage of HIV-1 Envelope V2/V3 Conformational Epitope-Specific Broadly Neutralizing Antibodies and Their Inferred Unmutated Common Ancestors
Authors: Bonsignori Mattia , Hwang Kwan-Ki , Chen Xi , Tsao Chun-Yen , Morris Lynn , Gray Elin , Marshall Dawn J. , Crump John A. , Kapiga Saidi H. , Sam Noel E. , Sinangil Faruk , Pancera Marie , Yongping Yang , Zhang Baoshan , Zhu Jiang , Kwong Peter D. , O'Dell Sijy , Mascola John R. , Wu Lan , Nabel Gary J. , Phogat Sanjay , Seaman Michael S. , Whitesides John F. , Moody M. Anthony , Kelsoe Garnett , Yang Xinzhen , Sodroski Joseph , Shaw George M. , Montefiori David C. , Kepler Thomas B. , Tomaras Georgia D. , Alam S. Munir , Liao Hua-Xin , Haynes Barton F. 
Published: July 2011

ABSTRACTV2/V3 conformational epitope antibodies that broadly neutralize HIV-1 (PG9 and PG16) have been recently described. Since an elicitation of previously known broadly neutralizing antibodies has proven elusive, the induction of antibodies with such specificity is an important goal for HIV-1 vaccine development. A critical question is which immunogens and vaccine formulations might be used to trigger and drive the development of memory B cell precursors with V2/V3 conformational epitope specificity. In this paper we identified a clonal lineage of four V2/V3 conformational epitope broadly neutralizing antibodies (CH01 to CH04) from an African HIV-1-infected broad neutralizer and inferred their common reverted unmutated ancestor (RUA) antibodies. While conformational epitope antibodies rarely bind recombinant Env monomers, a screen of 32 recombinant envelopes for binding to the CH01 to CH04 antibodies showed monoclonal antibody (MAb) binding to the E.A244 gp120 Env and to chronic Env AE.CM243; MAbs CH01 and CH02 also bound to transmitted/founder Env B.9021. CH01 to CH04 neutralized 38% to 49% of a panel of 91 HIV-1 tier 2 pseudoviruses, while the RUAs neutralized only 16% of HIV-1 isolates. Although the reverted unmutated ancestors showed restricted neutralizing activity, they retained the ability to bind to the E.A244 gp120 HIV-1 envelope with an affinity predicted to trigger B cell development. Thus, E.A244, B.9021, and AE.CM243 Envs are three potential immunogen candidates for studies aimed at defining strategies to induce V2/V3 conformational epitope-specific antibodies.
Construction of a Lytically Replicating Kaposi's Sarcoma-Associated Herpesvirus
Authors: Budt Matthias , Hristozova Tsvetana , Hille Georg , Berger Katrin , Brune Wolfram 
Published: July 2011

ABSTRACT
Karposi's sarcoma-associated herpesvirus (KSHV) is found predominantly in a latent state in most cell types, impeding investigations of the lytic replication cycle. Here, we engineered the cloned KSHV genome, bacterial artificial chromosome 36 (BAC36), to enforce constitutive expression of the main lytic switch regulator, the replication and transcription activator (RTA) (open reading frame 50 [ORF50]). The resulting virus, KSHV-lyt, activated by default the lytic cycle and replicated to high titers in various cells. Using KSHV-lyt, we showed that ORF33 (encoding a tegument protein) is essential for lytic KSHV replication in cell culture, but ORF73 (encoding the latent nuclear antigen [LANA]) is not. Thus, KSHV-lyt should be highly useful to study viral gene function during lytic replication.
Infection of Lymphoblastoid Cell Lines by Kaposi's Sarcoma-Associated Herpesvirus: Critical Role of Cell-Associated Virus
Authors: Myoung Jinjong , Ganem Don 
Published: July 2011

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) displays strong lymphotropism
in vivo
, but paradoxically, established B cell lines have largely been refractory to infection by soluble KSHV virions. Here we show that this block can be overcome by exposure to cell-associated virus. Doxycycline-inducible recombinant KSHV.219 (rKSHV.219)-harboring SLK (iSLK.219) cells were employed as KSHV donors. Cocultivation of lymphoid cell lines with reactivated iSLK.219 cells resulted in readily demonstrable viral entry into each cell line; similar observations were made in primary tonsillar B cell cultures. Moreover, infected lymphoid cells were able to outgrow upon puromycin selection, indicating development of persistent infection. Infected BJAB cells display signatures of latent infection, including classical latency-associated transcripts, a punctate pattern of LANA expression, and episomal maintenance of the KSHV genome. However, when lytically activated by various chemical stimuli, infected BJAB cells were able to produce only low levels of infectious virions. These data demonstrate that (i) cell-associated viruses can bypass viral entry blocks in most lymphoid cell lines, (ii) the determinants of cell-associated virus entry differ from those of soluble virion infection, and (iii) immortalized lymphoblastoid lines have partial postentry blocks to efficient lytic reactivation.
The Alpha Subunit of Eukaryotic Initiation Factor 2B (eIF2B) Is Required for eIF2-Mediated Translational Suppression of Vesicular Stomatitis Virus
Authors: Elsby Rachel , Heiber Joshua F. , Reid Peter , Kimball Scot R. , Pavitt Graham D. , Barber Glen N. 
Published: July 2011

ABSTRACT

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates
Saccharomyces cerevisiae
GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.
Spinoculation Triggers Dynamic Actin and Cofilin Activity That Facilitates HIV-1 Infection of Transformed and Resting CD4 T Cells
Authors: Guo Jia , Wang Weifeng , Yu Dongyang , Wu Yuntao 
Published: July 2011

ABSTRACT
Centrifugal inoculation, or spinoculation, is widely used in virology research to enhance viral infection. However, the mechanism remained obscure. Using HIV-1 infection of human T cells as a model, we demonstrate that spinoculation triggers dynamic actin and cofilin activity, probably resulting from cellular responses to centrifugal stress. This actin activity also leads to the upregulation of the HIV-1 receptor and coreceptor, CD4 and CXCR4, enhancing viral binding and entry. We also demonstrate that an actin inhibitor, jasplakinolide, diminishes spin-mediated enhancement. In addition, small interfering RNA (siRNA) knockdown of LIMK1, a cofilin kinase, decreases the enhancement. These results suggest that spin-mediated enhancement cannot be explained simply by a virus-concentrating effect; rather, it is coupled with spin-induced cytoskeletal dynamics that promote receptor mobilization, viral entry, and postentry processes. Our results highlight the importance of cofilin and a dynamic cytoskeleton for the initiation of viral infection. Our results also indicate that caution needs to be taken in data interpretation when cells are spinoculated; some of the spin-induced cellular permissiveness may be beyond the natural capacity of an infecting virus.
Molecular Evolution Analysis of the Human Immunodeficiency Virus Type 1 Envelope in Simian/Human Immunodeficiency Virus-Infected Macaques: Implications for Challenge Dose Selection
Authors: Varela Mariana , Landskron Lisa , Lai Rachel P. J. , McKinley Trevelyan J. , Bogers Willy M. , Verschoor Ernst J. , Dubbes Rob , Barnett Susan W. , Frost Simon D. W. , Heeney Jonathan L. 
Published: July 2011

ABSTRACT

Since the demonstration that almost 80% of human immunodeficiency virus type 1 (HIV-1) infections result from the transmission of a single variant from the donor, biological features similar to those of HIV mucosal transmission have been reported for macaques inoculated with simian immunodeficiency virus (SIV). Here we describe the early diversification events and the impact of challenge doses on viral kinetics and on the number of variants transmitted in macaques infected with the chimeric simian/human immunodeficiency virus SHIV
sf162p4
. We show that there is a correlation between the dose administered and the number of variants transmitted and that certain inoculum variants are preferentially transmitted. This could provide insight into the viral determinants of transmission and could aid in vaccine development. Challenge through the mucosal route with high doses results in the transmission of multiple variants in all the animals. Such an unrealistic scenario could underestimate potential intervention measures. We thus propose the use of molecular evolution analysis to aid in the determination of challenge doses that better mimic the transmission dynamics seen in natural HIV-1 infection.
Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence
Authors: Stein David A. , Perry Stuart T. , Buck Michael D. , Oehmen Christopher S. , Fischer Matthew A. , Poore Elizabeth , Smith Jessica L. , Lancaster Alissa M. , Hirsch Alec J. , Slifka Mark K. , Nelson Jay A. , Shresta Sujan , Früh Klaus 
Published: July 2011

ABSTRACT

The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5′ cyclization sequence (5′CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype
in vitro
. To determine if DC-3 siRNA could inhibit DENV
in vivo
, an “
in vivo
-ready” version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
Generation of Replication-Defective Virus-Based Vaccines That Confer Full Protection in Sheep against Virulent Bluetongue Virus Challenge
Authors: Matsuo Eiko , Celma Cristina C. P. , Boyce Mark , Viarouge Cyril , Sailleau Corinne , Dubois Eric , Bréard Emmanuel , Thiéry Richard , Zientara Stéphan , Roy Polly 
Published: July 2011

ABSTRACT

The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein
in trans
. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.
Expression Profiling of the Intermediate and Late Stages of Poxvirus Replication
Authors: Yang Zhilong , Reynolds Sara E. , Martens Craig A. , Bruno Daniel P. , Porcella Stephen F. , Moss Bernard 
Published: July 2011

ABSTRACT

The double-stranded DNA genome of vaccinia virus (VACV), the prototype poxvirus, contains approximately 200 open reading frames (ORFs) that are transcribed at early, intermediate, and late stages of infection. Previous high-throughput deep RNA sequencing allowed us to map 118 VACV early genes that are expressed before viral DNA replication and 93 postreplicative genes. However, the intermediate- and late-stage postreplicative genes could not be differentiated. Here, we synchronized infections with a reversible inhibitor of DNA replication and used a VACV mutant that conditionally transcribes late genes to sequence the two classes of mRNAs. In addition, each postreplicative ORF was individually expressed under conditions that distinguished intermediate and late classes. We identified 38 VACV genes that belong to the late class and 53 that belong to the intermediate class, with some of the latter continuing to be expressed late. These data allowed us to prepare a genome-wide early, intermediate, and late transcription map. Inspection of sequences upstream of these ORFs revealed distinctive characteristics of intermediate and late promoters and suggested that some promoters have intermediate and late elements. The intermediate genes encoded many DNA binding/packaging and core-associated proteins in addition to late transcription factors; the late genes encoded many morphogenesis and mature virion membrane proteins, including those involved in entry, in addition to early transcription factors. The top-ranked antigens for CD4
+
T cells and B cells were mainly intermediate rather than late gene products. The differentiation of intermediate and late genes may enhance understanding of poxvirus replication and lead to improvements in expression vectors and recombinant vaccines.
ChePep Controls Helicobacter pylori Infection of the Gastric Glands and Chemotaxis in the
Epsilonproteobacteria
Authors: Howitt Michael R. , Lee Josephine Y. , Lertsethtakarn Paphavee , Vogelmann Roger , Joubert Lydia-Marie , Ottemann Karen M. , Amieva Manuel R. 
Published: July 2011

ABSTRACT

Microbes use directed motility to colonize harsh and dynamic environments. We discovered that
Helicobacter pylori
strains establish bacterial colonies deep in the gastric glands and identified a novel protein, ChePep, necessary to colonize this niche. ChePep is preferentially localized to the flagellar pole. Although mutants lacking ChePep have normal flagellar ultrastructure and are motile, they have a slight defect in swarming ability. By tracking the movement of single bacteria, we found that ∆ChePep mutants cannot control the rotation of their flagella and swim with abnormally frequent reversals. These mutants even sustain bursts of movement backwards with the flagella pulling the bacteria. Genetic analysis of the chemotaxis signaling pathway shows that ChePep regulates flagellar rotation through the chemotaxis system. By examining
H. pylori
within a microscopic pH gradient, we determined that ChePep is critical for regulating chemotactic behavior. The
chePep
gene is unique to the
Epsilonproteobacteria
but is found throughout this diverse group. We expressed ChePep from other members of the
Epsilonproteobacteria
, including the zoonotic pathogen
Campylobacter jejuni
and the deep sea hydrothermal vent inhabitant
Caminibacter mediatlanticus
, in
H. pylori
and found that ChePep is functionally conserved across this class. ChePep represents a new family of chemotaxis regulators unique to the
Epsilonproteobacteria
and illustrates the different strategies that microbes have evolved to control motility.

IMPORTANCE
Helicobacter pylori
strains infect half of all humans worldwide and contribute to the development of peptic ulcers and gastric cancer.
H. pylori
cannot survive within the acidic lumen of the stomach and uses flagella to actively swim to and colonize the protective mucus and epithelium. The chemotaxis system allows
H. pylori
to navigate by regulating the rotation of its flagella. We identified a new protein, ChePep, which controls chemotaxis in
H. pylori
. ChePep mutants fail to colonize the gastric glands of mice and are completely outcompeted by normal
H. pylori
. Genes encoding ChePep are found only in the class
Epsilonproteobacteria
, which includes the human pathogen
Campylobacter jejuni
and environmental microbes like the deep-sea hydrothermal vent colonizer
Caminibacter mediatlanticus
, and we show that ChePep function is conserved in this class. Our study identifies a new colonization factor in
H. pylori
and also provides insight into the control and evolution of bacterial chemotaxis.