bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Unbiased Next-Generation Sequencing of RNA (RNA-seq) as a Diagnostic Method in Influenza Virus-Positive Respiratory Samples
Authors: Fischer Nicole , Indenbirken Daniela , Meyer Thomas , Lütgehetmann Marc , Lellek Heinrich , Spohn Michael , Aepfelbacher Martin , Alawi Malik , Grundhoff Adam 
Published: May 2015

ABSTRACT
Unbiased nontargeted metagenomic RNA sequencing (UMERS) has the advantage to detect known as well as unknown pathogens and, thus, can significantly improve the detection of viral, bacterial, parasitic, and fungal sequences in public health settings. In particular, conventional diagnostic methods successfully identify the putative pathogenic agent in only 30% to 40% of respiratory specimens from patients with acute respiratory illness. Here, we applied UMERS to 24 diagnostic respiratory specimens (bronchoalveolar lavage [BAL] fluid, sputum samples, and a swab) from patients with seasonal influenza infection and 5 BAL fluid samples from patients with pneumonia that tested negative for influenza to validate RNA sequencing as an unbiased diagnostic tool in comparison to conventional diagnostic methods. In addition to our comparison to PCR, we evaluated the potential to retrieve comprehensive influenza virus genomic information and the capability to detect known superinfecting pathogens. Compared to quantitative real-time PCR for influenza viral sequences, UMERS detected influenza viral sequences in 18 of 24 samples. Complete influenza virus genomes could be assembled from 8 samples. Furthermore, in 3 of 24 influenza-positive samples, additional viral pathogens could be detected, and 2 of 24 samples showed a significantly increased abundance of individual bacterial species known to cause superinfections during an influenza virus infection. Thus, analysis of respiratory samples from known or suspected influenza patients by UMERS provides valuable information that is relevant for clinical investigation.
Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines
Authors: Cheng Benson Yee Hin , Ortiz-Riaño Emilio , de la Torre Juan Carlos , Martínez-Sobrido Luis 
Published: May 2015

ABSTRACT

Several members of the
Arenaviridae
family cause hemorrhagic fever disease in humans and pose serious public health problems in their geographic regions of endemicity as well as a credible biodefense threat. To date, there have been no FDA-approved arenavirus vaccines, and current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the nucleoprotein (NP) and glycoprotein precursor (GPC) loci within the S segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than from the GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant trisegmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV (rLCMV) containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuated
in vivo
in a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of arenavirus live attenuated vaccines based on rearrangement of their viral genome.

IMPORTANCE
Several arenaviruses cause severe hemorrhagic fever in humans and also pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections and antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped to generate rLCMV/TransS. rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuated
in vivo
while conferring protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide a proof of concept for the rational development of safe and protective live attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic arenavirus infections in humans.
Two-Year Follow-Up of Macaques Developing Intermittent Control of the Human Immunodeficiency Virus Homolog Simian Immunodeficiency Virus SIVmac251 in the Chronic Phase of Infection
Authors: Shytaj Iart Luca , Nickel Gabrielle , Arts Eric , Farrell Nicholas , Biffoni Mauro , Pal Ranajit , Chung Hye Kyung , LaBranche Celia , Montefiori David , Vargas-Inchaustegui Diego , Robert-Guroff Marjorie , Lewis Mark G. , Sacha Jonah B. , Palamara Anna Teresa , Savarino Andrea 
Published: May 2015

ABSTRACT

Off-therapy control of viremia by HIV-infected individuals has been associated with two likely players: a restricted viral reservoir and an efficient cell-mediated immune response. We previously showed that a combination of highly suppressive antiretroviral therapy and two experimental drugs, i.e., auranofin and buthionine sulfoximine, was able to reduce the viral reservoir, elicit efficient cell-mediated antiviral responses, and induce intermittent posttherapy viral load control in chronically SIVmac251-infected macaques. We here show that the macaques that had received this drug combination and then stopped antiretroviral therapy were also able to maintain low numbers of activated CD4
+
T cells at viral rebound. Moreover, these macaques consistently displayed low-level simian immunodeficiency virus (SIV) diversity, which was in line with the strong and broadly reactive cell-mediated immune responses against conserved Gag antigens. Extended follow-up showed that the two macaques that had received the complete drug combination remained healthy and did not develop AIDS in 2 years of follow-up after therapy suspension. This disease-free survival is longer than twice the average time of progression to AIDS in SIVmac251-infected rhesus macaques. These results suggest that limited numbers of activated T cells at viral rebound and subsequent development of broadly reactive cell-mediated responses may be interrelated in reducing the viral reservoir.

IMPORTANCE
The HIV reservoir in CD4
+
T cells represents one main obstacle to HIV eradication. Recent studies, however, show that a drastic reduction of this reservoir is insufficient for inducing a functional cure of AIDS. In the present work, we thoroughly studied and subjected to long-term follow-up two macaques showing intermittent control of the virus following suspension of antiretroviral therapy plus an experimental antireservoir treatment, i.e., the gold salt auranofin and the investigational chemotherapeutic agent buthionione sulfoximine (BSO). We found that these drugs were able to decrease the number of activated CD4
+
T cells, which are preferential targets for HIV infection. Then, efficient immune responses against the virus were developed in the macaques, which remained healthy during 2 years of follow-up. This result may furnish another building block for future attempts to cure HIV/AIDS.
Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) and LMP2A in Nasopharyngeal Carcinoma Cells
Authors: Wasil Laura R. , Wei Leizhen , Chang Christopher , Lan Li , Shair Kathy H. Y. 
Published: May 2015

ABSTRACT
Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins 1 (LMP1) and LMP2A are consistently expressed in NPC and can cooperate in bitransgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were coexpressed in the EBV-negative NPC cell line HK1 and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A coexpression reduced γH2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5 μM) of the topoisomerase II inhibitor etoposide. Regulation of γH2AX occurred before the onset of caspase activation without modulation of other DNA damage signaling mediators, including ATM, Chk1, or Chk2, and additionally was suppressed by inducers of DNA single-strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A coexpressing cells recovered from cytotoxic doses of etoposide; however, LMP1 expression was sufficient for this effect. LMP1 and LMP2A coexpression did not enhance cell growth, with a moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells.

IMPORTANCE
NPC is characterized by clonal EBV infection and accounts for >78,000 annual cancer cases with increased incidence in regions where EBV is endemic, such as southeast Asia. The latent proteins LMP1 and LMP2A coexpressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A coexpression suppressed activation of the DNA damage response (DDR) protein γH2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A-coexpressing cells and indicating distinct LMP1-dependent effects in the restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the cooperative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.
Conservation of the STING-Mediated Cytosolic DNA Sensing Pathway in Zebrafish
Authors: Ge Rui , Zhou Yi , Peng Rui , Wang Rui , Li Mi , Zhang Yunbin , Zheng Chunfu , Wang Chen 
Published: May 2015

ABSTRACT

Zebrafish (
Danio rerio
) is a unique potential model animal for dissecting innate immune signaling. Here we demonstrate that herpes simplex virus 1 (HSV-1) could infect zebrafish at its different developmental stages and trigger the expression of type I interferons (IFNs) as well as interferon-stimulated genes (ISGs) in zebrafish larvae. Silencing of zSTING, but not zMAVS, markedly attenuates the DNA virus-induced antiviral responses. Notably, a conserved serine residue (S373) is essential for the action of zSTING. Unexpectedly, zebrafish cyclic GMP-AMP synthase (cGAS) is dispensable for the STING signaling, whereas zDHX9 and zDDX41 are potential sensors for HSV-1 infection
in vivo
. Taken together, this proof-of-concept study establishes the zebrafish larva as a feasible model for investigating the cytosolic DNA sensing mechanism, shedding light on the conservation of the STING antiviral signaling pathway.

IMPORTANCE
The zebrafish larva provides technical advantages for understanding host-pathogen interactions. In this study, we established the zebrafish larva as a useful model for studying HSV-1 infection. HSV-1 infection triggers strong type I interferon production, which depends on STING expression. In addition, STING-mediated antiviral signaling is conserved in zebrafish. Interestingly, zDHX9 and zDDX41 are indispensable for detecting HSV-1, while cGAS is dispensable. This proof-of-concept study indicates that the zebrafish represents an amenable model for the investigation of cytosolic DNA sensing mechanisms.
Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons
Authors: Wetmore Kelly M. , Price Morgan N. , Waters Robert J. , Lamson Jacob S. , He Jennifer , Hoover Cindi A. , Blow Matthew J. , Bristow James , Butland Gareth , Arkin Adam P. , Deutschbauer Adam 
Published: May 2015

ABSTRACT

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn
5
and
mariner
transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with
Escherichia coli
,
Phaeobacter inhibens
,
Pseudomonas stutzeri
,
Shewanella amazonensis
, and
Shewanella oneidensis
. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In
P. inhibens
, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative
d
-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling.

IMPORTANCE
A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.
Natural Bacterial Communities Serve as Quantitative Geochemical Biosensors
Authors: Smith Mark B. , Rocha Andrea M. , Smillie Chris S. , Olesen Scott W. , Paradis Charles , Wu Liyou , Campbell James H. , Fortney Julian L. , Mehlhorn Tonia L. , Lowe Kenneth A. , Earles Jennifer E. , Phillips Jana , Techtmann Steve M. , Joyner Dominique C. , Elias Dwayne A. , Bailey Kathryn L. , Hurt Richard A. , Preheim Sarah P. , Sanders Matthew C. , Yang Joy , Mueller Marcella A. , Brooks Scott , Watson David B. , Zhang Ping , He Zhili , Dubinsky Eric A. , Adams Paul D. , Arkin Adam P. , Fields Matthew W. , Zhou Jizhong , Alm Eric J. , Hazen Terry C. 
Published: May 2015

ABSTRACT

Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as
in situ
environmental sensors that respond to and capture perturbations caused by human impacts. These
in situ
biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive.

IMPORTANCE
Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.
Exploring risk of experiencing intimate partner violence after HIV infection: a qualitative study among women with HIV attending postnatal services in Swaziland
Authors: Mulrenan Claire , Colombini Manuela , Howard Natasha , Kikuvi Joshua , Mayhew Susannah H 
Published: May 2015

ObjectiveTo explore risks of experiencing intimate partner violence (IPV) after HIV infection among women with HIV in a postnatal care setting in Swaziland.DesignA qualitative semistructured in-depth interview study, using thematic analysis with deductive and inductive coding, of IPV experiences after HIV infection extracted from service-integration interview transcripts.SettingSwaziland.Participants19 women with HIV, aged 18–44, were purposively sampled for an in-depth interview about their experiences of services, HIV and IPV from a quantitative postnatal cohort participating in an evaluation of HIV and reproductive health services integration in Swaziland.ResultsResults indicated that women were at risk of experiencing IPV after HIV infection, with 9 of 19 disclosing experiences of physical violence and/or coercive control post-HIV. IPV was initiated through two key pathways: (1) acute interpersonal triggers (eg, status disclosure, mother-to-child transmission of HIV) and (2) chronic normative tensions (eg, fertility intentions, initiating contraceptives).ConclusionsThe results highlight a need to mitigate the risk of IPV for women with HIV in shorter and longer terms in Swaziland. While broader changes are needed to resolve gender disparities, practical steps can be institutionalised within health facilities to reduce, or avoid increasing, IPV pathways for women with HIV. These might include mutual disclosure between partners, greater engagement of Swazi males with HIV services, and promoting positive masculinities that support and protect women.Trial registration numberNCT01694862.
Serological investigation to identify risk factors for post-flood infectious diseases: a longitudinal survey among people displaced by Typhoon Morakot in Taiwan
Authors: Lin Chun-Yu , Chen Tun-Chieh , Dai Chia-Yen , Yu Ming-Lung , Lu Po-Liang , Yen Jeng-Hsien , Chen Yen-Hsu 
Published: May 2015

ObjectivesAfter Typhoon Morakot struck Taiwan in 2009, thousands of Taiwanese citizens were displaced to shelters for several weeks. Others were placed in urban communities where they had family members. This study aimed to investigate serological status in both groups and identify risk factors associated with seroconversion of infectious diseases.DesignA longitudinal survey.SettingAll experimental and clinical investigations were performed in a tertiary teaching hospital.ParticipantsA total of 288 displaced persons (96 males and 192 females) were recruited and complete follow-up data through two rounds of sampling were collected. The average age was 58.42 years (range 31–87 years).InterventionsFirst, serum specimens were collected between December 2009 and January 2010, 4–5 months after the typhoon. The second round of specimen collection was carried out after 6 months.Primary and secondary outcome measuresThe primary outcome measured was serological status of vaccine-preventable droplet-borne infectious diseases (ie, measles, mumps, rubella) and water-borne diseases (ie, amoebiasis and leptospirosis). The secondary outcome was identification of risk factors for seroconversion using univariate and multivariate analyses.ResultsComplete data were available for all 288 displaced persons (114 from the shelter group; 174 from the community group). Seroconversion ofEntamoeba histolyticawas observed in 128 (44.4%) participants, with a significantly higher rate in the shelter group than in the community group (56.1% vs 36.8%; p=0.001). There were 10 cases of rubella seroconversion. After adjusting for medical history, hypertension and hyperlipidaemia, shelter stay was associated with higher risk for seroconversion (OR=2.055, 95% CI 1.251 to 3.374; p=0.004). Amoebiasis was more evident in the shelter group, although the manifestations were mild.ConclusionsOur results suggested that (1) a clean water supply is essential postdisaster, especially in crowded shelters, and (2) vaccination programmes should be extended to populations at higher risk for post-disaster displacement or to those with weakened immune status.
Evaluation of a tobacco prevention programme among teenagers in Sweden
Authors: Hedman Linnéa , Andersson Martin , Stridsman Caroline , Rönmark Eva 
Published: May 2015

ObjectiveTo study the prevalence of tobacco use among teenagers, to evaluate a tobacco prevention programme and to study factors related to participation in the prevention programme.Design and settingPopulation-based prospective cohort study.MethodWithin the Obstructive Lung disease in Northern Sweden (OLIN) studies, a cohort study about asthma in schoolchildren started in 2006. All children aged 7–8 years in three municipalities were invited to a questionnaire survey and 2585 (96%) participated. The cohort was followed up at age 11–12 years (n=2612, 95% of invited) and 14–15 years (n=2345, 88% of invited). In 2010, some of the children in the OLIN cohort (n=447) were invited to a local tobacco prevention programme and 224 (50%) chose to participate.ResultsAt the age of 14–15 years, the prevalence of daily smoking was 3.5%. Factors related to smoking were female sex, having a smoking mother, participation in sports and lower parental socioeconomic status (SES). The prevalence of using snus was 3.3% and risk factors were male sex, having a smoking mother, having a snus-using father and non-participation in the prevention programme. In the prevention programme, the prevalence of tobacco use was significantly lower among the participants compared with the controls in the cohort. Factors related to non-participation were male sex, having a smoking mother, lower parental SES and participation in sports.ConclusionsThe prevalence of tobacco use was lower among the participants in the tobacco prevention programme compared with the non-participants as well as with the controls in the cohort. However, the observed benefit of the intervention may be overestimated as participation was biased by selection.