bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay
Authors: Blacksell Stuart D. , Lim Cherry , Tanganuchitcharnchai Ampai , Jintaworn Suthatip , Kantipong Pacharee , Richards Allen L. , Paris Daniel H. , Limmathurotsakul Direk , Day Nicholas P. J. 
Published: March 2016

ABSTRACT

The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for
Orientia tsutsugamushi
, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of
O. tsutsugamushi
developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.
Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients
Authors: Fauchier T. , Hasseine L. , Gari-Toussaint M. , Casanova V. , Marty P. M. , Pomares C. 
Published: March 2016

ABSTRACT

Pneumocystis
jirovecii
pneumonia (PCP) is an acute and life-threatening lung disease caused by the fungus
Pneumocystis jirovecii
. The presentation of PCP in HIV-positive patients is well-known and consists of a triad of dyspnea, fever, and cough, whereas the presentation of PCP in HIV-negative patients is atypical and consists of a sudden outbreak, O
2
desaturation, and a rapid lethal outcome without therapy. Despite the availability of direct and indirect identification methods, the diagnosis of PCP remains difficult. The cycle threshold (

C
T

) values obtained by quantitative PCR (qPCR) allow estimation of the fungal burden. The more elevated that the fungal burden is, the higher the probability that the diagnosis is pneumonia. The purposes of the present study were to evaluate the

C
T

values to differentiate colonization and pneumonia in a population of immunocompromised patients overall and patients stratified on the basis of their HIV infection status. Testing of bronchoalveolar lavage (BAL) fluid samples from the whole population of qPCR-positive patients showed a mean

C
T

value for patients with PCP of 28 (95% confidence interval [CI], 26 to 30) and a mean

C
T

value for colonized patients of 35 (95% CI, 34 to 36) (
P
< 10
−3
). For the subgroup of HIV-positive patients, we demonstrated that a

C
T

value below 27 excluded colonization and a

C
T

value above 30 excluded PCP with a specificity of 100% and a sensitivity of 80%, respectively. In the subgroup of HIV-negative patients, we demonstrated that a

C
T

value below 31 excluded colonization and a

C
T

value above 35 excluded PCP with a specificity of 80% and a sensitivity of 80%, respectively. Thus, qPCR of BAL fluid samples is an important tool for the differentiation of colonization and pneumonia in
P. jirovecii
-infected immunocompromised patients and patients stratified on the basis of HIV infection status with different

C
T

values.
A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production
Authors: Gebhard Leopoldo G. , Iglesias Néstor G. , Byk Laura A. , Filomatori Claudia V. , De Maio Federico A. , Gamarnik Andrea V. 
Published: March 2016

ABSTRACT
Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation.

IMPORTANCE
Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for dengue virus particle assembly. NS3 is a multifunctional enzyme that participates in different steps of the viral life cycle. Using reporter systems to dissect different viral processes, we identified a novel N-terminal unstructured region of the NS3 protein as crucial for production of viral particles. Based on our findings, we propose new ideas that include NS3 as a possible scaffold for the viral assembly process.
Both Epistasis and Diversifying Selection Drive the Structural Evolution of the Ebola Virus Glycoprotein Mucin-Like Domain
Authors: Ibeh Neke , Nshogozabahizi Jean Claude , Aris-Brosou Stéphane 
Published: March 2016

ABSTRACT
Throughout the last 3 decades, Ebola virus (EBOV) outbreaks have been confined to isolated areas within Central Africa; however, the 2014 variant reached unprecedented transmission and mortality rates. While the outbreak was still under way, it was reported that the variant leading up to this outbreak evolved faster than previous EBOV variants, but evidence for diversifying selection was undetermined. Here, we test this selection hypothesis and show that while previous EBOV outbreaks were preceded by bursts of diversification, evidence for site-specific diversifying selection during the emergence of the 2014 EBOV clade is weak. However, we show strong evidence supporting an interplay between selection and correlated evolution (epistasis), particularly in the mucin-like domain (MLD) of the EBOV glycoprotein. By reconstructing ancestral structures of the MLD, we further propose a structural mechanism explaining how the substitutions that accumulated between 1918 and 1969 distorted the MLD, while more recent epistatic substitutions restored part of the structure, with the most recent substitution being adaptive. We suggest that it is this complex interplay between weak selection, epistasis, and structural constraints that has shaped the evolution of the 2014 EBOV variant.

IMPORTANCE
The role that selection plays in the emergence of viral epidemics remains debated, particularly in the context of the 2014 EBOV outbreak. Most critically, should such evidence exist, it is generally unclear how this relates to function and increased virulence. Here, we show that the viral lineage leading up to the 2014 outbreak underwent a complex interplay between selection and correlated evolution (epistasis) in a protein region that is critical for immune evasion. We then reconstructed the three-dimensional structure of this domain and showed that the initial mutations in this lineage deformed the structure, while subsequent mutations restored part of the structure. Along this mutational path, the first and last mutations were adaptive, while the intervening ones were epistatic. Altogether, we provide a mechanistic model that explains how selection and epistasis acted on the structural constraints that materialized during the 2014 EBOV outbreak.
Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence
Authors: Ratinier Maxime , Shaw Andrew E. , Barry Gerald , Gu Quan , Di Gialleonardo Luigina , Janowicz Anna , Varela Mariana , Randall Richard E. , Caporale Marco , Palmarini Massimo 
Published: March 2016

ABSTRACT
Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence.

IMPORTANCE
Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by
Culicoides
biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.
Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis
Authors: Liu Long , Tian Jiao , Nan Hao , Tian Mengmeng , Li Yuan , Xu Xiaodong , Huang Baicheng , Zhou Enmin , Hiscox Julian A. , Chen Hongying 
Published: March 2016

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis.

IMPORTANCE
It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.
Mutagenesis of Coronavirus nsp14 Reveals Its Potential Role in Modulation of the Innate Immune Response
Authors: Becares Martina , Pascual-Iglesias Alejandro , Nogales Aitor , Sola Isabel , Enjuanes Luis , Zuñiga Sonia 
Published: March 2016

ABSTRACT

Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as “non-self” by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of
Alphacoronavirus
. Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response.

IMPORTANCE
The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described to date. The introduction of a mutation in zinc finger 1 of the ExoN domain of nsp14 led to production of a virus that induced a weak antiviral response, most likely due to the accumulation of lower levels of dsRNA in the late phases of infection. These observations allowed us to propose a novel role for CoV nsp14 ExoN activity in counteracting the antiviral response, which could serve as a novel target for the design of antiviral strategies.
Comparative Cell Biology and Evolution of Annexins in Diplomonads
Authors: Einarsson Elin , Ástvaldsson Ásgeir , Hultenby Kjell , Andersson Jan O. , Svärd Staffan G. , Jerlström-Hultqvist Jon 
Published: March 2016

Annexins are proteins that associate with phospholipids in a Ca
2+
-dependent fashion. These proteins have been intensely studied in animals and plants because of their importance in diverse cellular processes, yet very little is known about annexins in single-celled eukaryotes, which represent the largest diversity of organisms. The human intestinal parasite
Giardia intestinalis
is known to have more annexins than humans, and they contribute to its pathogenic potential. In this study, we investigated the annexin complement in the salmon pathogen
Spironucleus salmonicida
, a relative of
G. intestinalis
. We found that
S. salmonicida
has a large repertoire of annexins and that the gene family has expanded separately across diplomonads, with members showing sequence diversity similar to that seen across kingdom-level groups such as plants and animals.
S. salmonicida
annexins are prominent components of the cytoskeleton and membrane. Two annexins are associated with a previously unrecognized structure in the anterior of the cell.
Estimated incidence and risk factors of sudden unexpected death
Authors: Lewis Mary Elizabeth , Lin Feng-Chang , Nanavati Parin , Mehta Neil , Mounsey Louisa , Nwosu Anthony , Pursell Irion , Chung Eugene H , Mounsey J Paul , Simpson Ross J 
Published: March 2016

ObjectiveIn this manuscript, we estimate the incidence and identify risk factors for sudden unexpected death in a socioeconomically and racially diverse population in one county in North Carolina. Estimates of the incidence and risk factors contributing to sudden death vary widely. The Sudden Unexpected Death in North Carolina (SUDDEN) project is a population-based investigation of the incidence and potential causes of sudden death.MethodsFrom 3 March 2013 to 2 March 2014, all out-of-hospital deaths in Wake County, North Carolina, were screened to identify presumed sudden unexpected death among free-living residents between the ages of 18 and 64 years. Death certificate, public and medical records were reviewed and adjudicated to confirm sudden unexpected death cases.ResultsFollowing adjudication, 190 sudden unexpected deaths including 122 men and 68 women were identified. Estimated incidence was 32.1 per 100 000 person-years overall: 42.7 among men and 22.4 among women. The majority of victims were white, unmarried men over age 55 years, with unwitnessed deaths at home. Hypertension and dyslipidaemia were common in men and women. African-American women dying from sudden unexpected death were over-represented. Women who were under age 55 years with coronary disease accounted for over half of female participants with coronary artery disease.ConclusionsThe overall estimated incidence of sudden unexpected death may account for approximately 10% of all deaths classified as ‘natural’. Women have a lower estimated incidence of sudden unexpected death than men. However, we found no major differences in age or comorbidities between men and women. African-Americans and young women with coronary disease are at risk for sudden unexpected death.
Blood haemogram in Ovis aries and Capra hyrcus: effect of storage time
Authors: Fazio Francesco , Giangrosso Giuseppe , Marafioti Simona , Zanghì Elisa , Arfuso Francesca , Piccione Giuseppe 
Published: March 2016

The aim of the present study was to assess the effect of storage time at +4 °C on haematological profile in goat (n = 25) and sheep (n = 25). After collection, blood samples were immediately analyzed and then divided into four aliquots that were stored at 4 °C and tested at 24 h (T1), 48 h (T2), 72 h (T3), and 1 wk (T4), respectively. One-way repeated-measures analysis of variance (ANOVA) was used to determine statistically significant effect of storage conditions both in goats and in sheep. Our results showed that among the two species studied, goats showed highest blood stability after refrigeration at +4 °C. In goats, all hematological parameters, except PLT, showed no significant changes during all days of monitoring with respect to basal values (T0). In sheep, no significantly effect of storage time on RBC and WBC levels were found, whereas the other hematological parameters change significantly over the time. Our findings suggest that the blood storage time reported for goat may not be applied to sheep’s blood, which underscore the differences between these two species that are erroneously considered similar.
Screening Strategies for Colorectal Cancer: A Systematic Review of the Evidence
Authors: McLeod Robin S ,   members of the Canadian Task Force on Preventive Health Care
Published: March 2016

BACKGROUND: Colorectal cancer is the third most common cancer in Canada. It is well recognized that there are improved survival rates if the disease is treated in its early stages, and indeed this may be a preventable disease. This paper systematically reviews the effectiveness of specific screening techniques for colorectal cancer in asymptomatic individuals at normal or above average risk.METHODS: MEDLINE was searched for articles published between January 1966 and January 2001 by using the MESH terms 'screening' and 'colorectal neoplasia'. The reference sections of review articles published before January 2001 were checked, and content experts were surveyed. The evidence was evaluated using the standardized methodology of the Canadian Task Force on Preventive Health Care.RSULTS AND DISCUSSION: For individuals at normal risk, there is evidence to support the use of annual or biennial fecal occult blood testing and flexible sigmoidoscopy for asymptomatic individuals over age 50 years. The evidence regarding whether only one or both of fecal occult blood testing and sigmoidoscopy should be performed is unclear, as is the evidence regarding the use of colonoscopy as an initial screen. For individuals at above average risk, the evidence supports either genetic testing or flexible sigmoidoscopy of individuals at risk in familial adenomatous polyposis kindreds, and screening with colonoscopy of patients in kindreds with hereditary nonpolyposis colon cancer. The evidence regarding colonoscopy for individuals who have a family history of colorectal polyps or cancer but do not fit the criteria for hereditary nonpolyposis colon cancer is unclear. Development of better risk stratification for screening is a high research priority, and further research, including randomized, controlled trials, into the effectiveness and feasibility of other screening modalities is necessary.