bluuebunny/crossref_metadata_2025 huggingface dataset:
Site-Specific Clinical Evaluation of the Luminex xTAG Gastrointestinal Pathogen Panel for Detection of Infectious Gastroenteritis in Fecal Specimens
Authors: Patel Anami , Navidad Jose , Bhattacharyya Sanjib
Published: June 2014

We evaluate the clinical performance of the Luminex xTAG gastrointestinal (GI) pathogenin vitrodiagnostic (IVD) assay in a comparison between clinical and public health laboratories. The site reproducibility study showed 98.7% sensitivity with high positive and negative agreement values (96.2% and 99.8%, respectively), while assay performance against confirmatory methods resulted in 96.4% sensitivity with similar positive and negative agreement values (90.1% and 99.5%, respectively). High-throughput detection of multiple GI pathogens improved turnaround time, consolidated laboratory workflow, and simplified stool culture practices, thus reducing the overall cost and number of specimens processed.
The Pulmonary Localization of Virus-Specific T Lymphocytes Is Governed by the Tissue Tropism of Infection
Authors: Knudson Cory J. , Weiss Kayla A. , Hartwig Stacey M. , Varga Steven M.
Published: June 2014

The migration of pathogen-specific T cells into nonlymphoid tissues, such as the lung, is critical to control peripheral infections. Use of
in vivo
intravascular labeling of leukocytes has allowed for improved discrimination between cells located in the blood from cells present within peripheral tissues, such as the lung. This is particularly important in the lung, which is comprised of an intricate network of blood vessels that harbors a large proportion of the total blood volume at any given time. Recent work has demonstrated that >80% of antigen-specific effector CD8 T cells remain in the pulmonary vasculature following an intratracheal infection with a systemic viral pathogen. However, it remains unclear what proportion of effector CD8 T cells are located within lung tissue following a localized respiratory viral infection. We confirm that most effector and memory CD8 T cells are found in the vasculature after an intranasal infection with the systemic pathogens lymphocytic choriomeningitis virus (LCMV) or vaccinia virus (VACV). In contrast, following pulmonary viral infections with either respiratory syncytial virus (RSV) or influenza A virus (IAV), 80 to 90% of the antigen-specific effector CD8 T cells were located within lung tissue. Similarly, the majority of antigen-specific CD4 T cells were present within lung tissue during a pulmonary viral infection. Furthermore, a greater proportion of gamma interferon-positive (IFN-γ
+
) effector CD8 and CD4 T cells were located within lung tissue following a localized respiratory viral infection. Our results indicate that T cells exhibit significantly altered distribution patterns dependent upon the tissue tropism of the infection.

IMPORTANCE
The migration of T cells to nonlymphoid sites, such as the lung, is critical to mediate clearance of viral infections. The highly vascularized lung holds up to 40% of blood, and thus, the T cell response may be a reflection of lymphocytes localized to the pulmonary vasculature instead of lung tissue. We examined the localization of T cell responses within the lung following either a localized or systemic viral infection. We demonstrate that following intranasal infection with a systemic pathogen, most T cells are localized to the pulmonary vasculature. In contrast, T cells are primarily localized to lung tissue following a respiratory viral infection. Our results demonstrate vast differences in the localization of T cell responses within the lung parenchyma between pathogens that can replicate locally versus systemically and that intravascular antibody labeling can be utilized to assess the localization patterns of T cell responses in nonlymphoid organs.
Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4
Authors: van Doremalen Neeltje , Miazgowicz Kerri L. , Milne-Price Shauna , Bushmaker Trenton , Robertson Shelly , Scott Dana , Kinne Joerg , McLellan Jason S. , Zhu Jiang , Munster Vincent J.
Published: June 2014

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012. Recently, the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific interaction of the receptor-binding domain (RBD) of MERS-CoV spike protein and DPP4 was determined by crystallography. Animal studies identified rhesus macaques but not hamsters, ferrets, or mice to be susceptible for MERS-CoV. Here, we investigated the role of DPP4 in this observed species tropism. Cell lines of human and nonhuman primate origin were permissive of MERS-CoV, whereas hamster, ferret, or mouse cell lines were not, despite the presence of DPP4. Expression of human DPP4 in nonsusceptible BHK and ferret cells enabled MERS-CoV replication, whereas expression of hamster or ferret DPP4 did not. Modeling the binding energies of MERS-CoV spike protein RBD to DPP4 of human (susceptible) or hamster (nonsusceptible) identified five amino acid residues involved in the DPP4-RBD interaction. Expression of hamster DPP4 containing the five human DPP4 amino acids rendered BHK cells susceptible to MERS-CoV, whereas expression of human DPP4 containing the five hamster DPP4 amino acids did not. Using the same approach, the potential of MERS-CoV to utilize the DPP4s of common Middle Eastern livestock was investigated. Modeling of the DPP4 and MERS-CoV RBD interaction predicted the ability of MERS-CoV to bind the DPP4s of camel, goat, cow, and sheep. Expression of the DPP4s of these species on BHK cells supported MERS-CoV replication. This suggests, together with the abundant DPP4 presence in the respiratory tract, that these species might be able to function as a MERS-CoV intermediate reservoir.

IMPORTANCE
The ongoing outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) has caused 701 laboratory-confirmed cases to date, with 249 fatalities. Although bats and dromedary camels have been identified as potential MERS-CoV hosts, the virus has so far not been isolated from any species other than humans. The inability of MERS-CoV to infect commonly used animal models, such as hamster, mice, and ferrets, indicates the presence of a species barrier. We show that the MERS-CoV receptor DPP4 plays a pivotal role in the observed species tropism of MERS-CoV and subsequently identified the amino acids in DPP4 responsible for this restriction. Using a combined modeling and experimental approach, we predict that, based on the ability of MERS-CoV to utilize the DPP4 of common Middle East livestock species, such as camels, goats, sheep, and cows, these form a potential MERS-CoV intermediate host reservoir species.
MicroRNA miR-BART20-5p Stabilizes Epstein-Barr Virus Latency by Directly Targeting
BZLF1
and
BRLF1
Authors: Jung Yu-Jin , Choi Hoyun , Kim Hyoji , Lee Suk Kyeong
Published: June 2014

Epstein-Barr virus (EBV) is a human herpesvirus associated with various tumors. Rather than going through the lytic cycle, EBV maintains latency by limiting the expression of viral genes in tumors. Viral microRNAs (miRNAs) of some herpesviruses have been reported to directly target immediate early genes and suppress lytic induction. In this study, we investigated whether BamHI-A rightward transcript (BART) miRNAs targeted two EBV immediate early genes,
BZLF1
and
BRLF1
. Bioinformatic analysis predicted that 12 different BART miRNAs would target
BRLF1
. Of these, the results of a luciferase reporter assay indicated that only one interacted with the 3′ untranslated region (UTR) of
BRLF1
: miR-BART20-5p. miR-BART20-5p's effect on gene expression involved two putative seed match sites in the
BRLF1
3′ UTR, but a mutant version of the miRNA, miR-BART20-5pm, had no effect on expression. As expected from the fact that the entire 3′ UTR of
BZLF1
resides within the 3′ UTR of
BRLF1
, miR-BART20-5p interacted with the 3′ UTR of
BZLF1
as well.
BZLF1
and
BRLF1
mRNA and protein expression was suppressed in cells of an AGS cell line infected with the recombinant Akata strain of EBV (AGS-EBV) transfected with a miR-BART20-5p mimic. The expression of various EBV early proteins was also suppressed by the miR-BART20-5p mimic. In contrast,
BZLF1
and
BRLF1
expression in AGS-EBV cells transfected with a miR-BART20-5p inhibitor was enhanced. Furthermore, progeny virus production was suppressed by the miR-BART20-5p mimic and enhanced by the miR-BART20-5p inhibitor in AGS-EBV cells induced for the lytic cycle. Our data suggest that miR-BART20-5p plays a key role in latency maintenance in EBV-associated tumors by directly targeting immediate early genes.

IMPORTANCE
Herpesviruses maintain latency using various mechanisms and establish lifelong infection in the host. From time to time, herpesviruses are reactivated and express immediate early genes which trigger a lytic cascade, leading to the production of progeny viruses. Recently, some herpesviruses have been shown to use their own microRNAs (miRNAs) to downregulate immediate early genes to inhibit the lytic cycle. This study presents evidence that EBV also downregulates two immediate early genes by miR-BART20-5p to suppress the lytic cycle and progeny virus production. Overall, this is the first study to report the direct regulation of EBV immediate early genes by an EBV miRNA, implying its likely importance in latency maintenance in EBV-associated tumors.
Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor
Authors: Tawaratsumida Kazuki , Phan Van , Hrincius Eike R. , High Anthony A. , Webby Richard , Redecke Vanessa , Häcker Hans
Published: June 2014

Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production.

IMPORTANCE
Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense.
Kaposi's Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have Stage-Specific Immune Evasion Roles during Lytic Replication
Authors: Brulois Kevin , Toth Zsolt , Wong Lai-Yee , Feng Pinghui , Gao Shou-Jiang , Ensser Armin , Jung Jae U.
Published: June 2014

The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of
K3
or
K5
into a ΔK3 ΔK5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the
K
5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the
K
3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1α-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived
K3
and
K5
deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFP
high
and pK8.1-driven blue fluorescent protein-positive (BFP
+
) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages.

IMPORTANCE
Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.
Predicted Structure and Domain Organization of Rotavirus Capping Enzyme and Innate Immune Antagonist VP3
Authors: Ogden Kristen M. , Snyder Matthew J. , Dennis Allison F. , Patton John T.
Published: June 2014

Rotaviruses and orbiviruses are nonturreted
Reoviridae
members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted
Reoviridae
capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5′-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2′-
O
-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2′-
O
-methyltransferase catalytic tetrad, interact with
S
-adenosyl-
l
-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2′-5′ oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3.

IMPORTANCE
Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.
Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines
Authors: Kasloff Samantha B. , Pizzuto Matteo S. , Silic-Benussi Micol , Pavone Silvia , Ciminale Vincenzo , Capua Ilaria
Published: June 2014

Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas
in vivo
, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas.

IMPORTANCE
Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis
in vitro
and antitumor effects
in vivo
. These results suggest that slightly pathogenic IAVs may prove to be effective for oncolytic virotherapy of PDA and provide grounds for further studies to develop specific and targeted viruses, with the aim of testing their efficacy in clinical contexts.
Identification of B Cells as a Major Site for Cyprinid Herpesvirus 3 Latency
Authors: Reed Aimee N. , Izume Satoko , Dolan Brian P. , LaPatra Scott , Kent Michael , Dong Jing , Jin Ling
Published: June 2014

Cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV), is a member of theAlloherpesviridae, and is a recently discovered emerging herpesvirus that is highly pathogenic for koi and common carp. Our previous study demonstrated that CyHV-3 becomes latent in peripheral white blood cells (WBC). In this study, CyHV-3 latency was further investigated in IgM+WBC. The presence of the CyHV-3 genome in IgM+WBC was about 20-fold greater than in IgM−WBC. To determine whether CyHV-3 expressed genes during latency, transcription from all eight open reading frames (ORFs) in the terminal repeat was investigated in IgM+WBC from koi with latent CyHV-3 infection. Only a spliced ORF6 transcript was found to be abundantly expressed in IgM+WBC from CyHV-3 latently infected koi. The spliced ORF6 transcript was also detectedin vitroduring productive infection as early as 1 day postinfection. The ORF6 transcript fromin vitroinfection begins at −127 bp upstream of the ATG codon and ends +188 bp downstream of the stop codon, +20 bp downstream of the polyadenylation signal. The hypothetical protein of ORF6 contains a consensus sequence with homology to a conserved domain of EBNA-3B and ICP4 from Epstein-Barr virus and herpes simplex virus 1, respectively, both members of theHerpesviridae. This is the first report of latent CyHV-3 in B cells and identification of gene transcription during latency for a member of theAlloherpesviridae.

IMPORTANCEThis is the first demonstration that a member of theAlloherpesviridae, cyprinid herpesvirus 3 (CyHV-3), establishes a latent infection in the B cells of its host,Cyprinus carpio. In addition, this is the first report of identification of gene transcription during latency for a member ofHerpesviralesoutsideHerpesviridae. This is also the first report that the hypothetical protein of latent transcript of CyHV-3 contains a consensus sequence with homology to a conserved domain of EBNA-3B from Epstein-Barr virus and ICP4 from herpes simplex virus 1, which are genes important for latency. These strongly suggest that latency is evolutionally conserved across vertebrates.
Extraribosomal L13a Is a Specific Innate Immune Factor for Antiviral Defense
Authors: Mazumder Barsanjit , Poddar Darshana , Basu Abhijit , Kour Ravinder , Verbovetskaya Valentina , Barik Sailen
Published: June 2014

We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the
Pneumovirus
genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected.
In silico
RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3′untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (
g
amma-
a
ctivated
i
nhibitor of
t
ranslation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-γ)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial
v
irus-
a
ctivated
i
nhibitor of
t
ranslation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN-γ-independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense.

IMPORTANCE
The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3′ untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity.
Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex
Authors: Das Subhendu , Ortiz Daniel A. , Gurczynski Stephen J. , Khan Fatin , Pellett Philip E.
Published: June 2014

Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and
trans
-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis.

IMPORTANCE
Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.
Transfer of the Amino-Terminal Nuclear Envelope Targeting Domain of Human MX2 Converts MX1 into an HIV-1 Resistance Factor
Authors: Goujon Caroline , Moncorgé Olivier , Bauby Hélène , Doyle Tomas , Barclay Wendy S. , Malim Michael H.
Published: June 2014

The myxovirus resistance 2 (MX2) protein of humans has been identified recently as an interferon (IFN)-inducible inhibitor of human immunodeficiency virus type 1 (HIV-1) that acts at a late postentry step of infection to prevent the nuclear accumulation of viral cDNA (C. Goujon et al., Nature 502:559–562, 2013,http://dx.doi.org/10.1038/nature12542; M. Kane et al., Nature 502:563–566, 2013,http://dx.doi.org/10.1038/nature12653; Z. Liu et al., Cell Host Microbe 14:398–410, 2013,http://dx.doi.org/10.1016/j.chom.2013.08.015). In contrast, the closely related human MX1 protein, which suppresses infection by a range of RNA and DNA viruses (such as influenza A virus [FluAV]), is ineffective against HIV-1. Using a panel of engineered chimeric MX1/2 proteins, we demonstrate that the amino-terminal 91-amino-acid domain of MX2 confers full anti-HIV-1 function when transferred to the amino terminus of MX1, and that this fusion protein retains full anti-FluAV activity. Confocal microscopy experiments further show that this MX1/2 fusion, similar to MX2 but not MX1, can localize to the nuclear envelope (NE), linking HIV-1 inhibition with MX accumulation at the NE. MX proteins are dynamin-like GTPases, and while MX1 antiviral function requires GTPase activity, neither MX2 nor MX1/2 chimeras require this attribute to inhibit HIV-1. This key discrepancy between the characteristics of MX1- and MX2-mediated viral resistance, together with previous observations showing that the L4 loop of the stalk domain of MX1 is a critical determinant of viral substrate specificity, presumably reflect fundamental differences in the mechanisms of antiviral suppression. Accordingly, we propose that further comparative studies of MX proteins will help illuminate the molecular basis and subcellular localization requirements for implementing the noted diversity of virus inhibition by MX proteins.

IMPORTANCEInterferon (IFN) elicits an antiviral state in cells through the induction of hundreds of IFN-stimulated genes (ISGs). The human MX2 protein has been identified as a key effector in the suppression of HIV-1 infection by IFN. Here, we describe a molecular genetic approach, using a collection of chimeric MX proteins, to identify protein domains of MX2 that specify HIV-1 inhibition. The amino-terminal 91-amino-acid domain of human MX2 confers HIV-1 suppressor capabilities upon human and mouse MX proteins and also promotes protein accumulation at the nuclear envelope. Therefore, these studies correlate the cellular location of MX proteins with anti-HIV-1 function and help establish a framework for future mechanistic analyses of MX-mediated virus control.
Absence of Genetic Differences among G10P[11] Rotaviruses Associated with Asymptomatic and Symptomatic Neonatal Infections in Vellore, India
Authors: Libonati Margaret H. , Dennis Allison F. , Ramani Sasirekha , McDonald Sarah M. , Akopov Asmik , Kirkness Ewen F. , Kang Gagandeep , Patton John T.
Published: June 2014

Rotaviruses (RVs) are leading causes of severe diarrhea and vomiting in infants and young children. RVs with G10P[11] genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants responsible for differences in symptomology, the genome sequences of G10P[11] RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as those of the previously characterized symptomatic N155 Vellore isolate. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P[11] viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic infections and from neonates with asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P[11] RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms.

IMPORTANCE
Rotavirus (RV) strains have been identified that preferentially replicate in neonates, in some cases, without causing gastrointestinal disease. Surveillance studies have established that G10P[11] RVs are a major cause of neonatal infection in Vellore, India, with half of infected neonates exhibiting symptoms. We used Sanger and next-generation sequencing technologies to contrast G10P[11] RVs recovered from symptomatic and asymptomatic neonates. Remarkably, the data showed that the RNA genomes of the viruses were virtually indistinguishable and lacked any differences that could explain the diversity of clinical outcomes among infected Vellore neonates. The sequencing results also indicated that some symptomatic and some asymptomatic Vellore neonates were infected with other enteric viruses (Aichi virus, astrovirus, salvirus/klassevirus); however, none could be correlated with the presence of symptoms in neonates. Together, our findings suggest that other poorly defined factors, not connected to the genetic makeup of the Vellore G10P[11] viruses, influence whether neonates develop gastrointestinal disease symptoms.
To ‘Get by’ or ‘get help’? A qualitative study of physicians’ challenges and dilemmas when patients have limited English proficiency
Authors: Parsons Janet A , Baker Natalie A , Smith-Gorvie Telisha , Hudak Pamela L
Published: June 2014

Objective

Encounters between patients and physicians who do not speak the same language are relatively common in Canada, particularly in urban settings; this trend is increasing worldwide. Language discordance has important effects on health outcomes, including mortality. This study sought to explore physicians’ experiences of care provision in situations of language discordance in depth.

Design

Qualitative study based on individual interviews. Interview guides elicited physicians’ perspectives on how they determined whether communication could proceed unaided. A descriptive qualitative approach was adopted, entailing inductive thematic analysis.

Participants

22 physicians experienced in treating patients in situations of language discordance were recruited from the emergency and internal medicine departments of an urban tertiary-care hospital.

Setting

Large, inner-city teaching hospital in Toronto, Canada, one of the most linguistically diverse cities internationally.

Results

Determining when to ‘get by’ or ‘get help’ in order to facilitate communication was described as a fluid and variable process.Deciding which strategy to use depended on three inter-related factors: time/time constraints, acuity of situationandease of use/availability of translation aids. Participants reported at times feeling conflicted about their decisions, portraying some of these clinical encounters as a ‘troubling space’ in which they experienced one or moredilemmasrelated torealversusideal practice, responsibilityandinformed consent.

Conclusions

In situations of language discordance, a physician's decision to ‘get by’ (vs ‘get help’) rests on a judgement of whether communication can be considered ‘good enough’ to proceed and depends on the circumstances of the specific encounter. The tension set up between what is ‘ideal’ and what is practically possible can be experienced as a dilemma by physicians.The study's findings have implications for practice and policy not only in Canada but in other multilingual settings, and indicate that physicians require greater support.
Can you refuse these discounts? An evaluation of the use and price discount impact of price-related promotions among US adult smokers by cigarette manufacturers
Authors: Caraballo Ralph S , Wang Xu , Xu Xin
Published: June 2014

Objectives

The raising unit price of cigarette has been shown to be one of the most effective ways of reducing cigarette consumption and increasing rates of successful quitting. However, researchers have shown that price-sensitive smokers have used a variety of strategies to mitigate the effect of the rising price of cigarettes on their smoking habits. In particular, 23–34% of adult smokers in the US use cheaper brands, and 18–55% use coupons or promotions. Little is known about the discount use by type of brands. As such, the main purpose of this analysis is to evaluate the uses and price discount effects of these price-related discounts by manufacturers and major brands.

Setting

An analysis based on the cross-sectional 2009–2010 National Adult Tobacco Survey (NATS).

Participants

11 766 current smokers aged 18 or above in the USA.

Primary outcome measures

Price-related discount was defined as smokers who used coupons, rebates, buy-one-get-one-free, two-for-one or any other special promotions for their last cigarettes purchase.

Results

The use of price-related discounts and associated price impact vary widely by cigarette manufacturer and brand. Approximately one of three Camel, one of four Marlboro and one of eight Newport smokers used price-related discounts on their latest cigarette purchases. The average price reductions of discounts offered by Philip Morris (PM) or R.J. Reynolds (RJR) were around 29 cents per pack while that of Lorillard (Newport only) was 24 cents per pack. Cigarette brands that provided significant per pack price reductions include: PM Marlboro (28 cents), RJR brand Camel (41 cents), Doral (50 cents), Kool (73 cents) and Salem (80 cents), and Lorillard Newport (24 cents).

Conclusions

Policies that decrease price-minimisation strategies will benefit public health.
Failure to address potential bias in non-randomised controlled clinical trials may cause lack of evidence on patient-reported outcomes: a method study
Authors: Peinemann Frank , Labeit Alexander Michael , Thielscher Christian , Pinkawa Michael
Published: June 2014

Objectives

We conducted a workup of a previously published systematic review and aimed to analyse why most of the identified non-randomised controlled clinical trials with patient-reported outcomes did not match a set of basic quality criteria.

Setting

There were no limits on the level of care and the geographical location.

Participants

The review evaluated permanent interstitial low-dose rate brachytherapy in patients with localised prostate cancer and compared that intervention with alternative procedures such as external beam radiotherapy, radical prostatectomy and no primary therapy.

Primary outcome measure

Fulfilment of basic inclusion criteria according to a Participants, Interventions, Comparisons, Outcomes (PICO) framework and accomplishment of requirements to contain superimposed risk of bias.

Results

We found that 21 of 50 excluded non-randomised controlled trials did not meet the PICO inclusion criteria. The remaining 29 studies showed a lack in the quality of reporting. The resulting flaws included attrition bias due to loss of follow-up, lack of reporting baseline data, potential confounding due to unadjusted data and lack of statistical comparison between groups.

Conclusions

With respect to the reporting of patient-reported outcomes, active efforts are required to improve the quality of reporting in non-randomised controlled trials concerning permanent interstitial low-dose rate brachytherapy in patients with localised prostate cancer.
Non-pharmacological treatments for adult patients with functional constipation: a systematic review protocol
Authors: Chen Min , Zheng Hui , Li Juan , Huang Dequan , Chen Qin , Fang Jianqiao
Published: June 2014

Introduction

The aim of this review is to assess the effectiveness, efficacy and safety of non-pharmacological therapies for patients with functional constipation.

Methods and analysis

We will electronically search OVID MEDLINE, EMBASE, Cochrane library, CINAHL, AMED and ISI web of knowledge without any language restrictions. We will also try to obtain literatures from other sources, such as a hand search of library journals or conference abstracts. After searching and screening of the studies, we will run a meta-analysis of the included randomised-controlled trials. We will summarise the results as risk ratio for dichotomous data and standardised or weighted mean difference for continuous data.

Dissemination

This systematic review will summarise current evidence for using non-pharmacological therapies to treat functional constipation, and will be disseminated through peer-review publications or conference presentations.

Trial registration number

PROSPERO 2014: CRD42014006686.
Rationale and study design of the Adaptive study of IL-2 dose on regulatory T cells in type 1 diabetes (DILT1D): a non-randomised, open label, adaptive dose finding trial
Authors: Waldron-Lynch Frank , Kareclas Paula , Irons Kathryn , Walker Neil M , Mander Adrian , Wicker Linda S , Todd John A , Bond Simon
Published: June 2014

Introduction

CD4 T regulatory cells (Tregs) are crucial for the maintenance of self-tolerance and are deficient in many common autoimmune diseases such as type 1 diabetes (T1D). Interleukin 2 (IL-2) plays a major role in the activation and function of Tregs and treatment with ultra-low dose (ULD) IL-2 could increase Treg function to potentially halt disease progression in T1D. However, prior to embarking on large phase II/III clinical trials it is critical to develop new strategies for determining the mechanism of action of ULD IL-2 in participants with T1D. In this mechanistic study we will combine a novel trial design with a clinical grade Treg assay to identify the best doses of ULD IL-2 to induce targeted increases in Tregs.

Method and analysis

Adaptive study of IL-2 dose on regulatory T cells in type 1 diabetes (DILT1D) is a single centre non-randomised, single dose, open label, adaptive dose-finding trial. The primary objective of DILT1D is to identify the best doses of IL-2 to achieve a minimal or maximal Treg increase in participants with T1D (N=40). The design has an initial learning phase where pairs of participants are assigned to five preassigned doses followed by an interim analysis to determine the two Treg targets for the reminder of the trial. This will then be followed by an adaptive phase which is fully sequential with an interim analysis after each participant is observed to determine the choice of dose based on the optimality criterion to minimise the determinant of covariance of the estimated target doses. A dose determining committee will review all data available at the interim(s) and then provide decisions regarding the choice of dose to administer to subsequent participants.

Ethics and dissemination

Ethical approval for the study was granted on 18 February 2013.

Results

The results of this study will be reported through peer-reviewed journals, conference presentations and an internal organisational report.

Trial registration numbers

NCT01827735,ISRCTN27852285, DRN767.
Axl receptor tyrosine kinase expression in breast cancer
Authors: D'Alfonso Timothy M , Hannah Jeffrey , Chen Zhengming , Liu Yifang , Zhou Pengbo , Shin Sandra J
Published: June 2014

Aims

Triple-negative breast cancer comprises a clinically aggressive group of invasive carcinomas. We examined a published gene expression screen of a panel of breast cancer cell lines to identify a potential triple-negative breast cancer-specific gene signature, and attempted to verify our findings by performing immunohistochemical analysis on tissue microarrays containing a large cohort of invasive breast carcinomas.

Methods

The microarray dataset for a panel of human breast cancer cell lines was interrogated for triple-negative breast cancer-specific genes. Membranous immunohistochemical expression of the protein product of theAXLgene was assessed semiquantitatively in 569 invasive breast carcinomas grouped according to molecular subgroup by immunohistochemistry.

Results

AXLwas significantly upregulated in triple-negative/basal B cell lines compared with luminal or basal A cell lines. No significant difference was observed in the level of immunohistochemical expression of Axl protein between triple-negative breast cancers and other molecular subgroups (p=0.257). Axl expression was significantly associated with lymphovascular invasion (LVI) in all subgroups combined (p=0.033), and within the luminal A (p=0.002) and triple-negative breast cancer subgroups (p=0.026).

Conclusions

Despite preferential upregulation ofAXLin triple-negative/basal B cell lines, analysis of Axl protein expression in a large series of patients’ breast tumours revealed no association between Axl expression and triple-negative breast cancer or other subtype. The association of Axl expression with LVI supports previous work that implicates Axl as a promoter of invasiveness in breast cancer cell lines. Further studies are necessary to explore whether Axl expression of individual breast cancer tumours can be clinically useful.
ZERO-KNOWLEDGE BLACKBOX TESTING: WHERE ARE THE FAULTS?
Authors: WANG ERIC , CUI CEWEI , DANG ZHE , FISCHER THOMAS R. , YANG LINMIN
Published: June 2014

We derive a quantitative relationship between the maximal entropy rate achieved by a blackbox software system's specification graph, and the probability of faults Pnobtained by testing the system, as a function of the length n of a test sequence. By equating “blackbox” to the maximal entropy principle, we model the specification graph as a Markov chain that, for each distinct value of n, achieves the maximal entropy rate for that n. Hence the Markov transition probability matrices are not constant in n, but form a sequence of transition matrices T1,…, Tn. We prove that, for nontrivial specification graphs, the probability of finding faults goes asymptotically to zero as the test length n increases, regardless of the evolution of Tn. This implies that zero-knowledge testing is practical only for small n. We illustrate the result using a concrete example of a system specification graph for an autopilot control system, and plot its curve Pn.
Stochastic quantization and holographic Wilsonian renormalization group of massless fermions in AdS
Authors: Oh Jae-Hyuk
Published: June 2014

We have studied holographic Wilsonian renormalization group (HWRG) of free massless fermionic fields in AdS space and its stochastic quantization (SQ) by identifying the Euclidean action with its boundary on-shell action. The natural extension of the relation between stochastic two-point correlation function and double trace coupling obtained from HWRG computation conjectured in [J.-H. Oh and D. P. Jatkar, J. High Energy Phys. 1211, 144 (2012) arXiv:1209.2242] to fermionic fields is established. We have confirmed that the stochastic two-point function precisely captures the radial flow of the double trace coupling via this relation.