bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the Qiagen
artus
C. difficile QS-RGQ Kit for Detection of Clostridium difficile Toxins A and B in Clinical Stool Specimens
Authors: Jazmati Nathalie , Wiegel Pia , Ličanin Božica , Plum Georg
Published: March 2015

We compared the Qiagen
artus
C. difficile QS-RGQ kit, a new nucleic acid amplification test for the detection of
Clostridium difficile
toxins in stool specimens, with the Cepheid Xpert
C. difficile
test. The sensitivity, specificity, positive predictive value, and negative predictive value for the Qiagen
artus
C. difficile QS-RGQ test were 100%, 89.5%, 60.9%, and 100%, and those for the Cepheid Xpert
C. difficile
test were 100%, 90%, 62.2%, and 100%, respectively.
What Is the Origin of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolates from Humans without Livestock Contact? An Epidemiological and Genetic Analysis
Authors: Lekkerkerk W. S. N. , van Wamel W. J. B. , Snijders S. V. , Willems R. J. , van Duijkeren E. , Broens E. M. , Wagenaar J. A. , Lindsay J. A. , Vos M. C.
Published: March 2015

Fifteen percent of all methicillin-resistant
Staphylococcus aureus
(MRSA) clonal complex 398 (CC398) human carriers detected in The Netherlands had not been in direct contact with pigs or veal calves. To ensure low MRSA prevalence, it is important to investigate the likely origin of this MRSA of unknown origin (MUO). Recently, it was shown that CC398 strains originating from humans and animals differ in the presence of specific mobile genetic elements (MGEs). We hypothesized that determining these specific MGEs in MUO isolates and comparing them with a set of CC398 isolates of various known origin might provide clues to their origin. MUO CC398 isolates were compared to MRSA CC398 isolates obtained from humans with known risk factors, a MRSA CC398 outbreak isolate, livestock associated (LA) MRSA CC398 isolates from pigs, horses, chickens, and veal calves, and five methicillin-susceptible
Staphylococcus aureus
(MSSA) CC398 isolates of known human origin. All strains were
spa
typed, and the presence or absence of,
scn
,
chp
, φ3
int
, φ6
int
, φ7
int
,
rep7
,
rep27
, and
cadDX
was determined by PCRs. The MRSA CC398 in humans, MUO, or MRSA of known origin (MKO) resembled MRSA CC398 as found in pigs and not MSSA CC398 as found in humans. The distinct human MSSA CC398
spa
type, t571, was not present among our MRSA CC398 strains; MRSA CC398 was tetracycline resistant and carried no φ3 bacteriophage with
scn
and
chp
. We showed by simple PCR means that human MUO CC398 carriers carried MRSA from livestock origin, suggestive of indirect transmission. Although the exact transmission route remains unknown, direct human-to-human transmission remains a possibility as well.
Evaluation of Level of Agreement in Bordetella Species Identification in Three U.S. Laboratories during a Period of Increased Pertussis
Authors: Burgos-Rivera Brunilís , Lee Adria D. , Bowden Katherine E. , Faulkner Amanda E. , Seaton Brent L. , Lembke Bryndon D. , Cartwright Charles P. , Martin Stacey W. , Tondella M. Lucia
Published: March 2015

While PCR is the most common method used for detecting
Bordetella pertussis
in the United States, most laboratories use insertion sequence
481
(IS
481
), which is not specific for
B. pertussis
; therefore, the relative contribution of other
Bordetella
species is not understood. The objectives of this study were to evaluate the proportion of other
Bordetella
species misidentified as
B. pertussis
during a period of increased pertussis incidence, determine the level of agreement in
Bordetella
species detection between U.S. commercial laboratories and the CDC, and assess the relative diagnostic sensitivity of CDC's PCR assay when using a different PCR master mix. Specimens collected between May 2012 and May 2013 were tested at two U.S. commercial laboratories for
B. pertussis
and
B. parapertussis
detection. Every fifth specimen positive for IS
481
and/or IS
1001
with cycle threshold (

C
T

) values of ≤35 was sent to CDC for PCR testing that identifies
Bordetella
species. Specimens with indeterminate or negative results in the CDC PCR were tested using an alternate PCR master mix. Of 755 specimens, there was agreement in species identification for 83.4% (
n
= 630). Of the specimens with different identifications (
n
= 125), 79.2% (
n
= 99) were identified as indeterminate
B. pertussis
at CDC. Overall, 0.66% (
n
= 5) of the specimens were identified as
B. holmesii
or
B. bronchiseptica
at CDC. Of 115 specimens with indeterminate or negative results, 46.1% (
n
= 53) were
B. pertussis
positive when tested by an alternate master mix, suggesting a possible increase in assay sensitivity. This study demonstrates good agreement between the two U.S. commercial laboratories and CDC and little misidentification of
Bordetella
species during the 2012 U.S. epidemic.
Predominant Mycobacterium tuberculosis Families and High Rates of Recent Transmission among New Cases Are Not Associated with Primary Multidrug Resistance in Lima, Peru
Authors: Barletta Francesca , Otero Larissa , de Jong Bouke C. , Iwamoto Tomotada , Arikawa Kentaro , Van der Stuyft Patrick , Niemann Stefan , Merker Matthias , Uwizeye Cécile , Seas Carlos , Rigouts Leen
Published: March 2015

Sputum samples from new tuberculosis (TB) cases were collected over 2 years as part of a prospective study in the northeastern part of Lima, Peru. To measure the contribution of recent transmission to the high rates of multidrug resistance (MDR) in this area,
Mycobacterium tuberculosis
complex (MTBc) isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and 15-locus mycobacterial interspersed repetitive-unit (MIRU-15)-variable-number tandem repeat (VNTR) analysis. MDR was found in 6.8% of 844 isolates, of which 593 (70.3%) were identified as belonging to a known MTBc lineage, whereas 198 isolates (23.5%) could not be assigned to these lineages and 12 (1.4%) represented mixed infections. Lineage 4 accounted for 54.9% (
n
= 463) of the isolates, most of which belonged to the Haarlem family (
n
= 279). MIRU-15 analysis grouped 551/791 isolates (69.7%) in 102 clusters, with sizes ranging from 2 to 46 strains. The overall high clustering rate suggests a high level of recent transmission in this population, especially among younger patients (odds ratio [OR], 1.6;
P
= 0.01). Haarlem strains were more prone to cluster, compared to the other families taken together (OR, 2.0;
P
< 0.0001), while Beijing (OR, 0.6;
P
= 0.006) and LAM (OR, 0.7;
P
= 0.07) strains clustered less. Whereas streptomycin-resistant strains were more commonly found in clusters (OR, 1.8;
P
= 0.03), clustering rates did not differ between MDR and non-MDR strains (OR, 1.8;
P
= 0.1). Furthermore, only 16/51 MDR strains clustered with other MDR strains, suggesting that patients with primary MDR infections acquired the infections mostly from index cases outside the study population, such as retreated cases.
Bacteremia Caused by Kerstersia gyiorum
Authors: Bostwick A. Doran , Zhang Cecelia , Manninen Katja , Touchberry Joanne , Greene Shermalyn R. , Holland Thomas L.
Published: March 2015

Kerstersia
spp. are an unusual cause of human infections. We report the first known case of bacteremia and sepsis due to
Kerstersia gyiorum
, in a patient with chronic lower-extremity ulcers, and we review the literature on this uncommon pathogen.
Mycobacterium lepromatosis Infections in Nuevo León, Mexico
Authors: Vera-Cabrera Lucio , Escalante-Fuentes Wendy , Ocampo-Garza Sonia S. , Ocampo-Candiani Jorge , Molina-Torres Carmen A. , Avanzi Charlotte , Benjak Andrej , Busso Philippe , Singh Pushpendra , Cole Stewart T.
Published: March 2015

The frequency of infection caused by the recently described pathogen
Mycobacterium lepromatosis
is unknown. Here, we describe the demographics, clinical characteristics, and therapeutic outcomes of five lepromatous leprosy patients suffering from
M. lepromatosis
infection in Nuevo Léon, Mexico. Diagnosis was facilitated by a new highly specific PCR procedure.
Maintenance of Epstein-Barr Virus Latent Status by a Novel Mechanism, Latent Membrane Protein 1-Induced Interleukin-32, via the Protein Kinase Cδ Pathway
Authors: Lai Kun-Yi , Chou Ya-Ching , Lin Jiun-Han , Liu Yi , Lin Kai-Min , Doong Shin-Lian , Chen Mei-Ru , Yeh Te-Huei , Lin Sue-Jane , Tsai Ching-Hwa
Published: March 2015

Epstein-Barr virus (EBV), an oncogenic herpesvirus, has the potential to immortalize primary B cells into lymphoblastoid cell lines (LCLs)in vitro. During immortalization, several EBV products induce cytokines or chemokines, and most of these are required for the proliferation of LCLs. Interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, is upregulated after EBV infection, and this upregulation is detectable in all LCLs tested. EBV latent membrane protein 1 (LMP1) is responsible for inducing IL-32 expression at the mRNA and protein levels. Mechanistically, we showed that this LMP1 induction is provided by the p65 subunit of NF-κB, which binds to and activates the IL-32 promoter. Furthermore, the short hairpin RNA (shRNA)-mediated depletion of endogenous LMP1 and p65 in LCLs suppressed IL-32 expression, further suggesting that LMP1 is the key factor that stimulates IL-32 in LCLs via the NF-κB p65 pathway. Functionally, knockdown of IL-32 in LCLs elicits viral reactivation and affects cytokine expression, but it has no impact on cell proliferation and apoptosis. Of note, we reveal the mechanism whereby IL-32 is involved in the maintenance of EBV viral latency by inactivation of Zta promoter activity. This atypical cytoplasmic IL-32 hijacks the Zta activator protein kinase Cδ (PKCδ) and inhibits its translocation from the cytoplasm to the nucleus, where PKCδ binds to the Zta promoter and activates lytic cycle progression. These novel findings reveal that IL-32 is involved in the maintenance of EBV latency in LCLs. This finding may provide new information to explain how EBV maintains latency, in addition to viral chromatin structure and epigenetic modification.

IMPORTANCEEBV persists in two states, latency and lytic replication, which is a unique characteristic of human infections. So far, little is known about how herpesviruses maintain latency in particular tissues or cell types. EBV is an excellent model to study this question because more than 90% of people are latently infected. EBV can immortalize primary B cells into lymphoblastoid cell linesin vitro. Expression of IL-32, a novel atypical cytoplasmic proinflammatory cytokine, increased after infection. The expression of IL-32 was controlled by LMP1. In investigating the regulatory mechanism, we demonstrated that the p65 subunit of NF-κB is required for this upregulation. Of note, the important biological activity of IL-32 was to trap protein kinase Cδ in the cytoplasm and prevent it from binding to the Zta promoter, which is the key event for EBV reaction. So, the expression of LMP1-induced IL-32 plays a role in the maintenance of EBV latency.
Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3
Authors: Hurst-Hess Kelley R. , Kuo Lili , Masters Paul S.
Published: March 2015

Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis.

IMPORTANCE
The largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as to combat host innate immunity. We initiated a dissection of MHV nsp3 aimed at identifying those activities or structures in this huge molecule that are essential to replicase activity. We found that both PLP1 and ADRP could be entirely deleted, provided that the requirement for proteolytic processing by PLP1 was offset by an alternative mechanism. This demonstrated that neither PLP1 nor ADRP plays an essential role in coronavirus RNA synthesis.
Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction
Authors: Liu GuanQun , Park Hong-Su , Pyo Hyun-Mi , Liu Qiang , Zhou Yan
Published: March 2015

Retinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, using
in vitro
-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation.

IMPORTANCE
The IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.
Association of Kaposi's Sarcoma-Associated Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene Expression
Authors: Brulois Kevin , Wong Lai-Yee , Lee Hye-Ra , Sivadas Priyanka , Ensser Armin , Feng Pinghui , Gao Shou-Jiang , Toth Zsolt , Jung Jae U.
Published: March 2015

Transcription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes.
CpG Oligodeoxynucleotides Facilitate Delivery of Whole Inactivated H9N2 Influenza Virus via Transepithelial Dendrites of Dendritic Cells in Nasal Mucosa
Authors: Qin Tao , Yin Yinyan , Yu Qinghua , Huang Lulu , Wang Xiaoqing , Lin Jian , Yang Qian
Published: March 2015

The spread of the low-pathogenicity avian H9N2 influenza virus has seriously increased the risk of a new influenza pandemic. Although whole inactivated virus (WIV) vaccine via intranasal pathway is the effective method of blocking virus transmission, the mucosal barrier seems to be a major factor hampering its development. CpG oligodeoxynucleotides, a known adjuvant, can target downstream dendritic cells (DCs) and effectively enhance the mucosal and systemic immune responses. However, the ability of CpGs to assist H9N2 WIV in transepithelial transport remains unknown. Here,
in vitro
and
in vivo
, we showed that CpGs provided assistance for H9N2 WIV in recruiting DCs to the nasal epithelial cells (ECs) and forming transepithelial dendrites (TEDs) to capture luminal viruses. CD103
+
DCs participated in this process. Chemokine CCL20 from nasal ECs played a key role in driving DC recruitment and TED formation. Virus-loaded DCs quickly migrated into the draining cervical lymph nodes (CLNs) for antigen presentation. In addition, the competence of CpGs was independent of direct epithelial transport via the transcellular or paracellular pathway. Taken together, our data demonstrated that CpGs enhanced the transport of H9N2 WIV via TEDs of nasal DCs, which might be a novel mechanism for optimal adaptive immune responses.

IMPORTANCE
This paper demonstrates by both an
in vivo
and an
in vitro
coculture model that CpG oligodeoxynucleotides, known as an adjuvant generally targeting downstream immune responses, also are crucial for the transport of H9N2 WIV across nasal epithelial cells (ECs) via the uptake of transepithelial dendrites (TEDs). Our results prove for the first time to our knowledge that the immune-potentiating mechanism of CpGs is based on strengthening the transepithelial uptake of H9N2 WIV in nasal mucosa. These findings provide a fresh perspective for further improvement of intranasal influenza vaccines, which are urgently needed in the face of the potential threat of H9N2 influenza.
Increased Infectivity of Anchorless Mouse Scrapie Prions in Transgenic Mice Overexpressing Human Prion Protein
Authors: Race Brent , Phillips Katie , Meade-White Kimberly , Striebel James , Chesebro Bruce
Published: March 2015

Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous
in vivo
studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal.

IMPORTANCE
Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection.
Structure of Serotype 1 Reovirus Attachment Protein σ1 in Complex with Junctional Adhesion Molecule A Reveals a Conserved Serotype-Independent Binding Epitope
Authors: Stettner Eva , Dietrich Melanie H. , Reiss Kerstin , Dermody Terence S. , Stehle Thilo
Published: March 2015

Mammalian orthoreoviruses use glycans and junctional adhesion molecule A (JAM-A) as attachment receptors. We determined the structure of serotype 1 reovirus attachment protein σ1 alone and in complex with JAM-A. Comparison with the structure of serotype 3 reovirus σ1 bound to JAM-A reveals that both σ1 proteins engage JAM-A with similar affinities and via conserved binding epitopes. Thus, σ1–JAM-A interactions are unlikely to explain the differences in pathogenesis displayed by these reovirus serotypes.
Borna Disease Virus Phosphoprotein Modulates Epigenetic Signaling in Neurons To Control Viral Replication
Authors: Bonnaud Emilie M. , Szelechowski Marion , Bétourné Alexandre , Foret Charlotte , Thouard Anne , Gonzalez-Dunia Daniel , Malnou Cécile E.
Published: March 2015

Understanding the modalities of interaction of neurotropic viruses with their target cells represents a major challenge that may improve our knowledge of many human neurological disorders for which viral origin is suspected. Borna disease virus (BDV) represents an ideal model to analyze the molecular mechanisms of viral persistence in neurons and its consequences for neuronal homeostasis. It is now established that BDV ensures its long-term maintenance in infected cells through a stable interaction of viral components with the host cell chromatin, in particular, with core histones. This has led to our hypothesis that such an interaction may trigger epigenetic changes in the host cell. Here, we focused on histone acetylation, which plays key roles in epigenetic regulation of gene expression, notably for neurons. We performed a comparative analysis of histone acetylation patterns of neurons infected or not infected by BDV, which revealed that infection decreases histone acetylation on selected lysine residues. We showed that the BDV phosphoprotein (P) is responsible for these perturbations, even when it is expressed alone independently of the viral context, and that this action depends on its phosphorylation by protein kinase C. We also demonstrated that BDV P inhibits cellular histone acetyltransferase activities. Finally, by pharmacologically manipulating cellular acetylation levels, we observed that inhibiting cellular acetyl transferases reduces viral replication in cell culture. Our findings reveal that manipulation of cellular epigenetics by BDV could be a means to modulate viral replication and thus illustrate a fascinating example of virus-host cell interaction.

IMPORTANCE
Persistent DNA viruses often subvert the mechanisms that regulate cellular chromatin dynamics, thereby benefitting from the resulting epigenetic changes to create a favorable milieu for their latent and persistent states. Here, we reasoned that Borna disease virus (BDV), the only RNA virus known to durably persist in the nucleus of infected cells, notably neurons, might employ a similar mechanism. In this study, we uncovered a novel modality of virus-cell interaction in which BDV phosphoprotein inhibits cellular histone acetylation by interfering with histone acetyltransferase activities. Manipulation of cellular histone acetylation is accompanied by a modulation of viral replication, revealing a perfect adaptation of this “ancient” virus to its host that may favor neuronal persistence and limit cellular damage.
The 5′ Untranslated Region of a Novel Infectious Molecular Clone of the Dicistrovirus Cricket Paralysis Virus Modulates Infection
Authors: Kerr Craig H. , Wang Qing S. , Keatings Kathleen , Khong Anthony , Allan Douglas , Yip Calvin K. , Foster Leonard J. , Jan Eric
Published: March 2015

Dicistroviridae
are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species,
Cricket paralysis virus
(CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of
in vitro
-transcribed RNA from the CrPV clone into
Drosophila
Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult
Drosophila
flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5′ untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells.

IMPORTANCE
Dicistroviridae
, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in insect cells. Further insights into the viral molecular mechanisms are hampered due to a lack of an established infectious clone. We report the construction of the first infectious clone of the dicistrovirus, cricket paralysis virus (CrPV). We show that transfection of the CrPV clone RNA into
Drosophila
cells led to production of infectious particles that resemble natural CrPV virions and result in cytopathic effects and expression of CrPV proteins and RNA in infected cells. The CrPV clone should provide insights into the dicistrovirus life cycle and host-virus interactions in insect cells. Using this clone, we find that a 196-nucleotide duplication within the 5′ untranslated region of the CrPV clone increased viral translation in reporter constructs but decreased virus infectivity, thus revealing a balance that interplays between viral translation and replication.
Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits
Authors: Haagmans Bart L. , van den Brand Judith M. A. , Provacia Lisette B. , Raj V. Stalin , Stittelaar Koert J. , Getu Sarah , de Waal Leon , Bestebroer Theo M. , van Amerongen Geert , Verjans Georges M. G. M. , Fouchier Ron A. M. , Smits Saskia L. , Kuiken Thijs , Osterhaus Albert D. M. E.
Published: March 2015

The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.
Decoding Viral Infection by Ribosome Profiling
Authors: Stern-Ginossar Noam
Published: March 2015

Ribosome profiling is an emerging technique that uses deep sequencing to monitor translation in live cells. Studies using ribosome profiling have already provided novel insights into the identities and amounts of the proteins being produced in cells, as well as novel insights into the mechanism of protein synthesis and translation regulation. Application of ribosome profiling to cells infected with human cytomegalovirus and Kaposi's sarcoma-associated herpesvirus revealed unanticipated complexity in the coding capacity of herpesviruses. Here, I discuss these results and how the application of ribosome profiling to cells infected with other viruses can reveal novel insights into the process of infection.
Pandemic Swine H1N1 Influenza Viruses with Almost Undetectable Neuraminidase Activity Are Not Transmitted via Aerosols in Ferrets and Are Inhibited by Human Mucus but Not Swine Mucus
Authors: Zanin Mark , Marathe Bindumadhav , Wong Sook-San , Yoon Sun-Woo , Collin Emily , Oshansky Christine , Jones Jeremy , Hause Benjamin , Webby Richard
Published: March 2015

A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for the transmission of viruses between humans. Here we describe two pandemic H1N1 viruses, A/swine/Virginia/1814-1/2012 and A/swine/Virginia/1814-2/2012 (pH1N1
low
-1 and -2, respectively), that were isolated from swine symptomatic for influenza. The enzymatic activity of the NA of these viruses was almost undetectable, while the HA binding affinity for α2,6 sialic acids was greater than that of the highly homologous pH1N1 viruses A/swine/Pennsylvania/2436/2012 and A/swine/Minnesota/2499/2012 (pH1N1-1 and -2), which exhibited better-balanced HA and NA activities. The
in vitro
growth kinetics of pH1N1
low
and pH1N1 viruses were similar, but aerosol transmission of pH1N1
low
-1 was abrogated and transmission via direct contact in ferrets was significantly impaired compared to pH1N1-1, which transmitted by direct and aerosol contact. In normal human bronchial epithelial cells, pH1N1
low
-1 was significantly inhibited by mucus but pH1N1-1 was not. In Madin-Darby canine kidney cell cultures overlaid with human or swine mucus, human mucus inhibited pH1N1
low
-1 but swine mucus did not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a barrier for interspecies transmission between humans and swine for influenza viruses.

IMPORTANCE
A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for transmission of viruses from swine to humans. Here we show that a swine virus with extremely functionally mismatched HA and NAs (pH1N1
low
-1) cannot transmit via aerosol in ferrets, while another highly homologous virus with HA and NAs that are better matched functionally (pH1N1-1) can transmit via aerosol. These viruses show similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, but pH1N1
low
-1 is significantly inhibited by mucus in normal human bronchial epithelial cells whereas pH1N1-1 is not. Further, human mucus could inhibit these viruses, but swine mucus could not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a species barrier between humans and swine for influenza viruses.
Deep Sequencing and Phylogenetic Analysis of Variants Resistant to Interferon-Based Protease Inhibitor Therapy in Chronic Hepatitis Induced by Genotype 1b Hepatitis C Virus
Authors: Sato Mitsuaki , Maekawa Shinya , Komatsu Nobutoshi , Tatsumi Akihisa , Miura Mika , Muraoka Masaru , Suzuki Yuichiro , Amemiya Fumitake , Takano Shinichi , Fukasawa Mitsuharu , Nakayama Yasuhiro , Yamaguchi Tatsuya , Uetake Tomoyoshi , Inoue Taisuke , Sato Tadashi , Sakamoto Minoru , Yamashita Atsuya , Moriishi Kohji , Enomoto Nobuyuki
Published: March 2015

Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the
IFNL3
(rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the
IFNL3
TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy.

IMPORTANCE
In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.
Influenza Virus-Induced Caspase-Dependent Enlargement of Nuclear Pores Promotes Nuclear Export of Viral Ribonucleoprotein Complexes
Authors: Mühlbauer Dirk , Dzieciolowski Julia , Hardt Martin , Hocke Andreas , Schierhorn Kristina L. , Mostafa Ahmed , Müller Christin , Wisskirchen Christian , Herold Susanne , Wolff Thorsten , Ziebuhr John , Pleschka Stephan
Published: March 2015

Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection.

IMPORTANCE
In contrast to the process seen with most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than the cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral replication cycle. The report provides an intriguing example of how influenza virus exploits cellular structures and regulatory pathways, including intracellular transport mechanisms, to complete its replication cycle and maximize the production of infectious virus progeny.
Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells
Authors: Ternette Nicola , Block Peter D. , Sánchez-Bernabéu Álvaro , Borthwick Nicola , Pappalardo Elisa , Abdul-Jawad Sultan , Ondondo Beatrice , Charles Philip D. , Dorrell Lucy , Kessler Benedikt M. , Hanke Tomáš
Published: March 2015

Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv, which delivers HIV-1 conserved antigenic regions by using modified vaccinia virus Ankara (MVA). We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 h after MVA.HIVconsv infection. Four of the seven conserved peptides were monitored between 0 and 3.5 h of infection by using quantitative mass spectrometry (Q-MS), and their abundance in HLA class I associations reflected levels of the whole HIVconsv protein in the cell. While immunopeptides delivered by the incoming MVA vector proteins could be detected, all early HIVconsv-derived immunopeptides were likely synthesizedde novo. MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance.

IMPORTANCEThe vast changes in cellular antigen presentation after infection of cells with a vectored vaccine, as shown here for MVA.HIVconsv, highlight the complexity of factors that need to be considered for efficient antigen delivery and presentation. Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.
Murine Norovirus Replication Induces G0/G1Cell Cycle Arrest in Asynchronously Growing Cells
Authors: Davies Colin , Brown Chris M. , Westphal Dana , Ward Joanna M. , Ward Vernon K.
Published: March 2015

Many viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1phase and an accumulation of cells in the G0/G1phase. The accumulation in G0/G1phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1phase may be representative of other members of theCaliciviridae.

IMPORTANCENoroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1phase. Furthermore, we show that MNV replication is enhanced in the G1phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1phase for RNA virus replication.
A Virus-Like Particle System Identifies the Endonuclease Domain of Crimean-Congo Hemorrhagic Fever Virus
Authors: Devignot Stephanie , Bergeron Eric , Nichol Stuart , Mirazimi Ali , Weber Friedemann
Published: March 2015

Crimean-Congo hemorrhagic fever virus
(CCHFV; genus
Nairovirus
) is an extremely pathogenic member of the
Bunyaviridae
family. Since handling of the virus requires a biosafety level 4 (BSL-4) facility, little is known about pathomechanisms and host interactions. Here, we describe the establishment of a transcriptionally competent virus-like particle (tc-VLP) system for CCHFV. Recombinant polymerase (L), nucleocapsid protein (N) and a reporter minigenome expressed in human HuH-7 cells resulted in formation of transcriptionally active nucleocapsids that could be packaged by coexpressed CCHFV glycoproteins into tc-VLPs. The tc-VLPs resembled authentic virus particles in their protein composition and neutralization sensitivity to anti-CCHFV antibodies and could recapitulate all steps of the viral replication cycle. Particle attachment, entry, and primary transcription were modeled by infection of naive cells. The subsequent steps of genome replication, secondary transcription, and particle assembly and release can be obtained upon passaging the tc-VLPs on cells expressing CCHFV structural proteins. The utility of the VLP system was demonstrated by showing that the endonuclease domain of L is located around amino acid D693, as was predicted
in silico
by B. Morin et al. (PLoS Pathog 6:e1001038, 2010,
http://dx.doi.org/10.1371/journal.ppat.1001038
). The tc-VLP system will greatly facilitate studies and diagnostics of CCHFV under non-BSL-4 conditions.

IMPORTANCE
Crimean-Congo hemorrhagic fever virus (CCHFV) is an extremely virulent pathogen of humans. Since the virus can be handled only at the highest biosafety level, research is restricted to a few specialized laboratories. We developed a plasmid-based system to produce virus-like particles with the ability to infect cells and transcribe a reporter genome. Due to the absence of viral genes, the virus-like particles are unable to spread or cause disease, thus allowing study of aspects of CCHFV biology under relaxed biosafety conditions.
The impact of stopping high-energy oral nutritional supplements on eating behaviour and weight gain
Authors: Wright Charlotte M , Chillingworth Anna
Published: March 2015

Background and aims

Many children referred to a tertiary feeding clinic are already taking high-energy oral nutritional supplements (HEOS), but these often seem not clinically useful. We undertook a retrospective review of all children on HEOS at the time of referral to the clinic in order to describe their subsequent progress in terms of growth and feeding behaviour.

Results

A total of 48 children were on HEOS at referral and withdrawal of HEOS was attempted in 38 children, aged median 3.0 years (range 0.7–10 years) who were taking volumes equivalent to 2/3 of total daily energy requirements. The children tended to be very short and slim (median height SD score (SDS) −2.0 (range −5.7 to 1.9); body mass index −2.0 (−5.1 to 1.9)). Half had normal neurodevelopment (ND) but 4 (11%) had learning disability and 4 (11%) severe ND problems. By last follow-up after 0.86 (0–2.9) years, 30 (79%) had stopped all feeds. Those who stopped had a mean (SD) change in weight of 0.08 (0.6) SDS (range −0.88 to +1.59). Five children (17%) showed significant catch-up weight gain after stopping feeds, of whom three had been referred for weight faltering and possible tube feeding. Improvement in feeding behaviour was documented in 76% (29).

Conclusions

The use of HEOS in children suppresses appetite for solid food due to energy compensation. In some cases, HEOS may perpetuate or even cause weight faltering. It should not be assumed that failure to respond to HEOS is an indication for tube feeding.