bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT
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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACTCytotoxic 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

ABSTRACTMany 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

ABSTRACT

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 aimsMany 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.ResultsA 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).ConclusionsThe 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.