bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hepatitis E Virus (HEV) Genotype 3 Infection of Human Liver Chimeric Mice as a Model for Chronic HEV Infection
Authors: van de Garde Martijn D. B. , Pas Suzan D. , van der Net Guido , de Man Robert A. , Osterhaus Albert D. M. E. , Haagmans Bart L. , Boonstra Andre , Vanwolleghem Thomas 
Published: February 2016

ABSTRACT

Genotype 3 (gt3) hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities of
in vivo
HEV studies in a human liver chimeric mouse model (uPA
+/+
Nod-SCID-IL2Rγ
−/−
) next to the A549 cell culture system, using HEV RNA-positive EDTA-plasma, feces, or liver biopsy specimens from 8 immunocompromised patients with chronic gt3 HEV. HEV from feces- or liver-derived inocula showed clear virus propagation within 2 weeks after inoculation onto A549 cells, compared to slow or no HEV propagation of HEV RNA-positive, EDTA-plasma samples. These
in vitro
HEV infectivity differences were mirrored in human-liver chimeric mice after intravenous (i.v.) inoculation of selected samples. HEV RNA levels of up to 8 log IU HEV RNA/gram were consistently present in 100% of chimeric mouse livers from week 2 to week 14 after inoculation with human feces- or liver-derived HEV. Feces and bile of infected mice contained moderate to large amounts of HEV RNA, while HEV viremia was low and inconsistently detected. Mouse-passaged HEV could subsequently be propagated for up to 100 days
in vitro
. In contrast, cell culture-derived or seronegative EDTA-plasma-derived HEV was not infectious in inoculated animals. In conclusion, the infectivity of feces-derived human HEV is higher than that of EDTA-plasma-derived HEV both
in vitro
and
in vivo
. Persistent HEV gt3 infections in chimeric mice show preferential viral shedding toward mouse bile and feces, paralleling the course of infection in humans.

IMPORTANCE
Hepatitis E virus (HEV) genotype 3 infections are emerging in Western countries and are of great concern for immunosuppressed patients at risk for developing chronic HEV infection. Lack of adequate model systems for chronic HEV infection hampers studies on HEV infectivity and transmission and antiviral drugs. We compared the
in vivo
infectivity of clinical samples from chronic HEV patients in human liver chimeric mice to an
in vitro
virus culture system. Efficient
in vivo
HEV infection is observed after inoculation with feces- and liver-derived HEV but not with HEV RNA-containing plasma or cell culture supernatant. HEV in chimeric mice is preferentially shed toward bile and feces, mimicking the HEV infection course in humans. The observed
in vivo
infectivity differences may be relevant for the epidemiology of HEV in humans. This novel small-animal model therefore offers new avenues to unravel HEV's pathobiology.
Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation
Authors: Zhang Kuan , Brownlie Robert , Snider Marlene , van Drunen Littel-van den Hurk Sylvia 
Published: February 2016

ABSTRACT
VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status.

IMPORTANCE
In this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other alphaherpesviruses. Interestingly, lack of phosphorylation of VP8 by CK2 and US3 resulted in reduced incorporation of viral DNA into capsids during mutant BoHV-1 infection, as well as lower numbers of extracellular virions. Furthermore, mutant VP8 remained nuclear throughout infection, in contrast to WT VP8, which is nuclear at early stages and Golgi apparatus associated late during infection. This correlates with smaller amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, with the latter dependent on phosphorylation.
A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4
+
T Cells
Authors: Offersen Rasmus , Nissen Sara Konstantin , Rasmussen Thomas A. , Østergaard Lars , Denton Paul W. , Søgaard Ole Schmeltz , Tolstrup Martin 
Published: February 2016

ABSTRACT

Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a “shock-and-kill” approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703
ex vivo
exhibited increased secretion of interferon alpha (IFN-α) (
P
= 0.005) and CXCL10 (
P
= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56
dim
CD16
+
NK cells (
P
= 0.001) as well as higher proportions of CD107a-positive (
P
= 0.002) and IFN-γ-producing (
P
= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4
+
and CD8
+
T cells. Furthermore, CD4
+
T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (
P
= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4
+
T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (
P
= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4
+
T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials.

IMPORTANCE
We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel “dumbbell-shape” structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4
+
T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4
+
T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.
SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication
Authors: Su Chan-I , Tseng Chung-Hsin , Yu Chia-Yi , Lai Michael M. C. 
Published: February 2016

ABSTRACT

Small ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular gene
Ubc9
, which encodes the sole E2-conjugating enzyme required for SUMOylation. By
in vivo
and
in vitro
SUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication.

IMPORTANCE
SUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a “double indemnity” of NS5 to support DENV replication. Without SUMOylation, NS5 fails to maintain its protein stability, which consequently disrupts its function in viral RNA replication and innate immunity antagonism. DENV threatens billions of people worldwide, but no licensed vaccine or specific therapeutics are currently available. Thus, our findings suggest that rather than specifically targeting NS5 enzyme activity, NS5 protein stability is a novel drug target on the growing list of anti-DENV strategies.
Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity
Authors: Li Lian-Feng , Yu Jiahui , Li Yongfeng , Wang Jinghan , Li Su , Zhang Lingkai , Xia Shui-Li , Yang Qian , Wang Xiao , Yu Shaoxiong , Luo Yuzi , Sun Yuan , Zhu Yan , Munir Muhammad , Qiu Hua-Ji 
Published: February 2016

ABSTRACT

Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione
S
-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity.

IMPORTANCE
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.
The Attenuation Phenotype of a Ribavirin-Resistant Porcine Reproductive and Respiratory Syndrome Virus Is Maintained during Sequential Passages in Pigs
Authors: Khatun Amina , Shabir Nadeem , Seo Byoung-Joo , Kim Bum-Seok , Yoon Kyoung-Jin , Kim Won-Il 
Published: February 2016

ABSTRACT

In a previous study, ribavirin-resistant porcine reproductive and respiratory syndrome virus (PRRSV) mutants (RVRp13 and RVRp22) were selected, and their resistance against random mutation was shown in cultured cells. In the present study, these ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability during three pig-to-pig passages in comparison with modified live virus (MLV) (Ingelvac PRRS MLV). Pigs challenged with RVRp22 had significantly lower (
P
< 0.05) viral loads in sera and tissues than pigs challenged with MLV or RVRp13 at the first passage, and the attenuated replication of RVRp22 was maintained until the third passage. Viral loads in sera and tissues dramatically increased in pigs challenged with MLV or RVRp13 during the second passage. Consistently, all five sequences associated with the attenuation of virulent PRRSV in RVRp13 and MLV quickly reverted to wild-type sequences during the passages, but two attenuation sequences were maintained in RVRp22 even after the third passage. In addition, RVRp22 showed a significantly lower (
P
< 0.001) mutation frequency in nsp2, which is one of the most variable regions in the PRRSV genome, than MLV. Nine unique mutations were found in open reading frames (ORFs) 1a, 2, and 6 in the RVRp22 genome based on full-length sequence comparisons with RVRp13, VR2332 (the parental virus of RVRp13 and RVRp22), and MLV. Based on these results, it was concluded that RVRp22 showed attenuated replication in pigs; further, because of the high genetic stability of RVRp22, its attenuated phenotype was stable even after three sequential passages in pigs.

IMPORTANCE
PRRSV is a rapidly evolving RNA virus. MLV vaccines are widely used to control PRRS; however, there have been serious concerns regarding the use of MLV as a vaccine virus due to the rapid reversion to virulence during replication in pigs. As previously reported, ribavirin is an effective antiviral drug against many RNA viruses. Ribavirin-resistant mutants reemerged by escaping lethal mutagenesis when the treatment concentration was sublethal, and those mutants were genetically more stable than parental viruses. In a previous study, two ribavirin-resistant PRRSV mutants (RVRp13 and RVRp22) were selected, and their higher genetic stability was shown
in vitro
. Consequently, in the present study, both of the ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability
in vivo
. RVRp22 was found to exhibit higher genetic and phenotypic stability than MLV, and nine unique mutations were identified in the RVRp22 genome based on a full-length sequence comparison with the RVRp13, VR2332, and MLV genomes.
Viral Persistence Induces Antibody Inflation without Altering Antibody Avidity
Authors: Welten Suzanne P. M. , Redeker Anke , Toes René E. M. , Arens Ramon 
Published: February 2016

ABSTRACT

Antibodies are implicated in long-term immunity against numerous pathogens, and because of this property, antibody induction is the basis for many vaccines. Little is known about the influence of viral persistence on the evolving antibody response. Here, we examined the characteristics of antibody responses to persistent infection by employing the prototypic betaherpesvirus family member cytomegalovirus (CMV) in experimental mouse models. During the course of infection, mouse CMV (MCMV)-specific IgM and IgG responses are elicited; however, IgG levels gradually inflate in the persistent phase of infection while IgM levels are stably maintained. Whereas CD27-CD70 interactions are dispensable, the CD28/B7 costimulatory pathway is critical for the class switching of MCMV-specific IgM-to-IgG B cell responses, which corresponds to the CD28/B7-dependent formation of CD4
+
T follicular helper cells (T
FH
) and germinal center (GC) B cells. Furthermore, the initial viral inoculum dose dictates the height of the antibody levels during IgG antibody inflation and relates to the induction of long-lived plasma cells and memory B cells. Antibody avidity nonetheless is not altered after the establishment of viral persistence and occurs independently of the inoculum doses. However, repetitive challenge with intact viral particles, accompanied by increased GC reactivity, promotes the development of high-avidity IgG responses with neutralizing capacity. These insights can be used for the rational design of CMV-based vaccines aimed at inducing antibody responses.

IMPORTANCE
Antibodies provide long-term protection to different pathogens. However, how antibody responses develop during persistent virus infection is not entirely clear. Here, we characterize factors that influence the virus-specific antibody response to persistent CMV. This study describes that during persistent infection, CMV-specific IgM antibody levels are stably maintained while IgG2b and IgG2c levels gradually inflate over time. In contrast, the IgG avidity remains similar after the establishment of viral persistence. The induction of T follicular helper cells and GC B cells requires CD4
+
T cell help and CD28/B7 costimulation signals and is essential for the development of CMV-specific IgG antibody responses. Furthermore, neutralizing CMV-specific antibodies appear to develop late after infection, yet the neutralizing capacity can be improved upon repetitive viral challenge that is associated with increased GC reactivity. The results described here could inform the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.
Muscle injury is the principal injury type and hamstring muscle injury is the first injury diagnosis during top-level international athletics championships between 2007 and 2015
Authors: Edouard Pascal , Branco Pedro , Alonso Juan-Manuel 
Published: February 2016

BackgroundDuring top-level international athletics championships, muscle injuries are frequent.ObjectiveTo analyse the incidence and characteristics of muscle injuries and hamstring muscle injuries (hamstring injuries) occurring during top-level international athletics championships.MethodsDuring 16 international championships held between 2007 and 2015, national medical team and local organising committee physicians reported daily all injuries on a standardised injury report form. Only muscle injuries (muscle tears and muscle cramps) and hamstring injuries have been analysed.Results40.9% of all recorded injuries (n=720) were muscle injuries, with 57.5% of them resulting in time loss. The overall incidence of muscle injuries was higher in male athletes than female athletes (51.9±6.0 vs 30.3±5.0 injuries per 1000 registered athletes, respectively; RR=1.71; 95% CI 1.45 to 2.01). Muscle injuries mainly affected the thigh (52.9%) and lower leg (20.1%), and were mostly caused by overuse with sudden onset (38.2%) and non-contact trauma (24.6%). Muscle injury risk varied according to the event groups. Hamstring injuries represented 17.1% of all injuries, with a higher risk in male compared to female athletes (22.4±3.4 vs 11.5±2.6 injuries per 1000 registered athletes, respectively; RR=1.94; 95% CI 1.42 to 2.66).ConclusionsDuring international athletics championships, muscle injury is the principal type of injury, and among those, the hamstring is the most commonly affected, with a two times higher risk in male than female athletes. Athletes in explosive power events, male athletes and older male athletes, in specific were more at risk of muscle injuries and hamstring injuries. Injury prevention strategies should be sex-specific.