bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Catanionic Surfactant Vesicle Vaccines Protect against Francisella tularensis LVS and Confer Significant Partial Protection against F. tularensis Schu S4 Strain
Authors: Richard Katharina , Mann Barbara J. , Stocker Lenea , Barry Eileen M. , Qin Aiping , Cole Leah E. , Hurley Matthew T. , Ernst Robert K. , Michalek Suzanne M. , Stein Daniel C. , DeShong Philip , Vogel Stefanie N. 
Published: December 2013

ABSTRACTFrancisella tularensisis a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multipleF. tularensisstrains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of eitherF. tularensistype B live vaccine strain (F. tularensisLVS [LVS-V]) orF. tularensistype A Schu S4 strain (F. tularensisSchu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not expressF. tularensiscomponents, partially protected againstF. tularensisLVS, presumably through activation of the innate immune response, and yet it failed to protect against theF. tularensisSchu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.)F. tularensisLVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.)F. tularensisSchu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased afterF. tularensisLVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge withF. tularensisLVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effectiveF. tularensissubunit vaccine and may be applicable for use with other pathogens.
High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle
Authors: Plain Karren M. , Marsh Ian B. , Waldron Anna M. , Galea Francesca , Whittington Ann-Michele , Saunders Vanessa F. , Begg Douglas J. , de Silva Kumudika , Purdie Auriol C. , Whittington Richard J. 
Published: December 2013

ABSTRACT

Johne's disease (JD) is a chronic enteric disease caused by
Mycobacterium avium
subsp.
paratuberculosis
that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of
M. avium
subsp.
paratuberculosis
in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of
M. avium
subsp.
paratuberculosis
based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect
M. avium
subsp.
paratuberculosis
in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of
M. avium
subsp.
paratuberculosis
cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS
900
quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg
M. avium
subsp.
paratuberculosis
genomic DNA. Only
M. avium
subsp.
paratuberculosis
was detected from a panel of 51 mycobacterial isolates, including 10 with IS
900
-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from
M. avium
subsp.
paratuberculosis
-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611–622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.
Papillomavirus E6 PDZ Interactions Can Be Replaced by Repression of p53 To Promote Episomal Human Papillomavirus Genome Maintenance
Authors: Brimer Nicole , Vande Pol Scott B. 
Published: December 2013

ABSTRACT
Cancer-associated human papillomaviruses (HPVs) express E6 oncoproteins that target the degradation of p53 and have a carboxy-terminal PDZ ligand that is required for stable episomal maintenance of the HPV genome. We find that the E6 PDZ ligand can be deleted and the HPV genome stably maintained if cellular p53 is inactivated. This indicates that the E6-PDZ interaction promotes HPV genome maintenance at least in part by neutralization of an activity that can arise from residual undegraded p53.
Adenovirus E1B 19-Kilodalton Protein Modulates Innate Immunity through Apoptotic Mimicry
Authors: Radke Jay R. , Grigera Fernando , Ucker David S. , Cook James L. 
Published: December 2013

ABSTRACT

Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in
cis
restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation.

IMPORTANCE
We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not repress macrophage proinflammatory responses and enhanced some cytokine responses. Our results define a new function of the antiapoptotic, adenoviral protein E1B 19K, which we have termed “apoptotic mimicry.” Our studies suggest the possibility that the presence or absence of this E1B 19K function could alter the immunological outcome of both natural and therapeutic adenoviral infections. For example, emerging, highly immunopathogenic adenovirus serotypes might induce increased host inflammatory responses as a result of altered E1B 19K function or expression. It is also possible that engineered variations in E1B 19K expression/function could be created during adenovirus vector design that would increase the therapeutic efficacy of replicating adenovirus vectors for vaccines or oncolytic viral targeting of neoplastic cells.
Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase
Authors: Vörös Judit , Urbanek Annika , Rautureau Gilles Jean Philippe , O'Connor Maggie , Fisher Henry C. , Ashcroft Alison E. , Ferguson Neil 
Published: December 2013

ABSTRACT

Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies—broad-spectrum antivirals—are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in
Escherichia coli
, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of β-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity
in vitro
, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the
in vitro
priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV.

IMPORTANCE
The viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in
Escherichia coli
and also methods for purifying them in soluble forms that have activity
in vitro
. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.
Immunogenicity of Novel Mumps Vaccine Candidates Generated by Genetic Modification
Authors: Xu Pei , Chen Zhenhai , Phan Shannon , Pickar Adrian , He Biao 
Published: December 2013

ABSTRACTMumps is a highly contagious human disease, characterized by lateral or bilateral nonsuppurative swelling of the parotid glands and neurological complications that can result in aseptic meningitis or encephalitis. A mumps vaccination program implemented since the 1960s reduced mumps incidence by more than 99% and kept the mumps case numbers as low as hundreds of cases per year in the United States before 2006. However, a large mumps outbreak occurred in vaccinated populations in 2006 and again in 2009 in the United States, raising concerns about the efficacy of the vaccination program. Previously, we have shown that clinical isolate-based recombinant mumps viruses lacking expression of either the V protein (rMuVΔV) or the SH protein (rMuVΔSH) are attenuated in a neurovirulence test using newborn rat brains (P. Xu et al., Virology 417:126–136, 2011,http://dx.doi.org/10.1016/j.virol.2011.05.003; P. Xu et al., J. Virol. 86:1768–1776, 2012,http://dx.doi.org/10.1128/JVI.06019-11) and may be good candidates for vaccine development. In this study, we examined immunity induced by rMuVΔSH and rMuVΔV in mice. Furthermore, we generated recombinant mumps viruses lacking expression of both the V protein and the SH protein (rMuVΔSHΔV). Analysis of rMuVΔSHΔV indicated that it was stable in tissue culture cell lines. Importantly, rMuVΔSHΔV was immunogenic in mice, indicating that it is a promising candidate for mumps vaccine development.
Kaposi's Sarcoma-Associated Herpesvirus Induces the ATM and H2AX DNA Damage Response Early during
De Novo
Infection of Primary Endothelial Cells, Which Play Roles in Latency Establishment
Authors: Singh Vivek Vikram , Dutta Dipanjan , Ansari Mairaj Ahmed , Dutta Sujoy , Chandran Bala 
Published: December 2013

ABSTRACT

The DNA damage response (DDR) that evolved to repair host cell DNA damage also recognizes viral DNA entering the nucleus during infections. Here, we investigated the modulation of DDR signaling during
de novo
infection of primary endothelial cells by Kaposi's sarcoma-associated herpesvirus (KSHV). Phosphorylation of representative DDR-associated proteins, such as ataxia telangiectasia mutated (ATM) and H2AX, was induced as early as 30 min (0.5 h) postinfection and persisted during
in vitro
KSHV latency. Phosphorylated H2AX (γH2AX) colocalized at 30 min (0.5 h) with the KSHV genome entering the nuclei. Total H2AX protein levels also increased, and the increase was attributed to a decrease in degradative H2AX Lys48-linked polyubiquitination with a concomitant increase in Lys63-linked polyubiquitination that was shown to increase protein stability. ATM and H2AX phosphorylation and γH2AX nuclear foci were also induced by UV-inactivated KSHV, which ceased at later times of infection. Inhibition of ATM kinase activity by KU-55933 and H2AX knockdown by small interfering RNA significantly reduced the expression of the KSHV latency-associated nuclear antigen 1 (LANA-1; ORF73) and LANA-1 nuclear puncta. Knockdown of H2AX also resulted in a >80% reduction in the nuclear KSHV DNA copy numbers. Similar results were also observed in ATM-negative cells, although comparable levels of viral DNA entered ATM-negative and ATM-positive cell nuclei. In contrast, knockdown of CHK1 and CHK2 did not affect ORF73 expression. Collectively, these results demonstrate that KSHV induces ATM and H2AX, a selective arm of the DDR, for the establishment and maintenance of its latency during
de novo
infection of primary endothelial cells.

IMPORTANCE
Eukaryotic cells mount a DNA damage response (DDR) to sense and repair different types of cellular DNA damage. In addition, DDR also recognizes exogenous genetic material, such as the viral DNA genome entering the nucleus during infections. The present study was undertaken to determine whether
de novo
Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates DDR. Our results demonstrate that early during
de novo
infection of primary endothelial cells, KSHV induces a selective arm of DDR signaling, such as the ATM kinase and its downstream target, H2AX, which are essential for KSHV's latent gene expression and the establishment of latency. These studies suggest that targeting ATM and H2AX could serve as an attractive strategy to block the establishment of KSHV latent infection and the associated malignancies.
Divergent Antibody Subclass and Specificity Profiles but Not Protective HLA-B Alleles Are Associated with Variable Antibody Effector Function among HIV-1 Controllers
Authors: Lai Jennifer I. , Licht Anna F. , Dugast Anne-Sophie , Suscovich Todd , Choi Ickwon , Bailey-Kellogg Chris , Alter Galit , Ackerman Margaret E. 
Published: December 2013

ABSTRACTUnderstanding the coordination between humoral and cellular immune responses may be the key to developing protective vaccines, and because genetic studies of long-term HIV-1 nonprogressors have associated specific HLA-B alleles with spontaneous control of viral replication, this subject group presents an opportunity to investigate relationships between arms of the adaptive immune system. Given evidence suggesting that cellular immunity may play a role in viral suppression, we sought to determine whether and how the humoral immune response might vary among controllers. Significantly, Fc-mediated antibody effector functions have likewise been associated with durable viral control. In this study, we compared the effector function and biophysical features of HIV-specific antibodies in a cohort of controllers with and without protective HLA-B alleles in order to investigate whether there was evidence for multiple paths to HIV-1 control, or whether cellular and humoral arms of immunity might exhibit coordinated profiles. However, with the exception of IgG2 antibodies to gp41, HLA status was not associated with divergent humoral responses. This finding did not result from uniform antibody responses across subjects, as controllers could be regrouped according to strong differences in their HIV-specific antibody subclass specificity profiles. These divergent antibody profiles were further associated with significant differences in nonneutralizing antibody effector function, with levels of HIV-specific IgG1 acting as the major distinguishing factor. Thus, while HLA background among controllers was associated with minimal differences in humoral function, antibody subclass and specificity profiles were associated with divergent effector function, suggesting that these features could be used to make functional predictions. Because these nonneutralizing antibody activities have been associated with spontaneous viral control, reduced viral load, and nonprogression in infected subjects and protection in vaccinated subjects, understanding the specific features of IgGs with potentiated effector function may be critical to vaccine and therapeutic antibody development.IMPORTANCEIn this study, we investigated whether the humoral and cellular arms of adaptive immunity exhibit coordinated or compensatory activity by studying the antibody response among HIV-1 controllers with different genetic backgrounds.
Diverse Recombinant HIV-1 Envs Fail To Activate B Cells Expressing the Germline B Cell Receptors of the Broadly Neutralizing Anti-HIV-1 Antibodies PG9 and 447-52D
Authors: McGuire Andrew T. , Glenn Jolene A. , Lippy Adriana , Stamatatos Leonidas 
Published: December 2013

ABSTRACTBroadly neutralizing antibodies (bNAbs) against HIV-1 are generated during HIV-1-infection but have not yet been elicited by immunization with recombinant forms of the viral envelope glycoprotein (Env; the target of anti-HIV-1 neutralizing antibodies). A particular type of bNAb targets the CD4-binding site (CD4-BS) region of Env. These antibodies are derived from a limited number of VH/VL genes and can bind to and neutralize diverse HIV-1 strains. Recent reports have demonstrated the limited potential of Env to activate B cells expressing the germline B cell receptor (BCR) forms of anti-CD4-BS bNAbs. A potential reason for the lack of elicitation of anti-CD4-BS bNAbs by Env immunogens is the absence of stimulation of naive B cells expressing the germline BCRs of such antibodies. Several bNAbs have been isolated from HIV-1-infected subjects that target other structurally conserved regions of Env. How frequently Env immunogens stimulate the germline BCRs that give rise to bNAbs that target Env regions other than the CD4-BS is not well understood. Here, we investigated the interactions between diverse Envs and the BCRs of known bNAbs targeting not only the CD4-BS but also conserved elements of the second and third variable Env regions. Our results indicate that Env is generally ineffective in engaging germline BCRs of bNAbs irrespective of their epitope target. Potentially, this is the result of viral evolutionary mechanisms adopted to escape broadly neutralizing antibody responses. Our results also suggest that a single Env capable of activating germline BCRs that target distinct Env epitopes will be very difficult to identify or to design.IMPORTANCEBroadly neutralizing antibodies against HIV-1 are thought to be an important component of the immune responses that a successful vaccine should elicit. Broadly neutralizing antibodies are generated by a subset of those infected by HIV-1, but so far, they have not been generated by immunization with recombinant Envelope (Env, the target of anti-HIV-1 neutralizing antibodies). Here, we provide evidence that the inability of Env to elicit the production of broadly neutralizing antibodies is due to the inability of diverse Envs to engage the germline B cell receptor forms of known broadly neutralizing antibodies.
Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1
Authors: Schmeiser Stefanie , Mast Jan , Thiel Heinz-Jürgen , König Matthias 
Published: December 2013

ABSTRACT

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the
Flavivirus
and
Hepacivirus
genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.
Involvement of Viral MicroRNA in the Regulation of Antiviral Apoptosis in Shrimp
Authors: Huang Tianzhi , Cui Yalei , Zhang Xiaobo 
Published: December 2013

ABSTRACT

Viruses, in particular DNA viruses, generate microRNAs (miRNAs) to control the expression of host and viral genes. Due to their essential roles in virus-host interactions, viral miRNAs have attracted extensive investigations in recent years. To date, however, most studies on viral miRNAs have been conducted in cell lines. In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp
in vivo
. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis and Northern blotting, the expression of viral miRNAs was tissue specific
in vivo
. The results indicated that the viral miRNA WSSV-miR-N24 could target the shrimp caspase 8 gene, and this miRNA further repressed the apoptosis of shrimp hemocytes
in vivo
. As a result, the number of WSSV copies in shrimp
in vivo
was significantly increased compared with the control level (WSSV only). Therefore, our study presents the first report on the
in vivo
molecular events of viral miRNA in antiviral apoptosis.