| serology and cytokine profiles in patients infected with the newly discovered bundibugyo ebolavirus. | a new species of ebolavirus, bundibugyo ebolavirus, was discovered in an outbreak in western uganda in november 2007. to study the correlation between fatal infection and immune response in bundibugyo ebolavirus infection, viral antigen, antibodies, and 17 soluble factors important for innate immunity were examined in 44 patient samples. using luminex assays, we found that fatal infection was associated with high levels of viral antigen, low levels of pro-inflammatory cytokines, such as il-1α, i ... | 2011 | 22197674 |
| a small nonhuman primate model for filovirus-induced disease. | ebolavirus and marburgvirus are members of the filovirus family and induce a fatal hemorrhagic disease in humans and nonhuman primates with 90% case fatality. to develop a small nonhuman primate model for filovirus disease, common marmosets (callithrix jacchus) were intramuscularly inoculated with wild type marburgvirus musoke or ebolavirus zaire. the infection resulted in a systemic fatal disease with clinical and morphological features closely resembling human infection. animals experienced we ... | 2011 | 21959017 |
| real-time monitoring of cardiovascular function in rhesus macaques infected with zaire ebolavirus. | nine rhesus macaques were implanted with multisensor telemetry devices and internal jugular vein catheters before being infected with zaire ebolavirus. all animals developed viremia, fever, a hemorrhagic rash, and typical changes of ebola hemorrhagic fever in clinical laboratory tests. three macaques unexpectedly survived this usually lethal disease, making it possible to compare physiological parameters in lethally challenged animals and survivors. after the onset of fever, lethal illness was c ... | 2011 | 21987736 |
| impact of systemic or mucosal immunity to adenovirus on ad-based ebola virus vaccine efficacy in guinea pigs. | approximately 35% of the north american population and an estimated 90% of the sub-saharan african population have antibodies against adenovirus serotype 5 (adhu5) that are capable of neutralizing adhu5-based vaccines. in mice, intranasal delivery of adhu5 expressing the zaire ebolavirus glycoprotein human adenovirus serotype 5 (ad) containing the genes for the zaire ebolavirus glycoprotein (zgp) under the expressional control of a cytomegalovirus immediate early promoter (cmv)) can bypass syste ... | 2011 | 21987739 |
| Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis. | Virus entry into host cells is the first step of infection and a crucial determinant of pathogenicity. Here we show that Ebola virus-like particles (EBOV-VLPs) composed of the glycoprotein GP(1,2) and the matrix protein VP40 use macropinocytosis and clathrin-mediated endocytosis to enter cells. EBOV-VLPs applied to host cells induced actin-driven ruffling and enhanced FITC-dextran uptake, which indicated macropinocytosis as the main entry mechanism. This was further supported by inhibition of en ... | 2011 | 21987776 |
| unconventional secretion of ebola virus matrix protein vp40. | the ebola virus matrix protein vp40 plays an essential role in virus assembly and budding. in this study we reveal that transient vp40 expression results in the release into the culture medium of substantial amounts of soluble monomeric vp40 in addition to the release of virus-like particles containing an oligomeric form of this protein as previously described. we show that vp40 secretion is endoplasmic reticulum/golgi-independent and is not associated with cell death. soluble vp40 was observed ... | 2011 | 21987759 |
| Conserved proline-rich region of Ebola virus matrix protein VP40 is essential for plasma membrane targeting and virus-like particle release. | The matrix protein VP40 is essential for Ebola virus (EBOV) and Marburg virus assembly and budding at the plasma membrane. In this study we have investigated the effect of single amino acid substitutions in a conserved proline-rich region of the EBOV VP40 located in the carboxy-terminal part of the protein. We demonstrate that substitutions within this region result in an alteration of intracellular VP40 localization and also cause a reduction or a complete block of virus-like particle budding, ... | 2011 | 21987765 |
| single immunization with a monovalent vesicular stomatitis virus-based vaccine protects nonhuman primates against heterologous challenge with bundibugyo ebolavirus. | the recombinant vesicular stomatitis virus (rvsv) vector-based monovalent vaccine platform expressing a filovirus glycoprotein has been demonstrated to provide protection from lethal challenge with ebola (ebov) and marburg (marv) viruses both prophylactically and after exposure. this platform provides protection between heterologous strains within a species; however, protection from lethal challenge between species has been largely unsuccessful. to determine whether the rvsv-ebov vaccines have t ... | 2011 | 21987745 |
| filovirus outbreak detection and surveillance: lessons from bundibugyo. | the first outbreak of ebola hemorrhagic fever (ehf) due to bundibugyo ebolavirus occurred in uganda from august to december 2007. during outbreak response and assessment, we identified 131 ehf cases (44 suspect, 31 probable, and 56 confirmed). consistent with previous large filovirus outbreaks, a long temporal lag (approximately 3 months) occurred between initial ehf cases and the subsequent identification of ebola virus and outbreak response, which allowed for prolonged person-to-person transmi ... | 2011 | 21987748 |
| differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions. | clathrin-mediated endocytosis was previously implicated as one of the cellular pathways involved in filoviral glycoprotein mediated viral entry into target cells. here we have further dissected the requirements for different components of this pathway in ebola versus marburg virus glycoprotein (gp) mediated viral infection. although a number of these components were involved in both cases; ebola gp-dependent viral entry specifically required the cargo recognition proteins eps15 and dab2 as well ... | 2011 | 21855102 |
| the ebola virus vp24 protein prevents hnrnp c1/c2 binding to karyopherin α1 and partially alters its nuclear import. | the ebola virus (ebov) protein vp24 inhibits type i and ii interferon (ifn) signaling by binding to npi-1 subfamily karyopherin α (kpna) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated stat1 (phospho-stat1). this inhibits phospho-stat1 nuclear import. a biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex c1/c2 (hnrnp c1/c2) nuclear import as an additional target of vp24. co-immunoprecipitation studies demonstrate that hnrnp c1/c2 ... | 2011 | 21987768 |
| evolutionary maintenance of filovirus-like genes in bat genomes. | little is known of the biological significance and evolutionary maintenance of integrated non-retroviral rna virus genes in eukaryotic host genomes. here, we isolated novel filovirus-like genes from bat genomes and tested for evolutionary maintenance. we also estimated the age of filovirus vp35-like gene integrations and tested the phylogenetic hypotheses that there is a eutherian mammal clade and a marsupial/ebolavirus/marburgvirus dichotomy for filoviruses. | 2011 | 22093762 |
| characterization of filovirus protein-protein interactions in mammalian cells using bimolecular complementation. | the virion protein 40 (vp40) and nucleoprotein (np) of ebola (ebov) and marburg viruses (marv) play key roles during virion assembly and egress. the ability to detect interactions between vp40-vp40, vp40-np, and np-np and follow these complexes as they traffic through mammalian cells would enhance our understanding of the molecular events leading to filovirus assembly and budding, and provide new insights into filovirus replication and pathogenesis. here, we successfully employed a bimolecular c ... | 2011 | 21987757 |
| management of accidental exposure to ebola virus in the biosafety level 4 laboratory, hamburg, germany. | a needlestick injury occurred during an animal experiment in the biosafety level 4 laboratory in hamburg, germany, in march 2009. the syringe contained zaire ebolavirus (zebov) mixed with freund's adjuvant. neither an approved treatment nor a postexposure prophylaxis (pep) exists for ebola hemorrhagic fever. following a risk-benefit assessment, it was recommended the exposed person take an experimental vaccine that had shown pep efficacy in zebov-infected nonhuman primates (nhps) [12]. the vacci ... | 2011 | 21987751 |
| assessment of rodents as animal models for reston ebolavirus. | the emergence of reston ebolavirus (rebov) in domestic swine in the philippines has caused a renewed interest in rebov pathogenicity. here, the use of different rodent species as animal disease models for rebov was investigated. balb/c and stat1(-)(/-) mice, hartley guinea pigs, and syrian hamsters were inoculated intraperitoneally with rebov strain pennsylvania or reston08-a. although virus replication occurred in guinea pigs, hamsters, and stat1(-/-) mice, progression to disease was only obser ... | 2011 | 21987777 |
| characterization of zaire ebolavirus glycoprotein-specific monoclonal antibodies. | zaire ebolavirus (zebov) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. there are no effective therapeutic or prophylactic treatments available. the sole transmembrane glycoprotein (gp) is the key target for developing neutralizing antibodies. in this study, recombinant vsvδg/zebovgp was used to generate monoclonal antibodies (mabs) against the zebov gp. a total of 8 mabs were produced using traditional hybridoma cell fusion t ... | 2011 | 21925951 |
| therapeutics of ebola hemorrhagic fever: whole-genome transcriptional analysis of successful disease mitigation. | the mechanisms of ebola (ebov) pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in circulating cytokine levels, and development of coagulation abnormalities) is thought to play a major role. accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from ebov-infected cell ... | 2011 | 21987740 |
| vp24 is a molecular determinant of ebola virus virulence in guinea pigs. | in sharp contrast to human and nonhuman primates, guinea pigs and some other mammals resist ebola virus (ebov) replication and do not develop illness upon virus inoculation. however, serial passaging of ebov in guinea pigs results in a selection of variants with high pathogenicity. in this report, using a reverse genetics approach, we demonstrate that this dramatic increase in ebov pathogenicity is associated with amino acid substitutions in the structural protein vp24. we show that although rep ... | 2011 | 21987737 |
| filovirus infection of stat-1 knockout mice. | we evaluated the susceptibility to ebola and marburg virus infection of mice that cannot respond to interferon (ifn)-α/β and ifn-γ because of deletion of the stat-1 gene. a mouse-adapted zaire ebolavirus (zebov) caused rapidly lethal disease; wild-type zebov and sudan ebolavirus and 4 different marburg virus strains produced severe, but more slowly progressive illness; and reston ebolavirus caused mild disease that was late in onset. the virulence of each agent was mirrored by the pace and sever ... | 2011 | 21987780 |
| vesicular stomatitis virus-based ebola vaccines with improved cross-protective efficacy. | for ebola virus (ebov), 4 different species are known: zaire, sudan, côte d'ivoire, and reston ebolavirus. the newly discovered bundibugyo ebolavirus has been proposed as a 5th species. so far, no cross-neutralization among ebov species has been described, aggravating progress toward cross-species protective vaccines. with the use of recombinant vesicular stomatitis virus (rvsv)-based vaccines, guinea pigs could be protected against zaire ebolavirus (zebov) infection only when immunized with a v ... | 2011 | 21987743 |
| ebola virus entry requires the cholesterol transporter niemann-pick c1. | infections by the ebola and marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. filovirus entry is mediated by the viral spike glycoprotein (gp), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these c ... | 2011 | 21866103 |
| A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus. | Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, whi ... | 2011 | 21858240 |
| Role of VP30 phosphorylation in the Ebola virus replication cycle. | Ebola virus (EBOV) transcription is dependent on the phosphoprotein VP30, a component of the viral nucleocapsid. VP30 is phosphorylated at 2 serine residue clusters located at the N-terminal part of the protein. In this report, we have investigated the role of VP30 phosphorylation in EBOV replication using a reverse genetics approach. In effect, recombinant EBOVs with the VP30 serine clusters substituted either by nonphosphorylatable alanines or phosphorylation-mimicking aspartates were generate ... | 2011 | 21987772 |
| Genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles. | The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same ge ... | 2011 | 21900424 |
| drbp76 associates with ebola virus vp35 and suppresses viral polymerase function. | the zaire ebola virus (ebov) protein vp35 is multifunctional; it inhibits ifn-α/β production and functions as a cofactor of the viral rna polymerase. mass spectrometry identified the double stranded rna binding protein 76 (drbp76/nfar-1/nf90) as a cellular factor that associates with the vp35 c-terminal interferon inhibitory domain (iid). drbp76 is described to regulate host cell protein synthesis and play an important role in host defense. the vp35-iid-drbp76 interaction required the addition o ... | 2011 | 21987769 |
| Discovery of an ebolavirus-like filovirus in europe. | Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain. | 2011 | 22039362 |
| Knockdown of Ebola virus VP24 impairs viral nucleocapsid assembly and prevents virus replication. | The structural protein VP24 of Ebola virus (EBOV) is a determinant of virulence in rodent models and possesses an interferon antagonist function. In this study, we investigate the role of VP24 in EBOV replication using RNA interference by small interfering RNA to knock down the expression of this protein in virus-infected cells. We reveal that VP24 is required for assembly of viral nucleocapsids and that silencing of VP24 expression prevents the release of EBOV. | 2011 | 21987766 |
| Crystal structure of swine major histocompatibility complex class I SLA-1 0401 and identification of 2009 pandemic swine-origin influenza A H1N1 virus cytotoxic T lymphocyte epitope peptides. | The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1 0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIV(NW9); NSDTVGWSW) or Ebola virus (Ebola(AY9); ATAAATEAY) were determined in this study. The overall peptide-SLA-1 0401 structures res ... | 2011 | 21900158 |
| Lethality and pathogenesis of airborne infection with filoviruses in A129 a/ß -/- interferon receptor-deficient mice. | Normal immunocompetent mice are not susceptible to non-adapted filoviruses. There are therefore two strategies available to establish a murine model of filovirus infection: adaptation of the virus to the host or the use of genetically modified mice that are susceptible to the virus. A number of knockout (KO) strains of mice with defects in either their adaptive or innate immunity are susceptible to non-adapted filoviruses. In this study, A129 a/ß -/- interferon receptor-deficient KO mice, strain ... | 2012 | 21852521 |
| protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the syrian hamster model of lethal ebola virus infection. | outbreaks of filoviral hemorrhagic fever occur sporadically and unpredictably across wide regions in central africa and overlap with the occurrence of other infectious diseases of public health importance. | 2011 | 21987746 |
| the ebolavirus vp24 protein blocks phosphorylation of p38 mitogen-activated protein kinase. | type i interferon (ifn) signaling is mediated through several signaling pathways, including the janus kinase and signal transducer and activator (jak-stat) and p38 mitogen-activated protein (map) kinase pathways. the vp24 protein of ebolavirus is an ifn antagonist, blocking type i ifn signaling through the jak-stat pathway. here, we show that, in 293 cells, vp24 also interferes with the p38 map kinase pathway by blocking ifn-β-stimulated phosphorylation of p38-α. similar inhibition was not obser ... | 2011 | 21987775 |
| ebola reston virus infection of pigs: clinical significance and transmission potential. | in 2008, reston ebolavirus (rebov) was isolated from pigs during a disease investigation in the philippines. porcine reproductive and respiratory syndrome virus (prrsv) and porcine circovirus type 2 (pcv-2) infections were also confirmed in affected herds and the contribution of rebov to the disease outbreak remains uncertain. we have conducted experimental challenge studies in 5-week-old pigs, with exposure of animals to 10(6) tcid(50) of a 2008 swine isolate of rebov via either the oronasal or ... | 2011 | 21987755 |
| Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion. | Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2)) on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2) is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulp ... | 2011 | 22102923 |
| Host response dynamics following lethal infection of rhesus macaques with Zaire ebolavirus. | To gain further insight into the interdependent pathogenic processes in Ebola hemorrhagic fever (EHF), we have examined the dynamics of host responses in individual rhesus macaques infected with Zaire ebolavirus over the entire disease course. Examination of coagulation parameters revealed that decreased coagulation inhibitor activity triggered severe coagulopathy as indicated by prolonged coagulation times and decreased fibrinogen levels. This has been proposed as one of the significant mechani ... | 2011 | 21987781 |
| Ebola virion attachment and entry into human macrophages profoundly effects early cellular gene expression. | Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed t ... | 2011 | 22028943 |
| Ebola virus failure to stimulate plasmacytoid dendritic cell interferon responses correlates with impaired cellular entry. | We examined the ability of the Ebola virus to elicit an antiviral response from plasmacytoid dendritic cells (pDCs). Exposure of pDCs to Ebola virus did not result in significantly higher levels of interferon-a production than the levels in mock-infected cells. After inoculation with Ebola virus under the same conditions, conventional dendritic cells expressed viral proteins whereas pDCs did not, suggesting that the latter cells were not infected. Assessment of the entry of Ebola virus-like part ... | 2011 | 21987778 |
| impact of ebola mucin-like domain on antiglycoprotein antibody responses induced by ebola virus-like particles. | ebola virus (ebov) glycoprotein (gp), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield gp from neutralizing antibodies. to test whether the mucin-like domain inhibits the production and function of anti-gp antibodies, we vaccinated mice with ebola virus-like particles (vlps) that express vesicular stomatitis virus g, wild-type ebov gp (ebgp), ebov gp without its mucin-like domain (δmucgp), or ebov gp with ... | 2011 | 21987758 |
| Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection. | Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identificat ... | 2011 | 21866101 |
| The importance of the NP: VP35 ratio in Ebola virus nucleocapsid formation. | Ebola virus VP35 is a cofactor of the viral RNA polymerase complex and, together with NP and VP24, is an essential component for nucleocapsid formation. In the present study, we examined the interactions between VP35 and NP and found that VP35 interacts with helical NP-RNA complexes through the C-terminus of NP. We also found that coexpression of excess VP35 with NP reduced the yields of NP-RNA complexes purified by CsCl gradient ultracentrifugation and inhibited the formation of the NP-induced ... | 2011 | 21987764 |
| the ebola virus glycoprotein and hiv-1 vpu employ different strategies to counteract the antiviral factor tetherin. | the antiviral protein tetherin/bst2/cd317/hm1.24 restricts cellular egress of human immunodeficiency virus (hiv) and of particles mimicking the ebola virus (ebov), a hemorrhagic fever virus. the hiv-1 viral protein u (vpu) and the ebov-glycoprotein (ebov-gp) both inhibit tetherin. here, we compared tetherin counteraction by ebov-gp and vpu. we found that ebov-gp but not vpu counteracted tetherin from different primate species, indicating that ebov-gp and vpu target tetherin differentially. tethe ... | 2011 | 21987761 |
| sgp serves as a structural protein in ebola virus infection. | sgp, which is perceived as nonstructural, secretory glycoprotein, shares 295 amino acids at its n-terminal with gp(1,2), which include the specific residue necessary to interact with gp(2). in the present study, we tested whether the sgp protein of zaire ebolavirus (zebov) could substitute for gp(1) and form a complex with gp(2), thus serving as a structural protein. | 2011 | 21987767 |
| The Ebola virus soluble glycoprotein (sGP) does not affect lymphocyte apoptosis and adhesion to activated endothelium. | Ebola virus infection is associated with the release of a soluble glycoprotein (sGP) from infected cells. The sGP has been proposed to modulate Ebola virus pathogenesis in primates but little is known about the role of this protein during infection and disease manifestation. So far sGP has been shown to revert the effect of tumor necrosis factor a (TNF-a) on endothelial permeability, indicating that the function of sGP might be antiinflammatory. Since bystander apoptosis of lymphocytes has been ... | 2011 | 21987774 |
| Comparative analysis of Ebola virus glycoprotein interactions with human and bat cells. | Infection with Ebola virus (EBOV) causes hemorrhagic fever in humans with high case-fatality rates. The EBOV-glycoprotein (EBOV-GP) facilitates viral entry and promotes viral release from human cells. African fruit bats are believed not to develop disease upon EBOV infection and have been proposed as a natural reservoir of EBOV. We compared EBOV-GP interactions with human cells and cells from African fruit bats. We found that susceptibility to EBOV-GP-dependent infection was not limited to bat c ... | 2011 | 21987760 |
| risk factors for zaire ebolavirus--specific igg in rural gabonese populations. | in gabon, several ebolavirus outbreaks have occurred exclusively in the northeastern region. we conducted a large serosurvey to identify areas and populations at risk and potential demographic, clinical, and behavioral risk factors. | 2011 | 21987749 |
| induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the ebola virus glycoprotein lacking the mucin-like domain. | abstract: background: the genus ebolavirus includes five distinct viruses. four of these viruses cause hemorrhagic fever in humans. currently there are no licensed vaccines for any of them; however, several vaccines are under development. ebola virus envelope glycoprotein (gp1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (mld). we hypothesized that immunization with mld-deleted gp1,2 (gpdeltamld) would induce cross-species immunity by m ... | 2012 | 22273269 |
| filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences. | filoviruses are enveloped viruses that cause sporadic outbreaks of severe hemorrhagic fever (6, 8, 9, 16). previous studies revealed that endosomal cysteine proteases are host factors for ebolavirus zaire (7, 35). in this report, we show that infection mediated by glycoproteins from other phylogenetically-diverse filoviruses are also dependent on these proteases and provide additional evidence indicating they cleave gp1 and expose the binding domain for the critical host factor niemann-pick c1. ... | 2012 | 22238307 |