Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever

Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.     

Impact of habitat structure and fruit abundance on avian seed dispersal and fruit predation

So far, it is poorly understood how differential responses of avian seed dispersers and fruit predators to changes in habitat structure and fruit abundance along land-use gradients may translate into consequences for the seed dispersal of associated plants. We selected a gradient of habitat modification (forest, semi-natural, and rural habitat) characterized by decreasing tree cover and a high variation in local fruit availability. Along this gradient we quantified fruit removal by avian seed dispersers and fruit predators from 18 Sorbus aucuparia trees. We analyzed the relative importance of tree cover and fruit abundance in explaining species richness, abundance and fruit removal rates of both guilds from S. aucuparia trees. Species richness and abundance of seed dispersers decreased with decreasing tree cover, whereas fruit removal by seed dispersers decreased with decreasing fruit abundance independent of tree cover. Both variables had no effect on species richness, abundance and fruit removal by fruit predators. Consequently, seed dispersers dominated relative fruit removal in fruit-rich sites but the dispersal/predation ratio shifted in favor of predation in fruit-poor habitat patches. Our study demonstrates that variation in local habitat structure and fruit abundance can cause guild-specific responses. Such responses may result in a shift in fruit removal regimes and might affect the dispersal ability of dependent fruiting plants. Future studies should aim at possible consequences for plant recruitment and guild-specific responses of frugivores to disturbance gradients on the level of entire plant–frugivore associations.     

Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles

Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCV_TCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCV_TCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCV_TCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 10⁶ tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCV_TCP may prove valuable for gene delivery or vaccination approaches.Hepatitis C virus (HCV) is an enveloped plus-strand RNA virus of the genus Hepacivirus within the family Flaviviridae (34). The HCV genome is approximately 9.6 kb in length and consists of a single open reading frame encoding a polyprotein of ca. 3,000 amino acids and nontranslated regions (NTRs) located at the 5′ and 3′ termini. These NTRs are highly structured RNA segments encompassing critical cis-active RNA elements essential for genome replication and RNA translation (31). Viral proteins are expressed in a cap-independent manner by means of an internal ribosome entry site (IRES) located in the 5′ NTR. Co- and posttranslational cleavages liberate 10 viral proteins: core; envelope protein 1 (E1) and E2, representing the structural proteins that constitute the virion; a small membrane-associated ion channel protein designated p7 that is essential for virus assembly (16, 22, 43, 57); and six nonstructural (NS) proteins (NSs 2, 3, 4A, 4B, 5A, and 5B). HCV proteins NS3 to NS5B are both necessary and sufficient to establish membrane-bound replication complexes catalyzing RNA replication (13, 36). More recent data indicate that the NS2 protease that catalyzes cleavage at the NS2-NS3 site in addition participates in assembly and release of infectious viruses (22). Finally, ribosomal frame-shifting and internal translation initiation yield a group of additional proteins designated ARFP (alternative reading frame protein) or core+1 proteins. However, their function for the HCV replication cycle is currently not known.One hallmark of HCV is its high propensity to establish a persistent infection, which frequently causes progressive morbidity ranging from hepatic fibrosis to cirrhosis and hepatocellular carcinoma (20). Despite considerable progress in the treatment of HCV infection, the currently available therapy (a combination of pegylated interferon alpha with ribavirin) is not well tolerated and is efficacious in only ca. 50% of patients infected with the most prevalent genotype 1 (38). Therapeutic or prophylactic vaccines are not available. For these reasons and with currently ca. 170 million persistently infected individuals, HCV infection represents a considerable global health problem necessitating pertinent basic and applied research efforts.In recent years three major advances enabled analysis of the HCV replication cycle in tissue culture. First, Lohmann and colleagues developed subgenomic HCV replicons (36). These autonomously replicating RNA molecules carry all the genetic elements necessary for self-replication (the NTRs and NS3 to NS5B), including a selectable marker or a reporter gene in place of the viral structural proteins, and an internal IRES for expression of the HCV replicase genes (reviewed in reference 45). Second, HCV pseudotype particles, i.e., retroviral particles surrounded by an envelope carrying HCV E1-E2 complexes in place of their cognate envelope proteins, were established (3, 21). As these particles carry functional HCV glycoprotein complexes on their surface, HCV pseudotype particles have been instrumental for the analysis of E1-E2 receptor interactions and the early events of HCV infection (reviewed in reference 2). Finally, in 2005 fully permissive cell culture systems which are based on the JFH1 clone were described (33, 66, 72). This isolate replicates with unprecedented efficiency in transfected Huh7 human hepatoma cells and produces particles infectious both in vitro and in vivo, thus providing a model system reproducing the complete HCV replication cycle.Use of these novel models has considerably expanded our knowledge of viral and host cell factors involved in HCV replication (for a recent review, see reference 59). It is now known that similar to virtually all other plus-strand RNA viruses, HCV induces intracellular membrane alterations and replicates its genome in conjunction with vesicular membrane structures, the so-called “membranous web” (10, 13). Presumably as a consequence of this specific, rather secluded architecture of the membrane-associated replication machinery, all viral proteins involved in RNA replication, with the exception of NS5A function in cis, cannot be complemented in trans (1). Restricted trans-complementation of viral replicase proteins has been observed for other plus-strand RNA viruses as well, thus indicating that a rather “closed” replication machinery is a shared feature of these viruses (15, 27, 60). In contrast, for a number of plus-strand RNA viruses from diverse virus families like Picornaviridae (poliovirus), Alphaviridae (Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus), Coronaviridae (human coronavirus E229), and Flaviviridae (tick-borne encephalitis virus, Kunjin virus, West Nile virus, and yellow fever virus), assembly of progeny viruses can be achieved when structural proteins are expressed in trans and independent from the RNA molecule that encodes the replicase proteins. Similarly, Miyanari recently reported that HCV genomes with lethal mutations in core protein can be rescued by ectopic expression of functional core protein (39). This flexibility has been extensively used to create viral vectors for gene delivery as well as viral vector-based immunization approaches (32, 48, 49, 61, 68) (for a recent review on alphaviral vectors, the most frequently used among plus strand RNA vectors, see reference 37). In these systems the viral genome region encoding the structural proteins is replaced by a transgene. The resulting defective vector genomes are capable of RNA replication but due to the lack of structural proteins are unable to produce progeny virus particles. This defect is rescued by expression of the structural proteins in trans via helper viruses (28, 55) or, in some cases, by DNA constructs stably expressed in packaging cell lines (17). The resulting virus-like particles are infectious but support only single-round infection and are unable to spread, thus improving the safety of the viral transduction system.Given the success of plus-strand RNA vector technology for basic and applied clinical research, in this study we developed a trans-complementation system for HCV that provided new insights into the basic principles of HCV particle assembly.     

Ebola virus matrix protein VP40 uses the COPII transport system for its intracellular transport

The Ebola virus matrix protein VP40 plays an important role in virion formation and viral egress from cells. However, the host cell proteins and mechanisms responsible for intracellular transport of VP40 prior to its contribution to virion formation remain to be elucidated. Therefore we used coimmunoprecipitation and mass spectrometric analyses to identify host proteins interacting with VP40. We found that Sec24C, a component of the host COPII vesicular transport system, interacts specifically with VP40 via VP40 amino acids 303 to 307. Coimmunoprecipitation and dominant-negative mutant studies indicated that the COPII transport system plays a critical role in VP40 intracellular transport to the plasma membrane. Marburg virus VP40 was also shown to use the COPII transport system for intracellular transport. These findings identify a conserved intersection between a host pathway and filovirus replication, an intersection that can be targeted in the development of new antiviral drugs.     

Proliferative diabetic retinopathy is associated with a low level of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor. a pilot study.

Retinopathy is the most common microvascular diabetes complication and represents a major threat to the eyesight. The aim of this study was to address the role of pro- and anti-angiogenic molecules in diabetic retinopathy in the aqueous humor of the eye. Aqueous humor was collected at cataract surgery from 19 diabetic patients and from 13 age- and sex-matched normoglycemic controls. Levels of pro-angiogenic vascular endothelial growth factor (VEGF) and angiogenic inhibitor pigment epithelium-derived factor (PEDF) were determined. Angiogenic activity of the aqueous humor was quantified by measuring its effect on the migration of capillary endothelial cells. In the aqueous fluid, VEGF levels were increased in diabetics (mean values: 501 vs. 367 pg/ml; p = 0.05), compared to controls. PEDF was found to be decreased in diabetics (mean values: 2080 vs. 5780 ng/ml; p = 0.04) compared to controls. In seven diabetic patients with proliferative retinopathy, the most profound finding was a significant decrease of the PEDF level (mean value: 237 ng/ml), whereas VEGF levels were comparable to diabetic patients without proliferation (mean value: 3153; p = 0.003). Angiogenic activity in samples of patients from the control group was generally inhibitory due to PEDF, and inhibition was blocked by neutralizing antibodies to PEDF. Likewise, in diabetics without proliferation, angiogenic activity was also blocked by antibodies to PEDF. We will demonstrate here that the level of the natural ocular anti-angiogenic agent PEDF is inversely associated with proliferative retinopathy. PEDF is an important negative regulator of angiogenic activity of aqueous humor. Our data may have implications for the development of novel regimens for diabetic retinopathy.     

Nachweis und Chemische Trennung des Korrelationshemmstoffes und Seiner Hemmstoffvorstufe

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