Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice

Hantaviruses are the causative agents of HFRS and HCPS (hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome), two severe, and often fatal human diseases. Mortality from HFRS varies between hantaviruses; Hantaan and Dobrava show the highest, Seoul intermediate, and Puumala low mortality. Saaremaa, genetically closely related to Dobrava, is also known to induce HFRS, with low or no mortality. In this study, mice were inoculated with Dobrava and Saaremaa viruses to test for infectibility, lethality, viremia, nitric oxide production and antibody responses. Out of suckling mice intracerebrally inoculated with 50, 500 and 5,000 focus-forming units of Dobrava virus, respectively, 1/8, 2/8 and 7/8 died within 18-26 days. In all but one of the lethally infected mice high levels of replicating virus were detected, and most were positive for neutralizing antibodies and showed elevated levels of nitric oxide production. All suckling mice intracerebrally inoculated with 50, 500, or 5,000 focus-forming units of Saaremaa virus survived and all seroconverted. Clearly lower viral titers were observed for the Saaremaa virus-inoculated mice, also when sacrificed at day 18 after infection, compared to those in mice that died following Dobrava virus infection. Dobrava, Saaremaa, Puumala and Hantaan virus infections of adult mice were asymptomatic, and the anti-nucleocapsid protein IgG2a/IgG1-titer ratio was higher in mice inoculated with Dobrava virus than in those inoculated with Saaremaa virus. Elevated nitric oxide production was not detected in asymptomatically infected mice, and iNOS-/- mice, like normal mice, cleared viremia. In conclusion, we show that Dobrava virus and Saaremaa virus induce distinct differences in terms of survival, viremia, nitric oxide production and antibody responses in mice.     

A sensitive and quantitative assay for measuring cleavage of presenilin substrates.

The presenilin (PS) proteins are components of the gamma-secretase activity, which is central in the pathogenesis of Alzheimer's disease. Here we present a novel cell-based reporter gene assay for the quantification of PS-controlled gamma-secretase cleavage of the Alzheimer amyloid precursor protein (APP). We show that this assay offers several advantages, including increased sensitivity and specificity, improved quantification of cleavage, and simultaneous detection of all gamma-secretase cleavages in APP. Furthermore, the APP assay can be used in parallel with a similar assay that records gamma-secretase cleavage of a Notch receptor. The use of these assays to analyze the effects of two known gamma-secretase inhibitors and postulated PS active site mutants on APP and Notch processing demonstrated that inhibitors and mutants that differently affect Notch and APP cleavage can be identified rapidly. The possibility in using these assays for high throughput screening of candidate gamma-secretase inhibitors for APP and Notch in parallel opens up new vistas to systematically search for novel inhibitors that selectively block APP cleavage while not affecting Notch signaling.     

Interferon-gamma alters electrical activity and clock gene expression in suprachiasmatic nucleus neurons

The proinflammatory cytokine interferon (IFN-gamma) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN-gamma and the synergistically acting cytokine tumor necrosis factor-alpha acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN-gamma can be released chronically during infections, the authors studied the long-term effects of IFN-gamma on SCN neurons by treating dispersed rat SCN cultures with IFN-gamma over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN-gamma in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN-gamma can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN-gamma can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.     

Diving beetles (Dytiscidae) as predators of mosquito larvae (Culicidae) in field experiments and in laboratory tests of prey preference

Field experiments were performed in artificial ponds to evaluate how the density of predatory diving beetles (Dytiscidae) would affect the population levels of mosquito larvae (Culicidae). Mosquitoes colonizing the ponds were predominantly species of the genus Culex. In 2000, most of the dytiscids colonizing the ponds were small (Hydroporus spp.), and these predators had no impact on the size of larval mosquito populations, not even in ponds with added dytiscids. In 2001, larger beetles (Ilybius, Rhantus, and Agabus spp.) were more common, and there were significantly fewer mosquito larvae in ponds with the highest numbers of dytiscids. There was a negative correlation between numbers of diving beetles in the ponds and the mean body length of mosquito larvae. In neither year could dytiscid densities be maintained above a certain level owing to emigration. In laboratory tests, there were marked differences between three common dytiscid species in regard to preferences for Daphnia and Culex species as prey: Colymbetes paykulli Erichson chose mosquito larvae more often, whereas both Ilybius ater (De Geer) and I. fuliginosus (Fabricius) preferred Daphnia spp. All of the tested dytiscids consumed large numbers of prey. Since some dytiscid species can efficiently decrease populations of mosquito larvae, they are probably important in the natural control of these dipterans.     

γ‐Secretase modulators show selectivity for γ‐secretase–mediated amyloid precursor protein intramembrane processing

The aggregation of β‐amyloid peptide 42 results in the formation of toxic oligomers and plaques, which plays a pivotal role in Alzheimer''s disease pathogenesis. Aβ42 is one of several Aβ peptides, all of Aβ30 to Aβ43 that are produced as a result of γ‐secretase–mediated regulated intramembrane proteolysis of the amyloid precursor protein. γ‐Secretase modulators (GSMs) represent a promising class of Aβ42‐lowering anti‐amyloidogenic compounds for the treatment of AD. Gamma‐secretase modulators change the relative proportion of secreted Aβ peptides, while sparing the γ‐secretase–mediated processing event resulting in the release of the cytoplasmic APP intracellular domain. In this study, we have characterized how GSMs affect the γ‐secretase cleavage of three γ‐secretase substrates, E‐cadherin, ephrin type A receptor 4 (EphA4) and ephrin type B receptor 2 (EphB2), which all are implicated in important contexts of cell signalling. By using a reporter gene assay, we demonstrate that the γ‐secretase–dependent generation of EphA4 and EphB2 intracellular domains is unaffected by GSMs. We also show that γ‐secretase processing of EphA4 and EphB2 results in the release of several Aβ‐like peptides, but that only the production of Aβ‐like proteins from EphA4 is modulated by GSMs, but with an order of magnitude lower potency as compared to Aβ modulation. Collectively, these results suggest that GSMs are selective for γ‐secretase–mediated Aβ production.     

Hantavirus infections and their prevention.

Hantaviruses are rodent-borne bunyaviruses which cause haemorrhagic fever with renal syndrome and Hantavirus pulmonary syndrome in humans. This review covers the host interactions of the viruses, including the rodent reservoirs, the clinical outcome of human infections as well as the pathogenesis and laboratory diagnosis of infections. The current stage in prophylaxis and therapy of hantaviral diseases is described and different approaches in vaccine development are discussed.     

The Three Subtypes of Tick-Borne Encephalitis Virus Induce Encephalitis in a Natural Host,the Bank Vole (Myodes glareolus)

Tick-borne encephalitis virus (TBEV) infects bank voles (Myodes glareolus) in nature, but the relevance of rodents for TBEV transmission and maintenance is unclear. We infected colonized bank voles subcutaneously to study and compare the infection kinetics, acute infection, and potential viral persistence of the three known TBEV subtypes: European (TBEV-Eur), Siberian (TBEV-Sib) and Far Eastern (TBEV-FE). All strains representing the three subtypes were infective and highly neurotropic. They induced (meningo)encephalitis in some of the animals, however most of the cases did not present with apparent clinical symptoms. TBEV-RNA was cleared significantly slower from the brain as compared to other organs studied. Supporting our earlier findings in natural rodent populations, TBEV-RNA could be detected in the brain for up to 168 days post infection, but we could not demonstrate infectivity by cell culture isolation. Throughout all time points post infection, RNA of the TBEV-FE was detected significantly more often than RNA of the other two strains in all organs studied. TBEV-FE also induced prolonged viremia, indicating distinctive kinetics in rodents in comparison to the other two subtypes. This study shows that bank voles can develop a neuroinvasive TBEV infection with persistence of viral RNA in brain, and mount an anti-TBEV IgG response. The findings also provide further evidence that bank voles can serve as sentinels for TBEV endemicity.     

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/H?lln?s into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 microg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.