Rapid bedside inactivation of Ebola virus for safe nucleic acid tests

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available MagNA Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding MagNA Pure lysis/binding buffer directly into vacuum blood collection EDTA-tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months and Ebola virus RNA is preserved in the MagNA Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from MagNA Pure lysis/binding buffer-inactivated samples using the QIAamp Viral RNA mini kit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection

A Model System for In Vitro Studies of Bank Vole Borne Viruses

The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-β and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-β or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-β but not Mx2, while TBEV induced both IFN-β and Mx2

Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus

Acute hantavirus infection in humans triggers a rapid expansion and long-term persistence of NK cells

Vem är jag? : Elevers upplevelser av delaktighet och identitetsutveckling i gymnasiesärskolan utifrån tidigare skolgång.

Syftet med studien är att beskriva gymnasiesärskolelevers upplevelser utifrån tidigare skolgång, där de gått integrerat i grundskolan, samt synliggöra hur övergången till gymnasiesärskolan påverkar deras identitetsutveckling. Arbetet ger en översikt av tidigare forskning om utvecklingsstörning, styrdokument, specialpedagogiska perspektiv och identitet och delaktighet. Med hjälp av kvalitativa intervjuer vill jag se vad eleverna upplever som framgångsfaktorer för en lyckad skolgång. Hur eleverna upplever tillhörighet och delaktighet i särskolan, och hur övergången från en skolform till en annan kan påverka identitetsutvecklingen och vad skolformen innebär för elevernas framtid. Det teoretiska ramverket består av två teorier; det sociokulturella perspektivet och teorin om stigma. Dessa valdes eftersom de tydligt kan kopplas till den sociala miljöns betydelse för lärande, och hur den sociala identiteten hos personer med utvecklingsstörning skapas. Studien är kvalitativ och inspirerad av ett fenomenologiskt angreppssätt och bygger på semi-strukturerade intervjuer. I studien framkommer fyra områden som framgångsfaktorer för en lyckad individ integrerad skolgång; Den sociala miljön, lärarens kompetens, upprätthållandet av struktur och regler, samt organisationen av undervisningen. Det framkommer att den formella tillhörigheten till skolformen delar eleverna i två grupper, där upplevelserna skiljer sig åt mellan grupperna. Båda grupperna upplever fördelar med en tillrättalagd undervisning och trygghet i särskolan, men den formella tillhörigheten ses av den ena gruppen som negativt både för identiteten, självkänslan och skapar en begränsning av möjligheterna i framtiden. Genom att få möjligheten att ta del av gymnasiesärskoleelevers åsikter och perspektiv på sin skolgång, har jag fått insikten att de viktigaste frågorna för unga vuxna med utvecklingsstörning är frågor som rör tillhörighet, identitet och framtid. Dessa frågor kommer att vägleda mig i min roll som speciallärare på gymnasiesärskolan och jag hoppas att andra genom min studie kan få ta del av elevernas perspektiv och på så sätt vill jag ge elever med funktionsnedsättningar en röst i samhället. Min förhoppning är att mina insikter ska leda till att samarbetet mellan gymnasieskolan och gymnasiesärskolan stärks och att elevers åsikter alltid respekteras och prioriteras när åtgärder för elever i behov av särskilt stöd diskuteras

Lambda Interferon (IFN-λ) in Serum Is Decreased in Hantavirus-Infected Patients, and In Vitro-Established Infection Is Insensitive to Treatment with All IFNs and Inhibits IFN-γ-Induced Nitric Oxide Production▿

Hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), are known to be sensitive to nitric oxide (NO) and to pretreatment with type I and II interferons (alpha interferon [IFN-α]/IFN-β and IFN-γ, respectively). Elevated serum levels of NO and IFN-γ have been observed in HFRS patients, but little is known regarding the systemic levels of other IFNs and the possible effects of hantaviruses on innate antiviral immune responses. In Puumala virus-infected HFRS patients (n = 18), we report that the levels of IFN-α and IFN-β are similar, whereas the level of IFN-λ (type III IFN) is significantly decreased, during acute (day of hospitalization) compared to the convalescent phase. The possible antiviral effects of IFN-λ on the prototypic hantavirus Hantaan virus (HTNV) replication was then investigated. Pretreatment of A549 cells with IFN-λ alone inhibited HTNV replication, and IFN-λ combined with IFN-γ induced additive antiviral effects. We then studied the effect of postinfection treatment with IFNs. Interestingly, an already-established HTNV infection was insensitive to subsequent IFN-α, -β, -γ, and -λ stimulation, and HTNV-infected cells produced less NO compared to noninfected cells when stimulated with IFN-γ and IL-1β. Furthermore, less phosphorylated STAT1 after IFN treatment was observed in the nuclei of infected cells than in those of noninfected cells. The results suggest that hantavirus can interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of all IFNs. We believe that future studies addressing the mechanisms by which hantaviruses interfere with the activation and shaping of immune responses may bring more knowledge regarding HFRS and HCPS pathogenesis

Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

<div><p>Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.</p> </div

Hantaviruses inhibit staurosporine-induced apoptosis.

<p>Endothelial and epithelial cells were infected with hantavirus for three days or left uninfected (control). Cells were then exposed to the apoptosis-inducing drug staurosporine, and subsequently analyzed for induction of apoptosis. (<b>A and B</b>) Percentage of TUNEL-positive endothelial cells (A) and A549 cells (B) infected with ANDV or HTNV for three days and then treated with staurosporine for four hours. A low level of ANDV or HTNV (MOI 0.01 for both) was used for initial infection to allow for a level of approximately 20% infected cells at three days pi, the time at which cells were exposed to staurosporine. Amounts of TUNEL-positive infected and uninfected cells on the same slides were then determined using fluorescence microscopy. Staurosporine-treated uninfected cells represent maximal level of TUNEL-positive cells. Data shown represent the mean ± SEM of three independent experiments carried out in duplicate. One-way ANOVA was used for statistical evaluation; ** p<0.01; *** p<0.001. (<b>C</b>) Western blot analyses of pro-caspase 3 and caspase 3 from lysate of staurosporine-treated ANDV-infected and uninfected A549 cells. ANDV nucleocapsid protein (ANDV-N) was visualized with the mAb 7B3/F7. Calnexin was used as a control to show that similar amounts of protein were loaded in all wells. One representative experiment out of three is shown. Band intensity was analyzed by densitometry of cleaved, active, caspase 3 and full-length, inactive, pro-caspase 3. The caspase 3/pro-caspase 3-ratio was then calculated and compared between infected and uninfected cells. Staurosporine-treated uninfected cells represent the maximal caspase 3/pro-caspase 3 ratio at the indicated time-points after start of staurosporine-treatment. Data shown represent the mean ± SEM of three independent experiments. Two-way ANOVA was used for statistical evaluation; *** p<0.001. STS; staurosporine. (<b>D</b>) Caspase 3-activity after staurosporine-treatment of ANDV or HTNV infected A549 cells compared to that detected in uninfected A549 cells at the same time points. Levels of total protein in the samples were measured using a Bradford assay. Staurosporine-treated uninfected cells represent maximal level of caspase 3-activity/mg of total cellular protein. Data shown represent the mean ± SEM of three independent experiments carried out in duplicate. Two-way ANOVA was used for statistical evaluation; * p<0.05; ** p<0.01; *** p<0.001. STS; staurosporine. (<b>E</b>) Western blot analyses of full-length PARP and of caspase 3-cleaved PARP in staurosporine-treated uninfected and ANDV-infected A549 cells. ANDV-N was visualized with the mAb 7B3/F7. Calnexin was used as a control to show that similar amounts of protein were loaded in all wells. One representative experiment out of three is shown. Band intensity was analyzed by densitometry of caspase 3-cleaved PARP and full-length PARP. The caspase 3-cleaved PARP/PARP-ratio was then calculated and compared between infected and uninfected cells. Staurosporine-treated uninfected cells represent maximal caspase 3-cleaved PARP/full-length PARP-ratio at the indicated time-points after start of staurosporine-treatment. Data shown represent the mean ± SEM of three independent experiments. Two-way ANOVA was used for statistical evaluation; * p<0.05. STS; staurosporine.</p