Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

BACKGROUND Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM. METHODS We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. RESULTS A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. CONCLUSIONS The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

Seroprevalence of hepatitis E virus in domestic pigs and wild boars in Switzerland.

Hepatitis E is considered an emerging human viral disease in industrialized countries. Studies from Switzerland report a human seroprevalence of hepatitis E virus (HEV) of 2.6-21%, a range lower than in adjacent European countries. The aim of this study was to determine whether HEV seroprevalence in domestic pigs and wild boars is also lower in Switzerland and whether it is increasing and thus indicating that this zoonotic viral infection is emerging. Serum samples collected from 2,001 pigs in 2006 and 2011 and from 303 wild boars from 2008 to 2012 were analysed by ELISA for the presence of HEV-specific antibodies. Overall HEV seroprevalence was 58.1% in domestic pigs and 12.5% in wild boars. Prevalence in domestic pigs was significantly higher in 2006 than in 2011. In conclusion, HEV seroprevalence in domestic pigs and wild boars in Switzerland is comparable with the seroprevalence in other countries and not increasing. Therefore, prevalence of HEV in humans must be related to other factors than prevalence in pigs or wild boars.

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

Serum folate, cobalamin, homocysteine and methylmalonic acid concentrations in pigs with acute, chronic or subclinical Lawsonia intracellularis infection.

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy. The clinical presentation can be acute (i.e. proliferative hemorrhagic enteropathy, PHE), chronic (i.e. porcine intestinal adenomatosis, PIA) or subclinical. In humans with chronic enteropathies, low serum folate (vitamin B(9)) and cobalamin (vitamin B(12)) concentrations have been associated with increased serum concentrations of homocysteine and methylmalonic acid (MMA), which reflect the availability of both vitamins at the cellular level. The aim of this study was to evaluate serum folate, cobalamin, homocysteine and MMA concentrations in serum samples from pigs with PHE, PIA or subclinical L. intracellularis infection, and in negative controls. Serum folate, cobalamin, homocysteine and MMA concentrations differed significantly among pigs in the PHE, PIA, subclinical and negative control groups. Serum folate concentrations in the PHE and PIA groups were lower than in the subclinical and negative control groups, while serum cobalamin concentrations were lower in the PIA group than in other groups. Serum concentrations of homocysteine were higher in the PHE, PIA and subclinical groups than in the negative control group. Serum concentrations of MMA were higher in the subclinical and PIA groups than in the control group. These data suggest that pigs infected with L. intracellularis have altered serum cobalamin, folate, homocysteine and MMA concentrations.

Comparison of the diagnostic significance of clinical, radiographic and ultrasonographic results after an experimental aerosol infection of pigs with Actinobacillus pleuropneumoniae

Scoring schemes for clinical, ultrasonographic and radiographic findings in pigs were developed based upon a standardized animal model for Actinobacillus pleuropneumoniae infection.The results of these methods were compared to each other as well as with the corresponding pathomorphological findings during necropsy. Altogether 69 pigs of different breeding lines (Hampshire, Pietrain and German Landrace were examined. Positive correlations were found between the results of all three methods as well as with the necropsy scores (p <0.0001). Different pathomorphological findings were detected either by radiographic or by ultrasonographic examination dependent upon the type of lung tissue alterations: Alterations of the pleura as well as sequestration of lung tissue on the lung surface could be clearly identified during the ultrasonographic examination while deep tissue alterations with no contact to the lung surface could be detected reliably by radiographic examination. Both methods complement each other, and the application of a combined ultrasonographic and radiographic examination of the thorax allows a comprehensive inspection of the lung condition. Particularly during the acute phase of the disease the extent of lung tissue damage can be estimated more precisely than by clinical examination alone.

Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial.

BACKGROUND A recombinant, replication-competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Zaire Ebolavirus (rVSV-ZEBOV) is a promising Ebola vaccine candidate. We report the results of an interim analysis of a trial of rVSV-ZEBOV in Guinea, west Africa. METHODS For this open-label, cluster-randomised ring vaccination trial, suspected cases of Ebola virus disease in Basse-Guinée (Guinea, west Africa) were independently ascertained by Ebola response teams as part of a national surveillance system. After laboratory confirmation of a new case, clusters of all contacts and contacts of contacts were defined and randomly allocated 1:1 to immediate vaccination or delayed (21 days later) vaccination with rVSV-ZEBOV (one dose of 2 × 10(7) plaque-forming units, administered intramuscularly in the deltoid muscle). Adults (age ≥18 years) who were not pregnant or breastfeeding were eligible for vaccination. Block randomisation was used, with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 vs >20 individuals). The study is open label and masking of participants and field teams to the time of vaccination is not possible, but Ebola response teams and laboratory workers were unaware of allocation to immediate or delayed vaccination. Taking into account the incubation period of the virus of about 10 days, the prespecified primary outcome was laboratory-confirmed Ebola virus disease with onset of symptoms at least 10 days after randomisation. The primary analysis was per protocol and compared the incidence of Ebola virus disease in eligible and vaccinated individuals in immediate vaccination clusters with the incidence in eligible individuals in delayed vaccination clusters. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201503001057193. FINDINGS Between April 1, 2015, and July 20, 2015, 90 clusters, with a total population of 7651 people were included in the planned interim analysis. 48 of these clusters (4123 people) were randomly assigned to immediate vaccination with rVSV-ZEBOV, and 42 clusters (3528 people) were randomly assigned to delayed vaccination with rVSV-ZEBOV. In the immediate vaccination group, there were no cases of Ebola virus disease with symptom onset at least 10 days after randomisation, whereas in the delayed vaccination group there were 16 cases of Ebola virus disease from seven clusters, showing a vaccine efficacy of 100% (95% CI 74·7-100·0; p=0·0036). No new cases of Ebola virus disease were diagnosed in vaccinees from the immediate or delayed groups from 6 days post-vaccination. At the cluster level, with the inclusion of all eligible adults, vaccine effectiveness was 75·1% (95% CI -7·1 to 94·2; p=0·1791), and 76·3% (95% CI -15·5 to 95·1; p=0·3351) with the inclusion of everyone (eligible or not eligible for vaccination). 43 serious adverse events were reported; one serious adverse event was judged to be causally related to vaccination (a febrile episode in a vaccinated participant, which resolved without sequelae). Assessment of serious adverse events is ongoing. INTERPRETATION The results of this interim analysis indicate that rVSV-ZEBOV might be highly efficacious and safe in preventing Ebola virus disease, and is most likely effective at the population level when delivered during an Ebola virus disease outbreak via a ring vaccination strategy. FUNDING WHO, with support from the Wellcome Trust (UK); Médecins Sans Frontières; the Norwegian Ministry of Foreign Affairs through the Research Council of Norway; and the Canadian Government through the Public Health Agency of Canada, Canadian Institutes of Health Research, International Development Research Centre, and Department of Foreign Affairs, Trade and Development.

Impact of the Ebola epidemic on general and HIV care in Macenta, Forest Guinea, 2014.

OBJECTIVE The current Ebola epidemic massively affected the Macenta district in Forest Guinea. We aimed at investigating its impact on general and HIV care at the only HIV care facility in the district. DESIGN Prospective observational single-facility study. METHODS Routinely collected data on use of general hospital services and HIV care were linked to Ebola surveillance data published by the Guinea Ministry of Health. In addition, we compared retention among HIV-infected patients enrolled into care in the first semesters of 2013 and 2014. RESULTS Throughout 2014, service offer was continuous and unaltered at the facility. During the main epidemic period (August-December 2014), compared with the same period of 2013, there were important reductions in attendance at the primary care outpatient clinic (-40%), in HIV tests done (-46%), in new diagnoses of tuberculosis (-53%) and in patients enrolled into HIV care (-47%). There was a smaller reduction in attendance at the HIV follow-up clinic (-11%). Kaplan-Meier estimates of retention were similar among the patients enrolled into care in 2014 and 2013. In a multivariable Cox regression analysis, the year of enrolment was not associated with attrition (hazard ratio 1.02; 95% confidence interval: 0.72-1.43). CONCLUSION The Ebola epidemic resulted in an important decrease in utilization of the facility despite unaltered service offer. Effects on care of HIV-positive patients enrolled prior to the epidemic were limited. HIV care in such circumstances is challenging, but not impossible.

In vivo degradation of magnesium plate/screw osteosynthesis implant systems: Soft and hard tissue response in a calvarial model in miniature pigs.

Biodegradable magnesium plate/screw osteosynthesis systems were implanted on the frontal bone of adult miniature pigs. The chosen implant geometries were based on existing titanium systems used for the treatment of facial fractures. The aim of this study was to evaluate the in vivo degradation and tissue response of the magnesium alloy WE43 with and without a plasma electrolytic surface coating. Of 14 animals, 6 received magnesium implants with surface modification (coated), 6 without surface modification (uncoated), and 2 titanium implants. Radiological examination of the skull was performed at 1, 4, and 8 weeks post-implantation. After euthanasia at 12 and 24 weeks, X-ray, computed tomography, and microfocus computed tomography analyses and histological and histomorphological examinations of the bone/implant blocks were performed. The results showed a good tolerance of the plate/screw system without wound healing disturbance. In the radiological examination, gas pocket formation was found mainly around the uncoated plates 4 weeks after surgery. The micro-CT and histological analyses showed significantly lower corrosion rates and increased bone density and bone implant contact area around the coated screws compared to the uncoated screws at both endpoints. This study shows promising results for the further development of coated magnesium implants for the osteosynthesis of the facial skeleton.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

[Lung and pleural lesions before and after implementation of a national eradication program against enzootic pneumonia and actinobacillosis as well as changes of slaughter carcass organs in slaughter pigs in Switzerland].

In a representative cross-sectional study during 12 months of the years 2008/2009 in four abattoirs in Switzerland, lung and pleura lesions as well as lesions of slaughter carcasses and organs of 34 706 pigs were studied for frequency and type of macroscopic lesions. Of the 24276 examined pigs, 91.2% of the lungs, 94.4% of the heart and 95.5% of the livers showed no macroscopically visible lesions. Pigs that were produced for a label program had significantly less bronchopneumonia and pneumonia residuals, pleuritis and liver lesions due to echinococcosis. Pigs supervised by the Swiss Pig Health Service (SGD), showed significantly less bronchopneumonia and pneumonia residuals, diffuse pleuritis, pleuritis/pericarditis and milkspots compared to the non-SGD supervised farms. Thanks to the national eradication program for enzootic pneumonia (EP) and actinobacillosis, the health-status of lungs has been considerably improved and the prevalence of pleurisy decreased considerably. The results of this study indicate a good herd health in Swiss pig production.

Kapitänleutnant Frederik Siegfried Mörk [Illustration] : Gouverneur von Neu Guinea

Lippman Fraenckel

Delphyodon, eine neue Gattung der Apocynaceae aus Neu-Guinea

Von K. Schumann

Koloniales : eine Plauderei über Deutsch-Neu-Guinea

(von einem auf Java ansässigen Deutschen)