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

Spatio-temporal dynamics and aetiology of proliferative leg skin lesions in wild British finches

Proliferative leg skin lesions have been described in wild finches in Europe although there have been no large-scale studies of their aetiology or epizootiology to date. Firstly, disease surveillance, utilising public reporting of observations of live wild finches was conducted in Great Britain (GB) and showed proliferative leg skin lesions in chaffinches (Fringilla coelebs) to be widespread. Seasonal variation was observed, with a peak during the winter months. Secondly, pathological investigations were performed on a sample of 39 chaffinches, four bullfinches (Pyrrhula pyrrhula), one greenfinch (Chloris chloris) and one goldfinch (Carduelis carduelis) with proliferative leg skin lesions and detected Cnemidocoptes sp. mites in 91% (41/45) of affected finches and from all species examined. Fringilla coelebs papillomavirus (FcPV1) PCR was positive in 74% (23/31) of birds tested: a 394 base pair sequence was derived from 20 of these birds, from all examined species, with 100% identity to reference genomes. Both mites and FcPV1 DNA were detected in 71% (20/28) of birds tested for both pathogens. Histopathological examination of lesions did not discriminate the relative importance of mite or FcPV1 infection as their cause. Development of techniques to localise FcPV1 within lesions is required to elucidate the pathological significance of FcPV1 DNA detection.We thank the members of the public and BTO Garden BirdWatch participants who reported garden bird morbidity and mortality incidents and our colleagues, Katie Beckmann, Shaheed Macgregor, Ricardo Castro Cesar de Sa, Lydia Franklinos and Tim Hopkins from the Zoological Society of London; Kirsi Peck from the Royal Society for the Protection of Birds; BTO staff members in the Garden BirdWatch team; the staff at Abbey Veterinary Services and the Animal & Plant Health Agency (Daniel Hicks, Richard Irvine, Alejandro Núñez and Scott Reid) for their assistance with this investigation. This work was financially supported by the following organisations; Birdcare Standards Association, British Trust for Ornithology, British Veterinary Association Animal Welfare Foundation, CJ Wildbird Foods, Cranswick Pet Products, UK Department for the Environment Food & Rural Affairs and Welsh Government through the Animal & Plant Health Agency’s Diseases of Wildlife Scheme Scanning Surveillance Programme (Project ED1600), Esmée Fairbairn Foundation, Gardman Ltd, Institute of Zoology, Royal Society for the Protection of Birds and the Universities Federation for Animal Welfare. RAJW was supported by the Moncloa of Excellence PICATA programme and Crafoord Foundation Sweden (grant number 20160971). Molecular and sequencing costs were funded by the Spanish Ministry of Science and Innovation, (Ref: CGL2013-41642-P/BOS)

Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species

Evaluation of functional dynamics during osseointegration and regeneration associated with oral implants

The aim of this paper is to review current investigations on functional assessments of osseointegration and assess correlations to the peri-implant structure.The literature was electronically searched for studies of promoting dental implant osseointegration, functional assessments of implant stability, and finite element (FE) analyses in the field of implant dentistry, and any references regarding biological events during osseointegration were also cited as background information.Osseointegration involves a cascade of protein and cell apposition, vascular invasion, de novo bone formation and maturation to achieve the primary and secondary dental implant stability. This process may be accelerated by alteration of the implant surface roughness, developing a biomimetric interface, or local delivery of growth-promoting factors. The current available pre-clinical and clinical biomechanical assessments demonstrated a variety of correlations to the peri-implant structural parameters, and functionally integrated peri-implant structure through FE optimization can offer strong correlation to the interfacial biomechanics.The progression of osseointegration may be accelerated by alteration of the implant interface as well as growth factor applications, and functional integration of peri-implant structure may be feasible to predict the implant function during osseointegration. More research in this field is still needed. To cite this article: Chang P-C, Lang NP, Giannobile WV. Evaluation of functional dynamics during osseointegration and regeneration associated with oral implants. Clin. Oral Impl. Res . 21 , 2010; 1–12.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78668/1/j.1600-0501.2009.01826.x.pd

Evidence for Ljungan virus specific antibodies in humans and rodents, Finland.

Objectives Ljungan virus (LV) belongs to the genus Parechovirus. Human parechoviruses (HPeV) are common viruses causing diarrhea and gastroenteritis, and the first infection is commonly faced during the early childhood and 90% of humans acquire HPeV antibodies by the age of two. Ljungan virus, however, is known as a rodent-borne parechovirus first isolated (LV 87-012) near Ljungan river in Sweden from a wild bank vole (Myodes glareolus). Puumala hantavirus causes Nephropatia epidemica (NE) in humans and these cases are fairly common in Finland comprising approximately 800-3000 suspected NE cases per year depending somewhat on the size of rodent population. However, the abundance of Finnish patients diagnosed with NE, i.e., with a history of rodent contact, makes any connection between human disease and Ljungan virus infection likely to be found in the country. With this study, we sought to find evidence of Ljungan virus in Finnish humans and rodents. We aimed to develop a reliable method for serological screening of LV in humans and rodents, and to confirm the specificity of this method. Methods Initially, neutralization assays were designed and carried out followed by an immunofluorescent assay (IFA) for serology screening. The IFA and neutralization assays were set up using Vero-cells infected with Ljungan virus strain 145SLG kindly provided by Conny Tolf et al. (Uppsala University, Uppsala, Sweden). Serological assays were used for detecting Ljungan virus antibodies both in humans and rodents. Furthermore, 8 human sera used in the Ljungan virus neutralization assay were cross-checked for titres of neutralizing antibodies to six different human parechoviruses (HPeV 1-6), that are closely related to Ljungan virus. Neutralization effect of the antibodies to 42 different human picornaviruses against Ljungan virus (145SLG) was also studied. Results and Conclusion The study is ongoing, but preliminary data indicates that we have LV specific antibodies in Finland. Twenty-six sera out of 41 human serums were not able to neutralize Ljungan 145SLG virus. However, 15 human serum samples neutralized the Ljungan 145SLG virus. Wide titer range of the HPeV 1-6 antibodies were also detected in eight human serums studied in more closely. The preliminary IFA results are also supporting the neutralization results. In total, 8 out of the 50 (16%, 95% CI: 8.1–28.8%) rodent samples (Konnevesi, Finland, year 2008) were Ljungan antibody positive. Our data is first evidence of human Ljungan virus infections in Finland both in humans and rodents. This data can be used as a stepping stone for further studies of Ljungan virus: the broad seroprevalence study both in humans and rodents, and to investigate the role of Ljungan virus infection in Finland