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Bruning-Richardson, Anke/0000-0002-9862-9805; Mertens, Peter/0000-0002-3438-3738 WOS:000327877200004 PubMed: 24165208 Background: Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division. Methods: We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2. Results: Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects. Conclusions: We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells. BBSRC and DefraUK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC); Biotechnology and Biological Sciences Research CouncilUK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC) [BBS/E/I/00001720] Funding Source: researchfish The authors are grateful for the funding provided by the BBSRC and Defra.

Mukozal yolla uygulanan aşı hem sistemik hemde mukozal immün cevap oluşturabilmektedir. Genellikle, antijenlerin mukozal yoldan uygulaması zayıf immün cevap oluşturur. Bu nedenle, immün cevabın arttırılması için, adjuvant/ taşıyıcı sistemler gereklidir. Kitosan (CS), permeasyon ve hücreden alımı arttırıcı, antijen koruyucu, immün stimülatör aktivitesinin yanısıra, adjuvant ve biyoadhezif özellikleri nedeniyle de aşı taşıyıcı olarak üstünlük taşır. Benzer etkiler kitosan türevleri için de gösterilmektedir. Bu çalışmada, kitosan, polikatyonik türevi N-trimetil kitosan (TMC), ve poliamfolitik türevi mono-N-karboksimetil kitosan (MCC), mukozal immunizasyon için nanopartiküler taşıyıcı sistem hazırlanması için kullanılmıştır. Sitotoksisite çalışmaları (MTT tayini), çin hamster yumurtalık (CHO-K1) hücre dizisi üzerinde gerçekleştirilmiş olup, hücre yaşayabilirliği, en yüksekten en düşüğe olacak şekilde MCC, TMC/MCC kompleks, CS ve TMC sırasında gerçekleşmiştir. CS, TMC, MCC ve TMC/MCC kompleks nanopartikülleri iyonik jelasyon yöntemiyle hazırlanmış ve tetanoz toksoidi (TT) yüklenmiştir. Bu çalışmada, ilk kez MCC ve TMC/MCC kompleks nanopartikülleri geliştirilmiş ve antijen taşıyıcı sistem olarak değerlendirilmiştir. Yüksek yükleme etkinliğine sahip(>%90), partikül büyüklüğü 40-400 nm arasında değişen, MCC için negatif yüzey yükü, TMC, TMC/MCC kompleks ve CS için pozitif yüzey yüküne sahip nanopartiküller elde edilmiştir. Nanopartikülerdeki TT'nin yapısal bütünlüğü SDS-PAGE elektroforez analizi ile konfirme edilmiştir. FITC-BSA yüklü nanopartiküllerin hücrelerden etkin alımı J774A.1 hücreleri kulanılarak gösterilmiştir. TT yüklü formülasyonların immün yanıtı Balb/c fareleri üzerinde incelenmiştir. Pozitif yüklü CS ve TMC nanopartikülleri, negatif yüklü ve daha küçük partikül büyüklüğüne sahip MCC nanopartiküllerinden daha yüksek immün cevap oluşturmuştur. TMC/MCC kompleks nanopartiküllerinin, CS, TMC ve MCC nanopartiküllerine göre en yüksek immün cevabı oluşturduğu bulunmuştur. Kitosan ve türevleri ile hazırlanan biyoparçalanır nanopartiküller, antijenin partikül formunda sunumu, antijenin enzimatik degradasyondan koruması, nazal mukozadan alımını sağlamıştır ve sonuç olarak, nazal immünizasyon için umut verici adjuvan/taşıyıcı sistemler olarak önerilmektedirler. Mucosal application of a vaccine can effectively induce both systemic and mucosal immune responses. In general, mucosal applications of antigens result in poor immune responses. Therefore, adjuvant/delivery systems are required to enhance the immune response. Chitosan (CS) is a cationic biopolymer which exerts advantages as a vaccine carrier due to its adjuvant and bioadhesive properties as well as permeation and cellular uptake enhancement, antigen protection, immune stimulating activity. Similar effects are also shown by chitosan derivatives. In this study, chitosan, its polycationic derivative, N-trimethyl chitosan (TMC), and polyampholytic derivative, mono-N-carboxymethyl chitosan (MCC), were used to prepare nanoparticulate delivery systems for mucosal immunization. The cytotoxicity studies (MTT assay) on Chinese hamster ovary (CHO-K1) cell lines showed that cell viability was in the order of MCC, TMC/MCC complex, CS and TMC. CS, TMC, MCC and TMC/MCC complex nanoparticles were prepared using ionic gelation method and loaded with tetanus toxoid (TT). This was the first time that MCC and TMC/MCC complex nanoparticles were developed in this study and evaluated as antigen delivery system. Nanoparticles with high loading efficacy (>90%), particle size within the range of 40-400 nm, with a negative surface charge for MCC and positively surface charge for TMC, TMC/MCC complex and CS were obtained. The structural integrity of the TT in the nanoparticles was confirmed by SDS-PAGE electrophoresis analysis. The effective uptake of the FITC-BSA loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells. Immune response of the formulations loaded with TT was studied in vivo in Balb/c mice. CS and TMC nanoparticles which have positively charged surfaces induced higher immune responses when compared to those prepared with MCC which is negatively charged and has smaller particle size. TMC/MCC complex nanoparticles were found to induce the highest mucosal and systemic immunity response compared to CS, TMC or MCC alone. As a conclusion; chitosan and derivatives are promising polymers for mucosal vaccine delivery both as an adjuvant and a delivery system with their biocompatibility, high loading capacity and appropriate particle size. 130

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