Role of duck plague virus glycoprotein C in viral adsorption:Absence of specific interactions with cell surface heparan sulfate

Many mammalian herpes viruses utilize heparan sulfate （HS） moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C （gC） homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus （DPV） remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus （DPV-AgC-EGFP）. Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins （gB-DPV, gC-DPV, and gE-DPV） to HS was assessed using a prokaryotic expression system and HJTrapTM HeparJn HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-△gC-EGFP or wild-type strain Chinese virulent duck plague virus （DPV-CHv）. The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on Viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles

The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.

鸭肠炎病毒UL18基因的转录、表达特征及原核表达蛋白的初步应用

为了探讨鸭肠炎病毒（duck enteritis virus，DEV）UL18基因的特性和功能，本研究运用生物信息学软件对DEVUI，18基因及其编码蛋白进行了分析。构建原核表达质粒pET32a-UL18，并将其转化大肠杆菌BL21（DE3），经IPTG诱导后获得了大小约为55000的pET32a／DEV-UL18重组蛋白。将该重组蛋白纯化后免疫家免，获得了兔抗pET32a／DEV-UL18重组蛋白高免血清。采用荧光定量RT-PCR和Westernblot对DEV感染鸭胚成纤维细胞（DEF）后uL18基因的转录和表达情况进行检测，采用间接免疫荧光对DEV感染鸭的肠道和食道进行检测。生物信息学分析结果表明，DEVUL18基因大小969bp，编码1条由322个氨基酸残基组成的多肽，含15个潜在的磷酸化位点和2个N-糖基化位点，合15个潜在的B细胞表位；系统进化树分析表明，DEVUL18属于＆一疱疹病毒的1个成员，与MeHV-1亲缘关系最近；密码子偏爱性分析结果表明，若对DEVUL18基因进行外源表达，真核表达系统可能更容易，若选择原核表达系统，则需对宿主表达菌进行选择和条件优化。荧光定量RT-PCR和westernblot结果表明，DEV感染DEF后2h，UL18基因开始转录，感染后12h开始表达，感染24h后转录和表达产物急剧上升，到36和48h后转录和表达产物分别达最大值，之后逐渐下降。间接免疫荧光结果表明，被DEV感染鸭肠道、食道等组织能检测到明显的阳性信号，表明制备的DEVUL18原核表达蛋白及其兔抗多克隆抗体可用于DEV的诊断试剂材料。