目的 应用制备纯化的鸭坦布苏病毒(DTMUV)非结构蛋白1(NS1),制备其单克隆抗体(mAb)并鉴定抗体的特异性.方法 将纯化的NS1蛋白免疫BALB/c小鼠,聚乙二醇(PEG)处理小鼠脾细胞与Sp2/0细胞融合,经ELISA筛选和亚克隆得2株NS1单克隆抗体细胞株,命名为NS1B、NS1E.利用ELISA测定NS1 mAb效价,Western blot法和间接免疫荧光法(IFA)鉴定NS1 mAb特异性,试剂盒测定NS1 mAb亚类.最后,用NS1 mAb对感染DTMUV后6、12、24、36 h的BHK-21细胞进行IFA检测,观察NS1蛋白的表达.结果 NS1 mAb的亚型为IgG1,效价达到1∶1.28×104;表明制备的NS1 mAb能识别天然NS1蛋白;DTMUV感染BHK-21细胞12 h后NS1蛋白开始表达.结论 获得2株具有反应性、特异性且效价高的NS1mAb,可用于DTMUV感染检测.

Objective To measure the effect of duck viperin protein on the proliferation of duck Tembusu virus (DTMUV). Methods We analyzed the duck viperin gene using a bioinformatics. Plasmids pGEX-6P-viperin and pEGFP-N1-viperin were constructed and transformed into Rosseta and transfected into BHK-21 cells, respectively. BHK-21 cells transfected with plasmid pEGFP-N1-viperin and empty vector were infected with DTMUV. The content of DTMUV in cell precipitation and supernatant was analyzed by real-time fluorescent quantitative PCR. Finally, the effect of viperin on DTMUV proliferation and the binding proteins captured by EGFP monoclonal antibody were evaluated using mass spectrometry. Results Significant differences were observed between the expression levels of viperin gene in duck compared to other species. Duck viperin was found to inhibit the budding of DTMUV in BHK-21 cells. Further, we identified six proteins that might be involved in the inhibition of the proliferation and budding of DTMUV, possibly indicative of the viperin anti-Tembusu virus pathway in ducks. Conclusion Expression patterns of duck viperin reveal how the budding of duck Tembusu virus is inhibited.

Objective To obtain the envelope protein B2L of orf virus(ORFV) and prepare highly specific polyclonal antibody against B2L. Methods The B2L gene was amplified by PCR, and subcloned into the vectors pET-32a and p3×FLAG-CMV-14, pET-32a-B2L followed by expression of B2L protein in E.coli, and induction of the target protein by IPTG, purification by urea solution and identification via Western blot analysis. Furthermore, the BALB/c mice were immunized with B2L protein to prepare highly-specific anti-B2L polyclonal antibody. In addition, plasmid p3×FLAG-B2L was transfected into Vero cells and BHK-21 cells which was used to confirm its specificity and antigenicity by indirect immunofluorescence assay(IFA). Results Western blotting demonstrated that the B2L protein had high-level specificity. The results of IFA indicated that the anti-B2L specific polyclonal antibody had specificity and reactivity. Then Immunohistochemistry showed that it could neutralize natural ORFV. Conclusion Highly specific and purified polyclonal antibody against B2L protein of the ORFV was successfully prepared.

Apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and prunus necrotic ringspot virus (PNRSV) were identified in a crab apple tree by small RNA deep sequencing. The complete genome sequence of ACLSV isolate BJ (ACLSV-BJ) was 7554 nucleotides and shared 67.0%-83.0% nucleotide sequence identity with other ACLSV isolates. A phylogenetic tree based on the complete genome sequence of all available ACLSV isolates showed that ACLSV-BJ clustered with the isolates SY01 from hawthorn, MO5 from apple, and JB, KMS and YH from pear. The complete nucleotide sequence of ASGV-BJ was 6509 nucleotides (nt) long and shared 78.2%-80.7% nucleotide sequence identity with other isolates. ASGV-BJ and the isolate ASGV_kfp clustered together in the phylogenetic tree as an independent clade. Recombination analysis showed that isolate ASGV-BJ was a naturally occurring recombinant.

Objective To express prokaryotically the non-structural protein 1 (NS1) of duck Tembusu virus (DTMUV) and prepare NS1-specific polyclonal antibodies. Methods The NS1 gene of DTMUV strain AH-F10 was amplified by PCR, followed by subcloning and expression in the prokaryotic vector pET-32a. The recombinant NS1 protein was successfully expressed in Escherichia coli BL21 (DE3), and purified with hydroxymethyl urea and renatured by gradient centrifugation. The BALB/c mice were immunized with the purified recombinant NS1 protein to prepare polyclonal antibodies against the NS1 protein. Furthermore, the titer of the polyclonal antibodies was determined by agar diffusion test (AGP), and the specificity of the polyclonal antibodies was verified by Western blotting and indirect immunofluorescence assay (IFA). Results Polyclonal antibodies against NS1 protein in serum was successfully obtained with an AGP titer of 1:8. Western blotting and IFA demonstrated that the serum with polyclonal antibodies had a high-level specificity and reactivity to the NS1 protein of DTMUV. Conclusion Polyclonal antibodies against DTMUV NS1 protein were successfully prepared and validated in this study.

The complete genome sequence of a previously undescribed badnavirus isolated from a wisteria plant exhibiting mosaic and crinkle symptoms in Beijing, China, was determined. The circular double-stranded DNA genome of this virus was 7362 bp in size with four open reading frames (ORFs 1 to 4) on the plus strand. Sequence analysis showed that this virus shared the highest (69%) nucleotide (nt) sequence identity with pagoda yellow mosaic associated virus (PYMAV). In the RT-RNase H region of the ORF-3 encoded polyprotein, this virus shared 74% nt sequence identity with PYMAV. Phylogenetic analysis provided further evidence that the virus identified in this study is a member of a new species in the genus Badnavirus. The name wisteria badnavirus 1 (WBV1) is proposed for this new virus.

The first complete genome sequence of ISMV was determined by deep sequencing of a small RNA library constructed from ISMV-infected samples and rapid amplification of cDNA ends (RACE) PCR. The ISMV genome consists of 10,403 nucleotides excluding the poly(A) tail and contains a large open reading frame encoding a polyprotein of 3316 amino acids. Putative proteolytic cleavage sites were identified by BLAST analysis. The ISMV polyprotein showed highest amino acid sequence identity to that encoded by onion yellow dwarf virus. Phylogenetic analysis of the polyprotein amino acid sequence confirmed that ISMV forms a cluster with shallot yellow stripe virus, Cyrtanthus elatus virus A, narcissus degeneration virus and onion yellow dwarf virus. These results confirm that ISMV is a distinct member of the genus Potyvirus.