貉细小病毒LN10-1株分离鉴定及其免疫原性研究

Isolation,Identification and Immunogenic Evaluation of a Raccoon Dog Parvovirus:LN10-1

为研制貉细小病毒性肠炎疫苗,筛选出针对貉细小病毒性肠炎免疫原性好、安全高效的疫苗备选株,应用CRFK细胞从辽宁省发病貉的粪便中分离病毒,并通过形态学、血清学、分子生物学、动物回归及免疫接种等方法对分离株进行鉴定。鉴定结果表明成功分离出1株貉细小病毒,命名为LN10-1株。其VP2基因核苷酸序列与猕猴源猫泛白细胞综合征病毒株(BJ-22/2008/CHN株)相似性高达99.7%。VP2蛋白上决定宿主范围的2个氨基酸位点发生了突变。VP2基因种系发生分析显示,LN10-1株位于猫泛白细胞综合征病毒(Feline panleukopenia virus,FPLV)、蓝狐细小病毒(Blue fox parvovirus,BFPV)、水貂肠炎病毒(Mink enteritis virus,MEV)组成的食肉类动物细小病毒聚类分支与由犬细小病毒(Canine parvovirus,CPV)组成的聚类分支。由LN10-1株制备的灭活疫苗免疫结果显示,接种28d细小病毒中和抗体滴度可达到1∶256以上。推测LN10-1株可能正处于FPLV与CPV进化的中间状态,或是CPV适应新宿主(貉)而形成的一种新病毒,可以作为针对貉细小病毒性肠炎灭活疫苗的候选株。

To develop vaccine for raccoon dog enteritis caused by raccoon dog parvovirus, parvo- virus strains with high immunogenicity and high efficacy were selected as vaccine candidates. Live parvovirus was isolated from feces of clinical raccoon dog by Crandall-Reese feline kidney （CRFK） cells. The virus strains were isolated then identified with morphological methods, sero- logical methods, molecular biological methods, animal regression experiment and immunogenic evaluation. One RDPV strain named LN10-1 was isolated successfully. Its complete VP2 sequence of LN10-1 strain had the highest identity with FPLV monkey/BJ-22/2008/CHN （99.7 percent）. The two AA residues characterized as determination of host range were changed. The phylogenetic analysis of VP2 gene sequences revealed that the LN10-1 isolation was presented between the two genetic clusters, one was composed with CPV, and the other cluster was consist of carnivores parvovirus （FPLV, MEV and BFPV）. The Immunological assay result shows the neutralizing antibody titre of parvovirus is higher than 64 folds on 28 day post-immunization. It is supposed that LN10-1 is evolving between FPLV and CPV, or there may be a new virus that becomes adapted to its new host （raccoon dog）. And this strain could be used as a candidate inac-tivated vaccine for preventing haemorrhagic enteritis in Raccoon dogs.