ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, Uttar Pradesh, India.
Southern Research, Birmingham, AL, United States.
J Immunol Methods. 2022 Sep;508:113321. doi: 10.1016/j.jim.2022.113321. Epub 2022 Jul 14.
Classical swine fever (CSF) is a highly contagious notifiable disease of pigs caused by CSF virus of Flaviviridae family. Previously, lapinized vaccines were used for the disease control, which has now been replaced with cell culture vaccines. Determination of virus titre is the key factor for development and quality control testing of classical swine fever (CSF) cell culture vaccines. Since CSFV is a non- cytopathic virus, an accurate method for the titration of this virus in cell culture has not yet been reported. Here we present a full proof method of titration of CSF cell culture viruses employing Fluorescent Antibody Technique (FAT) in 24 well plate cover slip culture of PK-15 cells. CSFV monoclonal antibodies (Mab) used in the test bind to the CSF virus particles in the cell cytoplasm of the infected cells and the immune-fluorescence signal is produced by subsequent binding of FITC conjugate with Mab. In this newly developed method, apple green fluorescence is observed in the cytoplasm of the infected cells as the virus multiplies only in the cytoplasm. The nucleus as well as the uninfected cells cytoplasm is stained red without any traces of green fluorescence. Thus, the test clearly differentiates a CSFV infected cell from the uninfected cells in the vicinity, if any, and also from the uninfected controls. The test can also quantify the accurate titres of CSF live viruses in the cell culture vaccines and hence it has wide application in routine virus titration applied for manufacturing of CSF cell culture vaccines, determination of accurate multiplicity of infection (m.o.i.) during infection and quality control of vaccines by the testing laboratories.
古典猪瘟（Classical swine fever，CSF）是一种由黄病毒科瘟病毒属的古典猪瘟病毒引起的高度传染性猪病。以前，兔化疫苗曾被用于该病的控制，但现已被细胞培养疫苗所取代。病毒滴度的测定是细胞培养的古典猪瘟（Classical swine fever，CSF）疫苗开发和质量控制检测的关键因素。由于 CSFV 是一种非细胞病变病毒，因此尚未报道用于细胞培养中该病毒滴定的准确方法。在此，我们提出了一种使用 PK-15 细胞在 24 孔板盖玻片培养中进行荧光抗体技术（Fluorescent Antibody Technique，FAT）滴定 CSF 细胞培养病毒的完全有效方法。在试验中使用的 CSFV 单克隆抗体（Mab）与感染细胞的细胞质中的 CSF 病毒颗粒结合，随后 FITC 缀合物与 Mab 结合产生免疫荧光信号。在这种新开发的方法中，随着病毒仅在细胞质中繁殖，在感染细胞的细胞质中观察到苹果绿荧光。细胞核以及未感染细胞的细胞质被染成红色，没有任何绿色荧光的痕迹。因此，如果存在附近的未感染细胞，该试验可以清楚地区分 CSFV 感染细胞与未感染细胞，并且也可以与未感染的对照区分开来。该试验还可以定量测定细胞培养疫苗中 CSF 活病毒的准确滴度，因此在常规病毒滴定、感染期间准确感染倍数（multiplicity of infection，m.o.i.）的确定以及疫苗质量控制实验室的检测中具有广泛的应用。
