Development of indirect immunofluorescence assay for TCID50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells. (January 2018)

• Record Type: Journal Article
• Authors: Wang, Qing; Xie, Hualiang; Zeng, Weiwei; Wang, Linchuan; Liu, Chun; Wu, Jiexing; Wang, Yingying; Li, Yingying; Bergmann, Sven M.
• Is Part Of: Microbial pathogenesis. Volume 114(2018)
• Page Start: 68
• Page End: 74
• Publication Date: 2018-01
• Subjects: Immunofluorescence assay -- TCID50 -- qPCR -- Grass carp reovirus -- Genotype II -- Cyenopharyngodon idellus; Pathogenic microorganisms -- Periodicals; Pathology, Molecular -- Periodicals; Communicable Diseases -- microbiology -- Periodicals; Communicable Diseases -- parasitology -- Periodicals; Micro-organismes pathogènes -- Périodiques; Pathologie moléculaire -- Périodiques; Electronic journals; 616.9041
• DOI: 10.1016/j.micpath.2017.11.042
• Languages: English
• ISSNs: 0882-4010

Abstract

Abstract: Grass carp reovirus (GCRV) caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus, in southeast Asian. It was first isolated in 1983 in China, and clade analysis of the different GCRV isolates indicates there are at least three different genotypes I, II, and III. In recent years, GCRV genotype II has been determined as a dominant virus type which cause severe obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. TCID50 is one of standard method to quantity infectious virus particles. In the present study, an indirect immunofluorescence assay (IFA) was developed using antibody against a protein encoded by segment 10 of GCRV genotype II. Moreover, the specific assay to differentitate GCRV of different genotypes and a sensitive assay for determination of GCRV genotype II were developed respectively. The results showed the IFA only can recognize genotype II virus at the lowest initial concentration of 550 genomic copies/ml. Furthermore, comparison of results obtained from qPCR and the TCID50 assay combined IFA was conducted. The results indicated that TCID50 of GCRV isolates JX0901 and HZ08 differs with 2 log steps reduction in the numbers of viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is sensitive, specific, and the TCID50 combined with IFA will be a standardizable technique for the quantitation and detection of infectious GCRV in cell culture without cytolysis. Highlights: GCRV genotype II cause obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. The IFAs developed is sensitive and specific for GCRV genotype II. The TCID50 assay combined with IFA for virus quatification by titration of GCRV in cell culture was determined. A relation between isolate values quantified by the TCID50 assay and by qPCR was obtained.