Comparison of immune electron microscopy with direct negative staining for detection of Bovine Viral Diarrhoea Virus

Abstract

The objective of this study was to optimize an immune electron microscopy protocol for detection of Bovine Viral Diarrhoea Virus (BVDV) and to compare its detection limit with that of direct negative staining technique. Indian non-cytopathic BVDV isolate (Ind S1449) infected SFTR cell supernatant with a titre of 105.3 TCID 50 per ml was used. For IEM, various parameters including anti-BVDV polyclonal serum dilution, virus-antibody incubation temperature and time, and centrifugation time were optimized using the undiluted BVDV stock. Incubation with 1: 400 dilution of anti-BVDV polyclonal serum at 37°C for one hour was found to be optimum. To compare the detection limits of both the protocols, serial ten-fold dilutions of the virus stock (undiluted to 10−4 containing 105.3 to 101.3 TCID 50per ml, respectively) were used. Phosphotungstic acid stained grids were viewed under transmission electron microscope. BVDV was visualized as immune-aggregates of varying sizes distributed diffusely across IEM grids. In direct negative stained grids, the virus was detected mostly as individual particles. The detection limit of the IEM protocol was 103.3 TCID 50 per ml which was one hundred-fold more than that of direct negative staining protocol. The present study demonstrates that examination of suspected sample by immune electron microscopic procedures is feasible for BVDV detection and it can be used as routine diagnostic tool, especially for screening of cell culture supernatants infected with suspected clinical specimens. Suitability of this assay for direct screening for identification of persistently infected animals which are of great concern for control programmes, needs to be explored.