Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Abstract

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Virus isolation in cell culture for confirmatory diagnostic of rabies in bovine specimens

“…The T3 protocol (three passages of 48h each) failed in detecting one positive sample in BHK-21 cells and two in N2A. Previous studies have suggested a minimal incubation period of three (ANTÚNEZ et al, 2013) or 4-5 days (RUDD & TRIMARCHI, 1989), while others suggested that a 24h period would be adequate (BOURHY et al, 1989;CHITRA et al, 1988). These discrepancies may reflect different VI protocols or cells (cell lines, passage number, etc.…”

“…Hence, virus isolation (VI) in cell culture has been proposed as an alternative method to replace MIT as a confirmatory test for rabies. Isolation systems based on mouse neuroblastoma (N2A) and baby hamster kidney (BHK-21) cells have been evaluated regarding to sensitivity to RabV from different animal species (RUDD et al, 1980;1989;RUDD & TRIMARCHI, 1987;ANTÚNEZ et al, 2013). Considering the fastidious growth and the special culture medium requirements for N2A cells, the use of BHK-21 cells for VI as confirmatory rabies diagnosis would compare favorably since BHK-21 cells display a fast growth in vitro and are largely used in many laboratories.…”