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

Rabies is an acute viral disease that causes encephalitis and is universally fatal. The rabies virus is distributed worldwide, such that this disease constitutes an important public health problem. Prevention and control programs that involve the mass vaccination of domestic animals and the development of diagnostic laboratory methods with high sensitivity and specificity have been organised (Rupprecht et al. 2006, Bruckner 2009). The MIT is a technique used for rabies isolation and a large amount of virus can be isolated from a single mouse brain for strain identification purposes. This assay can be used in situations in which facilities for cell culture (CC) are not available. Once a validated and reliable CC unit is established in the laboratory, the mouse inoculation test (MIT) should be replaced with another assay to avoid the use of live animals. The CC method is less expensive and gives more rapid results (Favi et al. 1992, OIE 2011). The shell vial procedure is a modification of the standard CC procedure and is useful for the rapid detection of virus in vitro. This assay has been employed for the isolation of different microorganisms. The original shell vial protocol has been adapted for use with 24-well tissue culture plates and centrifugation [modified shell vial (MSV)] and has been employed for the detection of dengue and other arboviruses (Caceda & Kochel 2007). The purpose of this study was to replace the MIT with the MSV to reduce the time necessary for rabies virus isolation. Thirty brain specimens (25 positive and 5 negative) from rabies cases analysed by fluorescent antibody test (FAT) were studied. The samples originated from different animal species (22 dogs, 3 bovines, 1 cat, 1 goat, 2 humans and 1 bat) and were obtained through the epidemiological surveillance program of the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, in 2008. The rabies FAT was performed using the technique previously described by Bourhy et al. (1990) and the MIT was performed as reported by Koprowski (1996). For the MSV assay, the cover slips and tubes that are traditionally employed in the shell vial procedure were substituted by plastic 24-well plates. A 500 μL suspension of N2A cells (150-200,000 cells/mL) was added to each plate and 200 μL of the specimen supernatant (1:100 dilution) was used to inoculate the cell monolayers. The inoculated cells grown in the plastic 24-well plates were centrifuged for 1 h at 37oC and 3,000 rpm. The supernatant was discarded and 1 mL of Dulbecco’s modified Eagle’s medium containing 2% foetal calf serum was added to each well. After 24 h, 48 h or 72 h at 37oC in CO2, inoculated cells were detached and fixed on immunofluorescent slides. Then, rabies virus antigen was detected by direct FAT. Among the 30 samples previously analysed by FAT, 25 (96.2%) specimens were confirmed to be positive for rabies by the MSV test and the MIT. When the MSV assay and the MIT were compared, the MSV exhibited a 96.2% sensitivity (95% CI: 88.7-100), a specificity of 100%, a positive predictive value of 100% + Corresponding author: [email protected] Received 15 May 2012 Accepted 18 October 2012 Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation