Cultivation of PCV2 in swine testicle cells using the shell vial technique and monitoring of viral replication by qPCR and RT-qPCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Processo FAPESP: 06/57976-6

Processo FAPESP: 06/59002-9

Porcine circovirus type 2 (PCV2) is difficult to isolate. Currently, no published articles have used the shell vial technique to isolate PCV2. In addition, the action of D-glucosamine on swine testicle cells (ST) has not been evaluated properly. Thus, the aim of this study was to determine an optimal concentration of D-glucosamine and to test the shell vial technique for PCV2 propagation in ST cells. The optimal concentration of D-glucosamine was determined to be 100 mM. Because PCV2 is noncytopathic, the traditional adsorption was compared to the shell vial technique for 15 passages by qPCR, and RT-qPCR for passages 12 through 15. The quantities of viral DNA (P=0.013) and ORF1-mRNA detected with the shell vial technique were two-fold higher than the obtained with traditional adsorption. The levels of ORF2-mRNA were similar for both methods; however, by passage 15, a six-fold increase in levels was observed with the shell vial technique. Therefore, the shell vial technique was more efficient for the cultivation of PCV2, and qPCR/RT-qPCR can be used to monitor viral replication. In addition, a high viral load (>2.7 x 10(10) DNA copies/ml) and high levels of viral mRNA expression indicated that the ST cells were persistently infected. (C) 2013 Elsevier B.V. All rights reserved.