title: Cultivation of PCV2 in swine testicle cells using the shell vial technique and monitoring of viral replication by qPCR and RT-qPCR.
abstract: 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 100mM. 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 (>2.7×10(10) DNA copies/ml) and high levels of viral mRNA expression indicated that the ST cells were persistently infected.
authors.affiliation: Department of Microbiology and Immunology, Biosciences Institute, Univ. Estadual Paulista (UNESP), Botucatu 18618-970, São Paulo, Brazil. Electronic address: tfcruz@yahoo.com.br.
authors.forename: Taís F
authors.initials: TF
authors.lastname: Cruz
authors.affiliation: Department of Microbiology and Immunology, Biosciences Institute, Univ. Estadual Paulista (UNESP), Botucatu 18618-970, São Paulo, Brazil. Electronic address: jpessoa@ibb.unesp.br.
authors.forename: João P
authors.initials: JP
authors.lastname: Araujo
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D017925:Circovirus / Q000254:growth & development* / Q000502:physiology; D003470:Culture Media / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D005944:Glucosamine / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013552:Swine; D014776:Virus Cultivation / Q000379:methods; D014779:Virus Replication
chemical_list: D003470:Culture Media; D004279:DNA, Viral; D005944:Glucosamine
keywords: Centrifugal enhancement; Porcine circovirus 2; Quantitative PCR; Shell vial technique; Swine testicle cells