title: Evaluation of an integrated cell culture RT-PCR assay to detect and quantify infectious lymphocystis disease virus.
abstract: The lymphocystis disease virus (LCDV), a member of the Iridoviridae family, infects a wide range of fish species including gilthead seabream (Sparus aurata L.), the most important species cultured in the Mediterranean. LCDV is difficult to propagate in cell culture and does not produce clear and consistent cytopathic effects (CPE), especially in samples collected from subclinically infected fish. An integrated cell culture reverse transcription-polymerase chain reaction (ICC-RT-PCR) assay, followed by dot-blot hybridization of the RT-PCR products, was developed to improve the detection of infectious LCDV. The sensitivity of the ICC-RT-PCR assay, which can be performed in 7 d, was at least 100-fold higher than viral diagnosis obtained by CPE development. The developed assay thus allows the determination of infectious titres in samples with low viral loads, including those from asymptomatic carrier fish, in which no CPE was recorded after a 14-d incubation period. The ICC-RT-PCR assay enables rapid, specific and sensitive detection and quantification of infectious LCDV, and may be a valuable tool in the study of aspects of LCDV infection including transmission or epizootiology.
authors.affiliation: Universidad de Málaga, Departamento de Microbiología, 29071 Málaga, Spain. Electronic address: ejv@uma.es.
authors.forename: Estefania J
authors.initials: EJ
authors.lastname: Valverde
authors.affiliation: Universidad de Málaga, Departamento de Microbiología, 29071 Málaga, Spain. Electronic address: jjborrego@uma.es.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Borrego
authors.affiliation: Universidad de Málaga, Departamento de Microbiología, 29071 Málaga, Spain. Electronic address: dcastro@uma.es.
authors.forename: Dolores
authors.initials: D
authors.lastname: Castro
mesh_terms: D000818:Animals; D002353:Carrier State / Q000821:virology; D018929:Cell Culture Techniques; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D004266:DNA Virus Infections / Q000175:diagnosis / Q000821:virology; D015151:Immunoblotting / Q000379:methods; D007497:Iridoviridae / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D021541:Sea Bream / Q000821:virology*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods
keywords: Detection; ICC-RT-PCR; Infectivity; Lymphocystis disease virus; Quantification