title: A TaqMan real-time RT-PCR for quantifying Mourilyan virus infection levels in penaeid shrimp tissues.
abstract: A highly sensitive and specific TaqMan real-time quantitative RT-PCR (qRT-PCR) was developed to detect and quantify Mourilyan virus (MoV), a newly described bunya-like virus of penaeid shrimp. The PCR primers and TaqMan probe targeted a 67-nucleotide (nt) sequence in the MoV M RNA segment. Using dilution series of a 849 nt RNA transcribed in vitro from cDNA clone pMoV4.1, the assay could detect down to a single MoV RNA equivalent, reliably detected 10 RNA copies and had a log linear range up to 1 x 10(9) RNA copies. In experimentally infected Penaeus japonicus shrimp, the test was used to quantify increases in MoV loads over time in hemocytes, lymphoid organ and gills. Sequential increases in MoV RNA copy numbers occurred in lymphoid organ and gill tissues collected at 6, 24 and 48 h post-infection. However, RNA copy numbers decreased slightly in hemocytes sampled at 48 h compared to 24 h. The qRT-PCR data correlated well with amplicon yields generated using a conventional RT-nested PCR targeting the same MoV RNA segment. Moreover, histology and in situ hybridisation using shrimp cephalothorax sections identified increases in lymphoid organ spheroid numbers and confirmed that increases in MoV RNA detected in lymphoid organ tissue were due to expansion in the numbers of infected cells. The qRT-PCR assay should find use in high-throughput screening applications to detect MoV in broodstock and postlarvae used for culture or breeding purposes and for tracking changes in infection levels during shrimp grow-out.
authors.affiliation: CSIRO Food Futures Flagship, CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia 4067, Australia.
authors.forename: K V
authors.initials: KV
authors.lastname: Rajendran
authors.forename: Jeff A
authors.initials: JA
authors.lastname: Cowley
authors.forename: Russell J
authors.initials: RJ
authors.lastname: McCulloch
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Walker
mesh_terms: D000818:Animals; D005880:Gills / Q000821:virology; D006434:Hemocytes / Q000821:virology; D017403:In Situ Hybridization; D008221:Lymphoid Tissue / Q000821:virology; D033561:Penaeidae / Q000821:virology*; D012328:RNA Viruses / Q000302:isolation & purification* / Q000502:physiology; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D019562:Viral Load; D014779:Virus Replication
chemical_list: D012367:RNA, Viral