Physicochemical properties of double-stranded RNA used to discover a reo-like virus from blue crab Callinectes sapidus

Abstract

Mortality among blue crab Callinectes sapidus in soft shell production facilities is typically 25% or greater. The harvest, handling, and husbandry practices of soft shell crab production have the potential to spread or exacerbate infectious crab diseases. To investigate the possible role of viruses in soft shell crab mortalities, we took advantage of the physicochemical properties of doublestranded RNA (dsRNA) to isolate a putative virus genome. Further characterization confirmed the presence of a reo-like virus that possesses 12 dsRNA genome segments. The virus was present in > 50% of dead or dying soft shell crabs, but fewer than 5% of healthy hard crabs. Injection of the virus caused mortality and resulted in the appearance of viral RNA and virus inclusions in hemocytes. The genome of the virus was partially sequenced and the information used to develop a reverse transcription polymerase chain reaction (RT-PCR) assay that is able to detect the virus genome in as little as 7.5 pg of total RNA. The molecular tools developed during this study will allow us to quantify prevalence of the blue crab reo-like virus in captive (soft shell facilities, aquaculture operations) and wild populations and facilitate understanding of the role this virus has in blue crab life history.

Histopathological characterization and in situ detection of Callinectes sapidus reovirus

“…Callinectes sapidus reovirus (CsRV) was found in blue crabs captured from Chesapeake Bay in 2005 and was present in over 50% of dead/moribund soft shell crabs and 5% of wild crab populations sampled (Bowers et al, 2010). In laboratory infections of C. sapidus juveniles, this virus resulted in 100% mortality.…”

“…In laboratory infections of C. sapidus juveniles, this virus resulted in 100% mortality. CsRV was found to be $55 nm in diameter with a 22-24 kb genome consisting of 12 segments of dsRNA ranging from 1.1 to 4.0 kb and arranged in an 1/5/6 pattern (Bowers et al, 2010).…”

Disease, parasite, and commensal prevalences for blue crab Callinectes sapidus at shedding facilities in Louisiana, USA

“…Both A. michaelis and RLV are hypothesized to cause mass mortalities in shedding facilities, possibly due to crowding and cannibalism, which facilitate transmission (Messick & Sindermann 1992, Bowers et al 2010, 2011. A. michaelis causes muscle lysis in infected crabs (Messick & Sindermann 1992).…”

“…Detection of RLV was accomplished by extraction of total RNA from leg muscle and application of a modification of the RT-PCR assay described by Bowers et al (2010), with TaqMan® Fast Virus 1-Step Master Mix (Life Technologies), conducted on an Applied Biosystems Fast 7500 thermocycler. Standards for the RLV qPCR assay were prepared from a serial dilution of the RLV dsRNA genome (10 to 10 6 copies).…”

“…One walking leg was removed and frozen at −20°C for RLV reverse-transcription PCR at the University of Maryland Center for Environmental Science (see 'RLV RT-PCR detection'; Bowers et al 2010). Gill samples were frozen at −20°C for L. callinectes detection (see below).…”