Double-stranded RNA against WSSV genes provides antiviral protection in shrimp

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Double-stranded RNA against WSSV genes provides antiviral protection in shrimp

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Researchers explore solutions to major shrimp disease.

At the end of the experiments, most of the animals treated with dsRNA against WSSV genes survived and had low rates of WSSV infection.

At the end of the experiments, most of the animals treated with dsRNA against WSSV genes survived and had low rates of WSSV infection.

The white spot syndrome virus (WSSV) has caused major economic losses to shrimp farmers worldwide since the 1990s. Various field treatments and strategies for controlling WSSV have been tested, showing different degrees of efficacy. Some of these include immuno-stimulants, administration of recombinant viral proteins, manipulating water temperature, DNA vaccines and RNA interference (RNAi).

WSSV has up to 531 putative genes, some of which may be essential for WSSV infection/replication. Most of the WSSV genes have unknown roles in virus infection. Several studies have used RNAi against WSSV genes, encoding structural proteins involved in virion architecture and virus entry. Other studies have assessed the antiviral efficacy of non-structural WSSV proteins.

It is possible the antiviral efficacy of RNAi molecules depends on the targeted genes. Silencing WSSV genes with critical roles in virus replication might show a stronger antiviral effect and thus reduce shrimp mortality.

Study setup

The authors therefore established a study to evaluate the antiviral efficacy of double-stranded (ds)RNA against non-structural WSSV genes orf89 and wsv191 in Pacific white shrimp (Litopenaeus vannamei) and compare it to dsRNA against WSSV genes vp28 and vp26 under experimental conditions.

Juvenile shrimp from a hatchery in Sonora, Mexico, were tested WSSV-negative by polymerase chain reaction (PCR) analyses. The animals were maintained in artificial seawater with a salinity of 25 g/L, temperature of 27 ± 2 degrees-C and continuous aeration. Fifty percent water exchange was done every third day to maintain good water quality.

A WSSV inoculum prepared from naturally infected shrimp from Sinaloa, Mexico, was titrated in vivo. For each tenfold serial dilution (10-2-10-7), five shrimp were intramuscularly inoculated and individually kept in 12-L tanks with artificial seawater.

The shrimp were monitored twice daily for clinical signs of WSSV infection and mortality for 10 days. Infectivity and lethal titers were determined to be 105.6 SID50/mL and 105.6 L.D.50/mL, respectively. SID50 is the shrimp infectious dose that will result in 50 percent infected shrimp. L.D.50 is the single dose that will cause death in 50 percent of a group of test animals.

RNAi against four WSSV genes – vp26, vp28, vp191 and orf89 – was produced as dsRNA using a commercial transcription kit following the manufacturer instructions. Table 1 includes information on the specific primers and standard PCR conditions used to amplify the genes.

– See more at: http://advocate.gaalliance.org/double-stranded-rna-against-wssv-genes-provides-antiviral-protection-in-shrimp-2/#sthash.JkY4kR3n.dpuf

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