Stability of the WSSV ORF94 VNTR genotype marker during passage in marine shrimp, freshwater crayfish and freshwater prawns.

The white spot syndrome virus (WSSV) genome contains 3 variable number tandem repeat (VNTR) regions, located in open reading frame (ORF) 75, ORF94 and ORF125, which have been employed for molecular epizootiological studies. A previous report suggested that the ORF 94 VNTR is highly unstable, varying in the number of tandem repeats during single passages from shrimp to other crustaceans. As such rapid variations would have profound implications for the interpretation of molecular epizootiological data, we re-examined the stability of the ORF94 VNTR. Two WSSV isolates with different ORF94 VNTR genotypes (TRS5 and TRS7) were obtained from disease outbreaks in farmed black tiger shrimp Penaeus monodon in Indonesia. High titre stocks of each virus were produced by injection in specific pathogen-free (SPF) Pacific white shrimp Litopenaeus vannamei with filtered infected tissue extracts, and the genotypes were confirmed. Each stock (macerated tissue) was then used to feed SPF Pacific white shrimp, freshwater crayfish (Cherax sp.) and freshwater prawns Macrobrachium rosenbergii through 3 successive passages involving alternative hosts at each level. Taqman real-time PCR was conducted on samples from each group to confirm infection and quantify viral genetic loads. ORF94 VNTR genotype analysis conducted on samples from each of the 43 passage groups indicated no variations in the VNTR number in either genotype TRS5 or genotype TRS7. This finding is contrary to the previous report and suggests that ORF94 VNTR are stable during multiple passages in these crustaceans.