Real-time RT-PCR determination of viral copy number in Penaeus vannamei experimentally infected with Taura syndrome virus

• Published in: Aquaculture 2004-01, Vol.229 (1), p.1-10
• Main Authors: Nunan, L.M., Tang-Nelson, K., Lightner, D.V.
• Format: Article
• Language: English
• Subjects: Animal aquaculture; Animal productions; Aquaculture; Biological and medical sciences; copy number; Fundamental and applied biological sciences. Psychology; General aspects; Infections; Litopenaeus vannamei; Marine; Penaeus; Real-time PCR; Shellfish; Shrimp; Taura syndrome virus; TSV
• ISSN: 0044-8486; 1873-5622
• DOI: 10.1016/S0044-8486(03)00365-X

This study examined the viral copy number as determined by real-time RT-PCR, in different tissue samples from Penaeus vannamei exposed to Taura syndrome virus (TSV). Two routes of exposure, injection and per os, were investigated. Six different body parts from each shrimp were assessed for viral copy numbers. Eight shrimp were analyzed per treatment. In addition, eight specific pathogen free (SPF) P. vannamei were analyzed and served as a negative control. The tissue samples examined included: whole tail muscle, tail muscle (shell removed), gills, pleopods, head (cephalothorax with the hepatopancreas included) and tail fan. The results from these experiments showed a significant level of difference between the SPF and the injection treatments. As was expected, there was also a significant difference between the negative control and the per os treatment groups. There was no significant difference between the viral copy number contained in different body parts from the injection experiment. These results contrasted to the per os results, in which there was a statistically significant difference between tail/gills, tail/head, tail/tail fan, whole tail/tail fan and pleopods/tail fan. The tail samples had the lower viral copy numbers, as did the whole tail and pleopods when compared to the tail fan. The mean viral copy number per nanogram of total RNA ( x̄ cn/ng tRNA) extracted in the injection study ranged from 1.4×10 5 in the gills to 2.3×10 5 in the whole tail. In the per os experiment, the x̄ cn/ng of extracted tRNA ranged from 2.5×10 4 in the tail muscle to 4.3×10 5 in the head. When these values are converted to mean viral copy number per gram ( x̄ cn/g) of tissue, the values increased in range. In the injection study, the x̄ cn/g of tissue ranged from 1.2×10 9 in the tail fan to 7.4×10 9 in the head. These values contrast to the x̄ cn/g of tissue in the per os study in that the lowest value of 1.7×10 8 was in the tail muscle and the highest x̄, 1.7×10 10, was in the head. Overall, all body parts from infected shrimp, regardless of treatment type, quantitatively analyzed by real-time RT-PCR, determined the presence of TSV.