COMPARISON OF THE INTRA-HOST GENETIC VARIABILITY TAURA SYNDROME VIRUS (TSV) IN ACUTE AND CHRONIC INFECTIONS IN LITOPENAEUS VANNAMEI

Jana Moerbe Rocker and Jeffrey M. Lotz*
Department of Coastal Sciences
University of Southern Mississippi
Gulf Coast Research Laboratory
Ocean Springs, MS 39564 USA Geographic genetic variability has been noted in Taura Syndrome Virus (TSV) and this variability has been used in molecular epidemiological studies. However, little is known of the genetic variability that exists within a single host during an infection. Quasispecies is a term originally applied to a mathematical model describing the genetic variability of RNA viral particles within a host. Quasi-species result from a balance between a high mutation rate and selection against the assumed less fit mutations. Due to the lack of a proofreading mechanism in RNA-dependent RNA polymerase (RdRp), the replication of RNA is an inherently error-prone process Therefore, the RNA virus exists within its host as a heterogeneous mixture of sequences: the master sequence (the most common and fittest sequence) and various non-lethal but less fit mutants.

Our objective was to determine whether TSV exists as a quasi-species by characterizing the genetic variability within individual hosts with TSV infections. Further we compared the genetic variation between acutely infected shrimp and chronically infected shrimp. We approached characterizing the genetic variability by cloning and sequencing numerous TSV samples from one infected host. The assumption is that the relative abundance of sequences among the clones reflects the relative abundances of sequences within the host. In order to reduce any sequence errors during the PCR process we used high fidelity polymerases. We employed the CP2 gene, which is used for most geographical genetic characterization, for our quasi-species analysis. Five shrimp with acute infections and five shrimp with chronic infections were used in the analysis. We attempted to clone 25 hemolymph samples from each shrimp for a total of 250 clones. Each clone was sequenced and the sequences were analyzed.

We found that individual shrimp were carrying TSV with considerable sequence variation in the CP2 gene. The mean mutation frequency was 1.7 x 10-4. The mutation frequency in chronic infections (2.18 x 10-4) was higher than in acute infections (1.2 x 10-4).

DETECTION OF SHRIMP TAURA SYNDROME VIRUS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION USING A DESIGNED MULTICHANNEL PORTABLE TURBIDIMETER

Wansadaj Jaroenram*, Assawapong Sappat, Wansika Kiatpathomchai, Tanom Lomas, Adisorn Tuantranont and Timothy Flegel


CENTEX Shrimp and Department of Biotechnology, Faculty of Science, Mahidol University,
Rama 6 Road, Ratchathewi, Bangkok 10400 Thailand. Email kungbtram@gmail.com Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the synthesis of large amounts of DNA in a short time with high specificity. Since a white magnesium pyrophosphate (Mg2P2O7) precipitate is a characteristic by-product of LAMP reactions, a simple turbidimetric, end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) by spectroscopic measurement of the LAMP reaction precipitate. The device incorporated a heating block that maintained the optimal temperature of 63for the duration of the 25 LAMP reaction performed in a 0.2 ml tube. The temperature control and the turbidity measurements of this apparatus were sufficiently uniform for conducting LAMP reactions and monitoring the turbidity. The optimal conditions for TSV-LAMP was 6330 min. Using these conditions, LAMP-turbidity measurement revealed comparable sensitivity to that of LAMP-AGE, LAMP-LFD, nested PCR but showed 100 times greater than one step PCR. Cross reactions with other shrimp viruses as templates was not found, indicating that the LAMP methods were highly specific to TSV. Combining 10 min for nucleic acid preparation by a rapid method with 30 min for LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1 h compared to 4-8 h for the nested PCR. In addition, use of the turbidimeter yielded results immediately at the end of the LAMP reaction, without the need to open the reaction tube (i.e., avoidance of contamination) or to add further reagents. Thus, LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of shrimp viruses in the field without risk of amplicon contamination.