Infectious diseases are a threat to sustainab ility of shrimp aquaculture worldwide. The growth and profitability of shrimp industry has been punctuated with disease outbreak periodically. In the vast majority of cases, the etiologic agent of a disease is discovered only after the disease has spread beyond its initial geographical origin to across countries and even continents. Routinely the etiologic agent is identified by conducting an experimental bioassay using the suspected pathogen, histopathology, pathogen isolation, and genomic characterization. There is a need to expedite pathogen discovery in order to prevent worldwide spread of shrimp diseases.
We demonstrated the feasibility of combining conventional histopathology and next generation sequencing (NGS) to expedite pathogen discovery using known archived histology tissue blocks of White Spot Syndrome Virus (WSSV) , and Taura syndrome virus (TSV) , infected Penaeus vannamei shrimp (01 year old block for WSSV and 15 year old for TSV) . Th in (5 μm) sections of Davidson´s-fixed paraffin embedded (DFPE) archived shrimp tissues were stained with Hematoxylin and Eosin stain to confirm WSSV and TSV pathognomonic lesions. Parallel tissue sections were taken for isolation of nucleic acid, detecting WSSV and TSV by real-time PCR/ RT-PCR before taking the nucleic acids for NGS. For WSSV, DNA was also isolated from frozen tissue samples of the isolate corresponding to the archived histology block samples and NGS were carried out using the isolated DNA. NGS generated millions of short (~150 bases) sequence reads for both WSSV and TSV infected samples and the sequences were taken for further analysis. Upon subtracting the host genome sequence from the sequence pool, WSSV and TSV genome were reconstructed de novo . For WSSV, mutations (substitutions, deletions and insertions) detected in the de novo assembled genome sequence compared to the reference sequence in the NCBI database were confirmed by amplifying the corresponding regions from the original set of nucleic acid and sequencing the amplicon by Sanger sequencing method. The complete genomes of WSSV and TSV were reconstructed from ~2-year-old and 15 year-old archived tissue blocks. Phylogenetic analysis performed using TSV sequence derived from histology block showed that the de novo assembled genome clustered with homologous sequence deposited in the GenBank databases. This confirmed the authenticity of the genome sequence generated by NGS analysis from histology tissue blocks.
Until now, the use of nucleic acids derived from DFPE shrimp samples has not been tested for downstream high-throughput genomic analysis. The findings reported here demonstrate the utility of archived tissue samples for virus discovery and evolutionary studies involving viral disease in crustaceans. This opens an avenue to expedite pathogen discovery and enhance developing diagnostic tools to prevent the spread of viral diseases worldwide.
Key words: Penaeus vannamei , WSSV, TSV, Next Generation Sequencing, NGS