Aquaculture America 2021

August 11 - 14, 2021

San Antonio, Texas

EXPEDITING VIRAL DISCOVERY IN SHRIMP AQUACULTURE BY COMBINING CONVENTIONAL HISTOPATHOLOGY AND GENOMICS

 
Arun K. Dhar*, Roberto Cruz-Flores, Hung N. Mai
 
Aquaculture Pathology Laboratory
 School of Animal and C omparative B iomedical Sciences
University of Arizona, 1117 E  Lowell St. Tucson, Arizona, USA, 85721
* hungmai@arizona.edu
 

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