COMPLETE GENOME RECONSTRUCTION OF WHITE SPOT SYNDROME VIRUS FROM ARCHIVAL DAVIDSON-FIXED PARRAFFIN EMBEDDED SHRIMP TISSUE

Formalin-fixed paraffin-embedded tissues are a priceless resource for diagnostic laboratories worldwide. However, DNA extracted from these tissues is not optimal for most downstream molecular analysis due to fragmentation and chemical modification. Archival shrimp tissue presents additional challenges due to the use of Davidson´s fixative that contains acetic acid to soften the shrimp exoskeleton. Davidson´s fixative contributes to nucleic acid fragmentation via acid hydrolysis. In this study, a histological analysis was performed using an archived Davidson-fixed paraffin-embedded (DFPE) shrimp tissue that showed a Grade 4 level of WSSV infection. WSSV infection was further confirmed by qPCR, PCR and nested PCR. Using DNA isolated from DFPE sample, up to 1500 bp fragments could be amplified by nested PCR. Subsequently the complete genome of White Spot Syndrome Virus (WSSV) was reconstructed from an archived DFPE sample using Next Generation Sequencing (NGS).

DNA isolated from  DFPE tissue was sequenced by NGS using an Illumina MiSeq platform.  The complete genome reconstruction of WSSV (~305 Kbp) was achieved from both FF and DFPE tissue. Single nucleotide variants, insertion and deletions were compared between the genomes reconstructed from FF, DFPE and the reference WSSV genome sequence in NCBI database . Forty-one mutations were identified in the WSSV genomes from the FF and DFPE that differed from the reference genome. The presence of these mutations was confirmed by Sanger sequencing. This is the first study that has successfully sequenced the complete genome of any shrimp virus from archival DFPE tissue. These finding demonstrate that DFPE shrimp tissue represents an invaluable resource for prospective and retrospective studies, evolutionary studies and pathogen discovery.