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29 Apr 2016

Suppression Subtractive Hybridization to Investigate Viruses in the Lymphoid Organ of Penaeus Merguiensis and the Gills of Cherax Quadricarinatus

Rusaini, August 2013
James Cook University

Abstract
A polymerase chain reaction (PCR)-based cDNA subtraction technique termed suppression subtractive hybridization (SSH) was used to investigate the possible viral aetiology of cellular changes in decapod crustacea.  These included hypertrophied nuclei with marginated chromatin (signet rings)but without Cowdry type A (CA) intranuclear inclusion bodies in the gills of freshwater redclaw crayfish Cherax quadricarinatus (Chapter 4), spheroid cells in the lymphoid organ of banana prawn Penaeus merguiensis (Chapter 6) and to identify differential gene expression which were associated with these two cellular changes.  In both cases, viral genomes were not detected in SSH cDNA libraries, but multiple-transcripts were identified being induced in the hypertrophied nuclei population of redclaw crayfish and the hatchery population of banana prawn. These transcripts represented protein related to immunity, proteases and inhibitors, synthesis, processing and regulation-related proteins, structural and cytoskeletal related proteins, energy and metabolism factors, and ribosomal proteins, which are all known to be involved in biological process and defence mechanisms against infectious pathogens, in particular viral diseases.

To investigate the probability of the viral aetiology of the lesions in C. quadricarinatus (Chapter 5) and P. merguiensis(Chapter 6) due to virus with no poly(A) tail, reverse transcriptase (RT)-PCR using primers designed from Bunyaviridae were performed.  However, the results showed those were not the case. The PCR amplification using HPV140F/HPV140R primers revealed the absence of Penaeus merguiensis densovirus (PmergDNV) or related sequences in the redclaw populations (Chapter 5), but suggested the presence of PmergDNV in the hatchery population, while it was undetected in the wild population (Chapter 6).  This suggested that these spheroid cells may be formed as defensive response against the viral infection.

Further studies were conducted to uncover the cause of the signet ring changes in the gills of redclaw crayfish using several parvovirus primers for PCR amplifications (Chapter 5).  Instead of identifying the exogenous viral sequences, the presence of endogenous Brevidensovirus-like elements (EBreVEs) were detected for the first time in C. quadricarinatus.  Nine fragments that can be assembled into four consensus sequences were found from different sources of crayfish suggesting the widespread nature of these elements in C. quadricarinatus populations in northern Queensland, Australia.  The most remarkable feature of these elements is that they are located in the same region relative to IHNNV sequences and most likely have originated from the non-structural protein of ancestral virus.  Even though definitive insertion sites could not be determined, probably most of these elements, if not all, are randomly inserted within the mobile elements (microsatellites) of the host genomes.  In addition, the presence of these endogenous virus-like elements may have immunological function for the host through RNAi pathway against infection of the more closely related exogenous viruses.  Taken together, these studies have provided an insight into the host-viral interaction at the molecular level.  This knowledge may contribute to future research on crustacean immunity into establishing a holistic approach to combat the devastating impact of infectious diseases, in particular viral pathogens, in order to maintain production in crustacean aquaculture.

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