Understanding the Molecular Basis of Pathogenesis of White Spot Syndrome Virus

Abstract

The aquaculture industry has a substantial role to play in securing future food supply, but its expansion is currently limited by disease. In shrimp aquaculture White Spot Disease (WSD) outbreaks, caused by White Spot Syndrome Virus (WSSV), are the major limiter as they rapidly culminate in 100% mortality and lack prophylactics or therapeutics. Losses associated with WSD are estimated at $1 billion USD annually. All crustaceans are considered to be susceptible to WSSV but the degree of susceptibility varies dramatically between species from the highly susceptible penaeid shrimp to highly resistant European shore crab (Carcinus maenas), which is capable of harbouring the virus for long periods of time without any disease symptoms. This differential susceptibility between crustacean hosts offers the opportunity to investigate how they differ in their molecular responses to WSSV, in an attempt to identify the molecular mechanisms responsible for susceptibility to this pathogen. The thesis therefore aimed to investigate the host-WSSV molecular interactions occurring within two crustaceans with differing susceptibility to WSD in order to construct hypotheses for the likely molecular pathways that could be responsible for tolerance to WSD in crustaceans. Further, the thesis also explored the role of environmental abiotic factors in WSSV susceptibility. To address these aims I first conducted an in-depth literature review of the impacts of the environment on WSD outbreaks. I identified links between rapidly changing environmental conditions and WSD occurrence and substantial gaps in the study of many single and combined factors including dissolved oxygen, CO2, pH and nitrogen concentrations. Secondly, a WSSV infection trial was carried out in (highly susceptible) Penaeusvannamei and (mRNA and miRNA) transcriptomes produced. Transcriptome analysis of susceptible P. vannamei pointed towards increased susceptibility via rapid subversion of processes to enhance WSSV entry, evasion of cellular recycling components and cellular stress with a clear absence of significant immune responses during the early stages of the disease. During early time points, differentially expressed miRNAs were predicted to regulate cytoskeleton polymerisation, phagocytic activity, osmoregulation and metabolism with a hypothesised role in enhancing WSSV infection. The reduced immune responses observed in shrimp may be partially explained by differentially expressed miRNAs such as pPva-miR-H, which reduced immune- priming transcript expression (Dscam) and pPva-miR-K and –N, which regulated apoptosis at 24 and 36 hours post injection (hpi) to favour the virus. The ability of miRNAs to also enhance nucleotide and lipid metabolism from 24 hpi likely further supports virus replication. The P. vannamei transcriptome was then compared to an equivalent dataset in resistant C. maenas, which showed limited transcriptional responses during early time points. Critical alterations to transcripts involved in endocytosis and innate immunity, suggested that crabs may exhibit an enhanced ability to prevent WSSV entry and clear invading virions and that the associated transcripts may be key to resisting WSD. In particular, the significant upregulation of Cma-miR-92b, which may negatively regulate the translation of viral E3 ligase, immediate-early protein in WSSV-recalcitrant crabs should be explored further. For both susceptible and resistant crustaceans many novel miRNAs were identified, contributing to the current lack of crustacean genomic resources. These require further study to determine their significance in the WSSV infection. Based on the comparison of the transcriptome profiles between these two species with contrasting susceptibility to WSD, I propose the hypothesis that a two-pronged mechanism of reduced virion endocytosis (resulting from the inhibition of internal vesicle budding by dynamin-1), downregulation of transcripts that mediate cellular transport and increased viral clearance in crabs not replicating WSSV (by upregulation of apoptosis-associated transcripts) may underpin the mechanism of WSD resistance in crabs. In contrast, I hypothesise that the increased susceptibility of shrimp to WSD arises due to the suppression of both the innate immune responses during early infection and immune-priming molecule Dscam (by pPva-miR-H) during late infection. Further exploration of these key components in the molecular response to WSSV may lead to potential avenues to explore treatments for this devastating disease in aquaculture.