Advances in Viral Aquatic Animal Disease Knowledge: The Molecular Methods' Contribution

Viruses are pervasive components of aquatic ecosystems, and most of them are harmless to humans and animals; however, several aquatic viruses can infect animals, leading to diseases, especially when fish are confined, such as in aquaculture facilities. Traditional methods used to detect and study viruses have been widely applied to aquatic animals' viruses, leading to the successful isolation, identification and understanding of several of them. However, they have limits, which can be overcome by molecular methods, such as polymerase chain reaction (PCR)-based assays, sequencing and in situ hybridisation. A standard PCR, followed by the sequencing of purified amplicons, is an effective method for both identifying well-known viruses and discovering new ones. In situ hybridisation, in which a labelled probe binds to a nucleic acid sequence in tissue, is able to correlate the presence of viruses to lesions. Novel molecular isothermal methods, such as loop-mediated isothermal amplification (LAMP), were also developed and applied to viral aquatic animal diseases, bringing molecular diagnosis into the field. This review considers the scientific literature dealing with the molecular methods employed hitherto to study the most relevant finfish and shellfish viral pathogens, stressing their advantages and disadvantages. Aquaculture is the fastest-growing food-producing sector, with a global production of 122.6 million tonnes in 2020. Nonetheless, aquatic animal production can be hampered by the occurrence of viral diseases. Furthermore, intensive farming conditions and an increasing number of reared fish species have boosted the number of aquatic animals' pathogens that researchers have to deal with, requiring the quick development of new detection and study methods for novel unknown pathogens. In this respect, the molecular tools have significantly contributed to investigating thoroughly the structural constituents of fish viruses and providing efficient detection methods. For instance, next-generation sequencing has been crucial in reassignment to the correct taxonomic family, the sturgeon nucleo-cytoplasmic large DNA viruses, a group of viruses historically known, but mistakenly considered as iridoviruses. Further methods such as in situ hybridisation allowed objectifying the role played by the pathogen in the determinism of disease, as the cyprinid herpesvirus 2, ostreid herpesvirus 1 and betanodaviruses. Often, a combination of molecular techniques is crucial to understanding the viral role, especially when the virus is detected in a new aquatic animal species. With this paper, the authors would critically revise the scientific literature, dealing with the molecular techniques employed hitherto to study the most relevant finfish and shellfish viral pathogens.