Infectious salmon anemia (ISA) is a severe disease that affects farmed Atlantic salmon (Salmo salar), causing outbreaks in seawater in most salmon-producing countries worldwide, with particular aggressiveness in southern Chile. The etiological agent of this disease is a virus belonging to the Orthomyxoviridae family, named infectious salmon anemia virus (ISAV). Although it has been suggested that this virus can be vertically transmitted, even in freshwater, there is a lack of compelling experimental evidence to confirm this. Here we demonstrate significant putative viral loads in the ovarian fluid as well as in the eggs of two brood stock female adult specimens that harbored the virus systemically but without clinical signs. The target virus corresponded to a highly polymorphic region 3 (HPR-3) variant, which is known to be virulent in seawater and responsible for recent and past outbreaks of this disease in Chile. Additionally, the virus recovered from the fluid as well as from the interior of the eggs was fully infective to a susceptible fish cell line. To our knowledge, this is the first robust evidence demonstrating mother-to-offspring vertical transmission of the infective virus on the one hand and the asymptomatic transmission of a virulent form of the virus in freshwater fish on the other hand. IMPORTANCEThe robustness of the data presented here will contribute to a better understanding of the biology of the virus but most importantly will constitute a key management tool in the control of an aggressive agent constantly threatening the sustainability of the global salmon industry.

BackgroundThe detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters.ResultsThe in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus.ConclusionsHere, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile.

Genetic reassortment plays an important role in the evolution of several segmented RNA viruses and in the epidemiology of their associated diseases. In particular, orthomyxoviruses show rapid fluctuation in the proportion of viral variants coexisting in an infected individual, especially under strong selective pressure. This is particularly relevant in salmon production carried out under confined and stressful conditions where one of the most feared pathogenic agents is the Infectious Salmon Anemia Virus, an orthomyxovirus family member whose biological behavior is only recently beginning to be understood. Pathogenicity of the virus has been mainly associated with deletions of the HPR region in coding segment 6 and the presence or absence of a specific insertion in a key region in coding segment 5. In this study we report, for the first time in Chile, the coexistence of two variants in fully asymptomatic fish. Of five samples analyzed, two were identified as the non-pathogenic variant, HPR0, and two as the highly pathogenic HPR7b variant, though with no clinical signs detectable in the fish. Interestingly, one of the samples unequivocally carried both variants, again without any clinical signs. Considering that in none of the samples the typical insertion in coding segment 5 was detected, it is our impression that this may represent a shift from the non-pathogenic HPR0 variant towards the highly infective HPR7b variant. If this were the case, the transition may be triggered first by deleting the corresponding sequence of the HPR region of segment 6, followed by the putative insertion in segment 5 to generate a virulent strain.

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