Assessments of epidemic spread in aquaculture: comparing different scenarios of infectious bacteria incursion through spatiotemporal hybrid modeling

The sustainable development of the aquaculture sector is at risk due to the significant challenges posed by many emerging infectious diseases. While disease prevention and control measures are becoming increasingly critical, there is a dearth of studies on the epidemiological aspects of disease transmission in aquatic ecosystems. This study aims to forecast the spread of a bacterial disease between fish farms in two regions, Romsdalsfjord in Norway and Gujwa in South Korea by applying a DTU-DADS-Aqua spatiotemporal hybrid simulation model. The simulation model assessed the pattern of disease transmission between fish farms under different degrees of transmission power based on the distance between farms (ScalingInf), host susceptibility (RelSusceptibility), the origin site of disease, and the capacity of culling fish. The distance between fish farms was found to have significant associations with disease transmission. In most simulation conditions, the disease transmission between different bay management areas (BMAs) was not evident in Romsdalsfjord. In the Guwja region, where there are relatively narrow distances between fish farms, the spread of infectious disease was greatly affected by ScalingInf. The impact of RelSusceptibility on disease transmission patterns is a critical factor to consider in simulation modeling. When RelSusceptibility ranges from 0.5–1, there is little impact on the likelihood of disease transmission. Conversely, lower ranges (0.2 and 0.05) of RelSusceptibility result in a significant decrease in the area affected by the spread of disease. Eradication measures could control the patterns of infectious disease transmission, but the effectiveness of the depopulation strategy can be dramatically changed depending on the geographical environment. In conclusion, through a comparative analysis of the disease transmission and management scenarios, this study demonstrates the potential use of existing simulation models in predicting the spread of infectious diseases under different epidemiological circumstances and quarantine actions.publishedVersio

Molecular and antigenic characterization of Piscine orthoreovirus (PRV) from rainbow trout (Oncorhynchus mykiss)

Piscine orthoreovirus (PRV-1) causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Recently, a novel PRV (formerly PRV-Om, here called PRV-3), was found in rainbow trout (Oncorhynchus mykiss) with HSMI-like disease. PRV is considered to be an emerging pathogen in farmed salmonids. In this study, molecular and antigenic characterization of PRV-3 was performed. Erythrocytes are the main target cells for PRV, and blood samples that were collected from experimentally challenged fish were used as source of virus. Virus particles were purified by gradient ultracentrifugation and the complete coding sequences of PRV-3 were obtained by Illumina sequencing. When compared to PRV-1, the nucleotide identity of the coding regions was 80.1%, and the amino acid identities of the predicted PRV-3 proteins varied from 96.7% (λ1) to 79.1% (σ3). Phylogenetic analysis showed that PRV-3 belongs to a separate cluster. The region encoding σ3 were sequenced from PRV-3 isolates collected from rainbow trout in Europe. These sequences clustered together, but were distant from PRV-3 that was isolated from rainbow trout in Norway. Bioinformatic analyses of PRV-3 proteins revealed that predicted secondary structures and functional domains were conserved between PRV-3 and PRV-1. Rabbit antisera raised against purified virus or various recombinant virus proteins from PRV-1 all cross-reacted with PRV-3. Our findings indicate that despite different species preferences of the PRV subtypes, several genetic, antigenic, and structural properties are conserved between PRV-1 and-3

Long-term persistence of piscine orthoreovirus-1 (PRV-1) infection during the pre-smolt stages of Atlantic salmon in freshwater

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Piscine Orthoreovirus (PRV)-3, but Not PRV-2, Cross-Protects against PRV-1 and Heart and Skeletal Muscle Inflammation in Atlantic Salmon

Heart and skeletal muscle inflammation (HSMI), caused by infection with Piscine orthoreovirus-1 (PRV-1), is a common disease in farmed Atlantic salmon (Salmo salar). Both an inactivated whole virus vaccine and a DNA vaccine have previously been tested experimentally against HSMI and demonstrated to give partial but not full protection. To understand the mechanisms involved in protection against HSMI and evaluate the potential of live attenuated vaccine strategies, we set up a cross-protection experiment using PRV genotypes not associated with disease development in Atlantic salmon. The three known genotypes of PRV differ in their preference of salmonid host species. The main target species for PRV-1 is Atlantic salmon. Coho salmon (Oncorhynchus kisutch) is the target species for PRV-2, where the infection may induce erythrocytic inclusion body syndrome (EIBS). PRV-3 is associated with heart pathology and anemia in rainbow trout, but brown trout (S. trutta) is the likely natural main host species. Here, we tested if primary infection with PRV-2 or PRV-3 in Atlantic salmon could induce protection against secondary PRV-1 infection, in comparison with an adjuvanted, inactivated PRV-1 vaccine. Viral kinetics, production of cross-reactive antibodies, and protection against HSMI were studied. PRV-3, and to a low extent PRV-2, induced antibodies cross-reacting with the PRV-1 σ1 protein, whereas no specific antibodies were detected after vaccination with inactivated PRV-1. Ten weeks after immunization, the fish were challenged through cohabitation with PRV-1-infected shedder fish. A primary PRV-3 infection completely blocked PRV-1 infection, while PRV-2 only reduced PRV-1 infection levels and the severity of HSMI pathology in a few individuals. This study indicates that infection with non-pathogenic, replicating PRV could be a future strategy to protect farmed salmon from HSMI

Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss)

Abstract Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection

Experimental transmission of piscine orthoreovirus-1 (PRV-1) in different life stages of Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)

Piscine orthoreovirus -1 (PRV-1) causes the disease heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, and the virus has been detected in wild anadromous Atlantic salmon and brown trout. However, the infection prevalence, viral kinetics, and disease severity in different life stages of Atlantic salmon and brown trout are unknown. The current study aimed to evaluate and compare susceptibility to PRV-1 infection and development of HSMI in different life stages of anadromous Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). We challenged Atlantic salmon and brown trout fry, parr, and post-smolts with PRV-1 by bath, cohabitation, or IP injection. The kinetics of viral infection and disease development were evaluated by RT-qPCR, in situ hybridization, and histology. Our results indicated that PRV-1 infection prevalence and viral kinetics depend on the developmental stage and challenge method in both Atlantic salmon and brown trout. All developmental stages of Atlantic salmon and brown trout can be infected with PRV-1. However, brown trout showed a lower infection prevalence, with positive cases exhibiting only mild infections without any pathological changes in the target organs, while all life stages of Atlantic salmon developed heart lesions characteristic of HSMI. These results strongly suggest that brown trout are less susceptible to PRV-1 infection than Atlantic salmon and further confirm the species-specific susceptibility and disease development for PRV-1 infection

Molecular characterization of Piscine orthoreovirus (PRV) and its pathogenesis in salmonids

Piscine orthoreovirus (PRV) is a virus of salmonid fish. PRV has been associated with different diseases in various salmonid fish species, including heart and skeletal muscle inflammation (HSMI) in Atlantic salmon and more recently a disease resembling HSMI in rainbow trout. In general, PRV appears to be widely distributed, with detection in diseased as well as non-diseased fish. For some of the diseases, a causative relationship has been established, confirming PRV as the etiological agent of the disease, whereas for others the disease association is questioned. The combination of multiple salmonid species and discovery of multiple PRV variants, compose a complex landscape to study disease association. This calls for basic studies revealing the characteristics of the virus, combined with targeted and controlled experiments to settle disease association. The present thesis focuses on PRV-1 infection of Atlantic salmon and PRV-3 in Rainbow trout. The work encompasses the basic characterization of PRV and provides important information on the pathogenesis and disease association. The first study addressed potential virulence differences between PRV-1 strains infecting Atlantic salmon. A full genome sequence analysis of different PRV-1 strains was conducted. The analysis of HSMI-associated PRV-1 strains and low virulent North American pacific coast (NAPC) isolates revealed connection foremost with genomic segments S1 and M2 and the HSMI trait. Reassortment of these two genomic segments and/or possibly the accumulation of mutations have contributed to the evolution of the virulent strains. Some of the PRV-1 isolates showed segment reassortments, indicating that this mechanism contributes to PRV evolution. PRV strains from archived samples, revived by in vivo propagation and full-genome sequenced, confirmed the presence of different variants of PRV during the pre-HSMI period in Norway. The HSMI associated genotypes are adapted to farmed Atlantic salmon, as indicated by the sequences of S1 and M2 segments being stable for the last 20 years. The second study looked into viral kinetics, the differential peak of PRV RNA and protein during HSMI development. Following experimental infections, irrespective of the challenge method, PRV-1 infection of erythrocytes has a clear peak. The virus particles release from erythrocytes into plasma in large numbers in the peak period and spread to other organs. Cardiomyocytes are particularly permissive for infection with PRV-1, and the resulting immune response to the infected cells causes the typical HSMI histopathological changes, i.e. epicarditis, myocarditis in spongy and compact cardiomyocytes. The immune response clears the virus from the cardiomyocytes. However, the virus persists in erythrocytes as observed by detection of PRV RNA by RT-qPCR and in situ staining. The third study focused on a new PRV variant infecting rainbow trout, which had been associated with an HSMI-like disease. Genetic and antigenic characterization of the virus were conducted to study the relation to PRV-1. Based on full genome sequence analysis, the virus was assessed as a new PRV variant and named PRV-3. The overall nucleotide identity to PRV-1 was 80%. Western blot analysis showed cross-reaction with antibodies raised against PRV-1 proteins for all homolog PRV-3 proteins tested. The antigenic analysis did not indicate that PRV-3 was a new serotype. The protein structure and functions are conserved between PRV-1 and PRV-3. The screening for PRV-3 by RT-qPCR revealed the presence of this virus in Denmark, Italy, Germany, and Scotland. The fourth study continued to focus on PRV-3 and aimed to look at the relationship between PRV-3 and the HSMI-like disease in rainbow trout. A ten-week long cohabitation challenge experiment was conducted to prove a causal relationship with the disease. The virus was purified and shown to be morphologically indistinguishable from PRV-1 in transmission electron microscopy (TEM). The study showed that PRV-3 infection causes inflammation and pathological changes in the heart of both shedders and cohabitants. The viral infection induced innate antiviral immune responses, as measured by gene expression analysis. The PRV-3 infection in rainbow trout showed differential viral kinetics compared to PRV-1 in Atlantic salmon. PRV-3 is cleared in rainbow trout, whereas PRV-1 persists in erythrocytes of Atlantic salmon. To summarize, 1) We have shown that PRV-1 was present in Norway before the report of HSMI appeared. The M2/S1 segment pair links to the evolution of virulence in PRV-1 in Atlantic salmon. 2) The PRV peak in erythrocytes coincides in time with infection of heart and liver. PRV-1 is cleared from cardiomyocytes, but persist in erythrocytes. 3) The genetic and antigenic characterization of PRV from rainbow trout showed close relation to PRV-1. The new virus is a distinct PRV subtype, named as PRV-3. 4) PRV-3 is present in rainbow trout and brown trout in other European countries. 5) Finally, we proved that PRV-3 causes heart pathology in rainbow trout using purified PRV-3 in a challenge study.Indian council of agricultural research (ICAR) through NS-International fellowship program, the research council of Norway (NFR) and European unions’ Horizon 2020 research and innovation program under grant agreement No. 652831 (AQUAEXCEL2020

Dissemination of Piscine orthoreovirus-1 (PRV-1) in Atlantic Salmon (Salmo salar) during the Early and Regenerating Phases of Infection

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Erythroid Progenitor Cells in Atlantic Salmon (Salmo salar) May Be Persistently and Productively Infected with Piscine Orthoreovirus (PRV)

Piscine orthoreovirus (PRV-1) can cause heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus targets erythrocytes in the acute peak phase, followed by cardiomyocytes, before the infection subsides into persistence. The persistent phase is characterized by high level of viral RNA, but low level of viral protein. The origin and nature of persistent PRV-1 are not clear. Here, we analyzed for viral persistence and activity in various tissues and cell types in experimentally infected Atlantic salmon. Plasma contained PRV-1 genomic dsRNA throughout an 18-week long infection trial, indicating that viral particles are continuously produced and released. The highest level of PRV-1 RNA in the persistent phase was found in kidney. The level of PRV-1 ssRNA transcripts in kidney was significantly higher than that of blood cells in the persistent phase. In-situ hybridization assays confirmed that PRV-1 RNA was present in erythroid progenitor cells, erythrocytes, macrophages, melano-macrophages and in some additional un-characterized cells in kidney. These results show that PRV-1 establishes a productive, persistent infection in Atlantic salmon and that erythrocyte progenitor cells are PRV target cells.publishedVersio

Melanized focal changes in skeletal muscle in farmed Atlantic salmon after natural infection with Piscine orthoreovirus (PRV)

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