Effect of a novel DNA vaccine against pancreas disease caused by salmonid alphavirus subtype 3 in Atlantic salmon (Salmo salar).

Pancreas disease (PD) caused by salmonid alphavirus subtype 3 (SAV3) is a serious disease with large economic impact on farmed Norwegian Atlantic salmon production despite years of use of oil-adjuvanted vaccines against PD (OAVs). In this study, two commercially available PD vaccines, a DNA vaccine (DNAV) and an OAV, were compared in an experimental setting. At approximately 1040° days (dd) at 12 °C post immunization, the fish were challenged with SAV3 by cohabitation 9 days after transfer to sea water. Sampling was done prior to challenge and at 19, 54, and 83 days post-challenge (dpc). When compared to the OAV and control (Saline) groups, the DNAV group had significantly higher SAV3 neutralizing antibody titers after the immunization period, significantly lower SAV3 viremia levels at 19 dpc, significantly reduced transmission of SAV3 to naïve fish in the latter part of the viremic phase, significantly higher weight gain post-challenge, and significantly reduced prevalence and/or severity of SAV-induced morphologic changes in target organs. The DNAV group had also significantly higher post-challenge survival compared to the Saline group, but not to the OAV group. The data suggest that use of DNAV may reduce the economic impact of PD by protecting against destruction of the pancreas tissue and subsequent growth impairment which is the most common and costly clinical outcome of this disease.

Atlantic salmon endothelial cells from the heart were more susceptible than fibroblasts from the bulbus arteriosus to four RNA viruses but protected from two viruses by dsRNA pretreatment.

Heart diseases caused by viruses are major causes of Atlantic salmon aquaculture loss. Two Atlantic salmon cardiovascular cell lines, an endothelial cell line (ASHe) from the heart and a fibroblast cell line (BAASf) from the bulbus arteriosus, were evaluated for their response to four fish viruses, CSV, IPNV, VHSV IVa and VHSV IVb, and the innate immune agonist, double-stranded RNA mimic poly IC. All four viruses caused cytopathic effects in ASHe and BAASf. However, ASHe was more susceptible to all four viruses than BAASf. When comparing between the viruses, ASHe cells were found to be moderately susceptible to CSV and VHSV IVb, but highly susceptible to IPNV and VHSV IVa induced cell death. All four viruses were capable of propagating in the ASHe cell line, leading to increases in virus titre over time. In BAASf, CSV and IPNV produced more than one log increase in titre from initial infection, but VHSV IVb and IVa did not. When looking at the antiviral response of both cell lines, Mx proteins were induced in ASHe and BAASf by poly IC. All four viruses induced Mx proteins in BAASf, while only CSV and VHSV IVb induced Mx proteins in ASHe. IPNV and VHSV IVa suppressed Mx proteins expression in ASHe. Pretreatment of ASHe with poly IC to allow for Mx proteins accumulation protected the culture from subsequent infections with IPNV and VHSV IVa, resulting in delayed cell death, reduced virus titres and reduced viral proteins expression. These data suggest that endothelial cells potentially can serve as points of infections for viruses in the heart and that two of the four viruses, IPNV and VHSV IVa, have mechanisms to avoid or downregulate antiviral responses in ASHe cells. Furthermore, the high susceptibility of the ASHe cell line to IPNV and VHSV IVa can make it a useful tool for studying antiviral compounds against these viruses and for general detection of fish viruses.

Screening of Fish Cell Lines for Piscine Orthoreovirus-1 (PRV-1) Amplification: Identification of the Non-Supportive PRV-1 Invitrome.

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.

VER-155008 induced Hsp70 proteins expression in fish cell cultures while impeding replication of two RNA viruses.

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 °C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 µM VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

Fish viruses stored in RNAlater can remain infectious and even be temporarily protected from inactivation by heat or by tissue homogenates.

RNAlater is a commonly used transport and storage solution for samples collected for fish health investigations, particularly those potentially involving viruses. However, the infectivity of fish viruses after storage in RNAlater have not been determined. Nevertheless, knowledge of pathogen infectivity of preserved samples is crucial for ensuring safe transport and storage protocols. Therefore, the infectivity of three fish RNA viruses in RNAlater was examined at four temperatures: -80 °C, 4 °C, room temperature (RT, approximately 22 °C) and 37 °C. The viruses were viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV) and chum salmon reovirus (CSV). Overall, three consistent outcomes were observed. First, all three viruses remained infectious in RNAlater at RT or lower. High log titres of these viruses remained over 30 d of storage in either RNAlater or PBS. Second, RNAlater delayed the thermal inactivation of these viruses when compared to PBS at 37 °C. For VHSV, the titre remained high in RNAlater after one day of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For IPNV, the titre remained high in RNAlater after 30 d of incubation at 37 °C, but was inactivated to below threshold in PBS over the same period. For CSV, the titre was slightly higher in RNAlater than PBS at 37 °C over 7 d, and by day 30, only samples stored in RNAlater proved infectious at titres above the detection threshold. Third, RNAlater delayed the inactivation of these viruses when they were stored together with head kidney homogenates. For VHSV, infectious virus was recovered from samples stored at 4 °C in RNAlater by day 7 of incubation, whereas it was inactivated to below threshold in PBS over the same period. For both IPNV and CSV, infectious virus was recovered from samples stored at 37 °C in RNAlater for 7 d, but not so in PBS. In summary, fish viruses can remain infectious and are even temporarily protected from inactivation while in RNAlater. This makes RNAlater a potentially useful solution for the transport of fish viruses. At the same time, precautionary measures must be taken when transporting potentially infectious samples in RNAlater.

Development of an Atlantic salmon heart endothelial cell line (ASHe) that responds to lysophosphatidic acid (LPA).

As diseases and abnormalities of the heart can interfere with the aquaculture of Atlantic salmon, the heart was investigated as a source of cell lines that could be used to study the cellular basis of these conditions. An Atlantic salmon heart endothelial cell line, ASHe, was developed and characterized for growth properties, endothelial cell characteristics, and responsiveness to lysophosphatidic acid (LPA). AHSe cells stained negative for senescence associated ß-galactosidase and grew well in 10 and 20% FBS/L15 at high cell density, but not in L15 medium supplemented with calf serum. It displayed many endothelial cell-like characteristics including a cobblestone morphology, capillary-like structures formation on Matrigel, and expression of von Willebrand factor and endothelial cell-related tight junction proteins ZO-1, claudin 3, and claudin 5. ASHe cells responded to the cardiovascular modulator, LPA, in two contrasting ways. LPA at 5 and 25 µM inhibited the ability of ASHe cells to heal a wound but stimulated their proliferation, especially as evaluated by colony formation in low-density cultures. The enhancement of proliferation by LPA parallels what has been observed previously in mammalian endothelial cell cultures exposed to LPA, whereas the LPA slowing of ASHe cell migration contrasted with the LPA-enhanced migration of some mammalian cells. Therefore, this cell line is a potentially useful model for future comparative studies on piscine and mammalian cardiovascular cell biology and for studies on diseases of Atlantic salmon in aquaculture.

Bringing plant-based veterinary vaccines to market: Managing regulatory and commercial hurdles.

The production of recombinant vaccines in plants may help to reduce the burden of veterinary diseases, which cause major economic losses and in some cases can affect human health. While there is abundant research in this area, a knowledge gap exists between the ability to create and evaluate plant-based products in the laboratory, and the ability to take these products on a path to commercialization. The current report, arising from a workshop sponsored by an Organisation for Economic Co-operation and Development (OECD) Co-operative Research Programme, addresses this gap by providing guidance in planning for the commercialization of plant-made vaccines for animal use. It includes relevant information on developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval with a focus on Canadian regulations.