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Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

University of Guelph

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.

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

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.

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.

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.

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

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.

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

Fusarium solani haplotype 12-b and aortic and branchial arteritis in Hippocampus erectus Perry.

Aquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation.

Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines.

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p < .05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1.

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.

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

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Magnesium is involved in a variety of physiological processes in marine animals and is known to be deleterious in both excess and deficiency. The effects of magnesium concentration ranging from 700 mg/L (low), 1344 mg/L (control), and 2000 mg/L (high) on size and pulse rate in upside-down jellyfish (Cassiopea andromeda) medusae were examined in two separate 28-day trials. Exposure to low magnesium resulted in significantly (p < .05) higher pulse rates and decreased bell diameter and also produced oral arm degradation. Exposure to high magnesium resulted in significantly (p < .05) lower pulse rates and decreased bell diameter as well as oral arm cupping. In both low and high magnesium, almost all specimens changed color from pale blue on Day 1, to brown by Day 28, suggesting a loss of zooxanthellae. The decrease in bell diameter and color change was more pronounced and occurred more rapidly in low magnesium. The results of both trials demonstrate the deleterious effects of high and low magnesium on C. andromeda and emphasize the importance of monitoring magnesium concentration to maintain healthy display animals in public aquaria.

Magnesium concentration influences size and pulse rate in the upside-down jellyfish, Cassiopea andromeda.

The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Worldwide efforts are being made to develop vaccines to mitigate this pandemic. We engineered two recombinant Newcastle disease virus (NDV) vectors expressing either the full-length SARS-CoV-2 spike protein (NDV-FLS) or a version with a 19 amino acid deletion at the carboxy terminus (NDV-Δ19S). Hamsters receiving two doses (prime-boost) of NDV-FLS developed a robust SARS-CoV-2-neutralizing antibody response, with elimination of infectious virus in the lungs and minimal lung pathology at five days post-challenge. Single-dose vaccination with NDV-FLS significantly reduced SARS-CoV-2 replication in the lungs but only mildly decreased lung inflammation. NDV-Δ19S-treated hamsters had a moderate decrease in SARS-CoV-2 titers in lungs and presented with severe microscopic lesions, suggesting that truncation of the spike protein was a less effective strategy. In summary, NDV-vectored vaccines represent a viable option for protection against COVID-19.

Intranasal vaccination with a Newcastle disease virus-vectored vaccine protects hamsters from SARS-CoV-2 infection and disease.

A study was conducted on 500 juvenile rainbow trout (122 ± 4 g) fed either a control diet or a treatment diet containing 300 mg/kg of a microencapsulated blend of organic acids and essential oils to elucidate effects on intestinal morphology and microbiome. Proximal intestinal villi length was significantly increased in fish fed the treatment diet. Despite no differences in gut inflammation scores, edema, lamina propria inflammation and apoptosis were completely absent in the distal intestine of fish fed the treatment diet. Next-generation sequencing of the 16S rDNA showed no differences in alpha and beta diversity, and gut bacteria were mainly composed of Firmicutes, Bacteroidetes and Proteobacteria. On the genus level, LefSe analysis of indicator OTUs showed , , ,  and  were associated with the control diet whereas ,  and  were associated with the treatment diet.  and  spp. are opportunistic pathogens and several strains have been found to be resistant to antibiotics. The increase in villi length and reduction of specific pathogens indicates that feeding a microencapsulated blend of organic acids and essential oils improves gut health and may serve as a part of an effective strategy to reduce antibiotic use in aquaculture.

Dietary Microencapsulated Blend of Organic Acids and Plant Essential Oils Affects Intestinal Morphology and Microbiome of Rainbow Trout ().

Previously, rainbow trout fed deoxynivalenol (DON) or partially fed (pair-fed) for 4 weeks before and during experimental infection with Flavobacterium psychrophilum had significantly decreased mortality rates. Similar results were obtained in the present study after 12 days, but not after 6 days, feeding 5 ppm DON or pair-fed before infection. Furthermore, feeding 250 ppm chloroquine (CQ) also reduced mortality (p = .052) compared with controls and may have promise for treatment of some fish disease. Parallel groups of fish were maintained on the respective treatments for 15 days, with an additional group that was fasted, but were not infected to monitor autophagy. Fish that were fasted or fed DON had significantly increased LC3II in the liver and fasted fish had significantly decreased LC3II in muscle compared with controls using western blot. There was no difference in LC3II signal in the spleen of any treatment group. Fish that were fasted or pair-fed had significant up-regulation of the Atg genes atg4, atg7, lc3, gabarap and atg12 in muscle using quantitative PCR. Less alteration of Atg expression was seen in liver. Fish treated with CQ had significantly increased expression of atg4, becn1, lc3 and atg12 in the liver. Fish fed DON for 15 days had few alterations of Atg genes in either the liver or muscle. It is still not clear if autophagy is responsible for the resistance of rainbow trout fed DON, CQ or pair-fed before F. psychrophilum infection.

Phuc H. Pham

Department of Pathobiology

University of Guelph

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Phuc H. Pham

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathobiology

University of Guelph

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Department of Pathobiology