Risk of spread of Megalocytivirus pagrus1 (infectious spleen and kidney necrosis virus) from frozen fillets

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Becker, Paul M. Hick, Dorothea Megarani, Hannah Siler, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6816932/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The emergence of infectious spleen and kidney necrosis virus (ISKNV) is a significant threat to global aquatic food security by causing large scale mortality in the aquaculture of tilapia ( Oreochromis niloticus ) and mandarin fish ( Siniperca chuatsi ). ISKNV (Genotype II) is a genogroup of Megalocytivirus pagrus1 , along with RSIV (Genotype I) and TRBIV (Genotype III). Their recent listing as WOAH-notifiable diseases highlights the need to assess ISKNV spread pathways to support quantitative risk assessments to prevent exotic pathogen incursions. The objectives were to evaluate the risk of ISKNV introduction from the trade in frozen seafood products by determining viability after freezing and the median infectious dose (ID 50 ). An albino rainbow shark ( Epalzeorhynchos frenatum ) challenge model was used with juvenile fish held at 27°C in an aerated freshwater flow-through aquaculture system. Six donor fish were injected with ISKNV with tissues collected after clinical signs appeared and used immediately or stored at –20 °C for seven days. Tissue pools (challenge inocula) were prepared consisting of snout, eyes, and brain (Pool A), caudal skin and muscle (Pool B), or peritoneal viscera (Pool C). Naïve albino rainbow sharks were challenged by intraperitoneal (IP) injection with a clarified tissue homogenate or by bath immersion for one hour. Each treatment group was completed in triplicate, with the frozen immersion bath treatment repeated six times. Fish were sampled for the detection of ISKNV by qPCR at the time of morbidity/death or on day 14. Negative control fish all survived without detection of ISKNV. All tissue pools caused infection and disease via IP injection or immersion, whether used fresh or frozen, showing ISKNV remains infectious after seven days at −20 °C. From dose titration experiments by IP injection, the ID 50 was 42 ISKNV genome equivalents (95% CI: 19-98) estimated by probit regression. This study is the first to investigate the potential for ISKNV spread via frozen fish fillets, a commodity frequently traded in international markets. The findings provide evidence to inform import risk assessments and highlight the need for further investigation into spread pathways involving uncooked, frozen fish products. Biological sciences/Microbiology/Virology Biological sciences/Zoology/Ichthyology RSIV transboundary disease risk Iridoviridae frozen seafood ornamental fish epidemiology ID50 food security Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Globally, aquatic foods from wild-harvest and farmed sources are one of the most heavily traded food commodities, with an estimated annual value of $ 190 billion 1 . Generally, it is recognized that aquatic foods are vital to food and nutrition security by providing 15% of the global animal protein supply, and this proportion reaches over 50% in some Asian and African countries 2 , 3 . However, significant and enduring challenges to the global aquatic food supply are threatening food and nutrition security, which include the COVID-19 pandemic, the war in Ukraine, escalating energy costs, climate change, and disease outbreaks 2 – 6 . A recent economic analysis of the impact of the COVID-19 pandemic showed that the global seafood trade markets have a lower resilience to trade disruptions compared to other sectors within the agri-food industry 6 . By 2030, economic forecasts have predicted that hundreds of millions of people living in South Asia and Africa will be unable to meet their aquatic food consumption needs due to expected population growth and the lingering effects of the COVID-19 pandemic 6 . The synergistic negative impacts of climate change on aquatic species distributions and abundance, and on the frequency and severity of aquatic disease outbreaks have long been recognized as a key threat to food security 7 . Internationally relevant animal diseases are regulated through the World Organization for Animal Health (WOAH) to support the fair and safe trade of live animals and animal products and to protect animal health and welfare. Frozen seafood products for human consumption are generally considered to pose a substantially lower risk for pathogen transmission compared to live animal movements 8 . However, evidence suggests that transboundary spread has occurred for WOAH-notifiable pathogens through this pathway. Infected viscera from imported rainbow trout ( Oncorhynchus mykiss ) were the likely source of viral haemorrhagic septicaemia virus (VHSV) to the United Kingdom in 2006 9 . Imported uncooked prawn products for human consumption being used as fishing bait have been implicated in the introduction of white spot syndrome virus (WSSV) to Australia 10 . First identified in the early 1990s as the cause of high mortality in marine finfish and initially listed by WOAH as red seabream iridovirus (RSIV) 11 , megalocytiviruses have since been subject to biosecurity and regulatory measures due to their potential impact on aquaculture and wild fish populations 12 . Previously, the exemplar virus species was known as infectious spleen and kidney necrosis viru s (ISKNV) with three recognized genotypes — ISKNV (Genotype II), RSIV (Genotype I), and turbot reddish body iridovirus (TRBIV; Genotype III) 13 — each of which is further divided into two clades 14 . A recent taxonomic revision assigned the binomial name Megalocytivirus pagrus1 to infections caused by any of the genotypes 15 , which enabled a revision to the Aquatic Animal Code to consider the three genotypes as being internationally notifiable to WOAH 12 . Disease associated with M. pagrus1 infections occurs most frequently in Asia 16 in numerous species of freshwater and marine fishes, often resulting in high mortality but subclinical phases can also develop (see recent reviews 17 – 19 ). The three genotypes of M. pagrus1 have a broad and overlapping host range with non-specific clinical signs and significant disease outbreaks have commonly occurred in many important aquaculture species, such as barramundi ( Lates calcarifer ) 20 , 21 , red sea bream ( Pagrus major ), rock bream ( Oplegnathus fasciatus ), tilapia ( Oreochromis niloticus ) 22 – 24 , and grouper ( Epinephelus spp.) 25 . The transboundary spread of the ISKNV genotype has been linked to the international trade of ornamental fish between several continents 26 – 30 . A significant threat to global aquatic food security is the emergence of ISKNV, causing mass mortalities in tilapia aquaculture in Southeast Asia, USA, South America, and Africa 22 – 24 , 31 . Global tilapia aquaculture has grown 11% annually since 1990 to reach 5.2 million tonnes valued at $ 14 billion USD 3 , 32 . Fresh and frozen tilapia fillets are major commodities in international trade, but tariff disputes between the USA (the largest importer) and China (the leading producer and exporter) have reduced profit margins, creating opportunities for other countries such as Colombia, Brazil, and Mexico to expand their exports to the U.S. market 33 . The ISKNV DNA has been detected in frozen seafood imported to Australia, which is considered free of megalocytiviruses 34 , though it is untested if the virus in these products is infectious. As there are no effective vaccines or treatments for ISKNV, risk management and prevention are done through trade restrictions, pathogen surveillance, and biosecurity controls, such as effective disinfection 35 , 36 . Understanding ISKNV spread through frozen fish products and the live fish trade remains a critical knowledge gap in managing disease risks across local, national, and international levels. Therefore, the objectives of this study were to (1) evaluate spread pathways for M. pagrus 1 (ISKNV) through the trade in frozen seafood products and to (2) determine the median infectious dose (ID 50 ) for ISKNV. Results For Trials 1 and 2, all albino rainbow sharks ( Epalzeorhynchos frenatum ) sampled immediately prior to experimentation and negative control groups at the end of the observation periods were confirmed to be negative for ISKNV by qPCR (see Tables 1 and 2 for negative control results). Further, no ectoparasites were observed, and no histopathological changes that would indicate a prior ISKNV infection were observed (Fig. 2 A, 2 C, 2 E present exemplar sections from control fish). Table 1 Quantity of ISKNV in inocula and bioassay outcomes for Trial 1 to evaluate the spread risk of ISKNV from different infected tissue pools with and without freezing at -20°C for 7 days. Treatment group Challenge method ISKNV challenge dose, range a Proportion of fish positive from bioassay b Range of mean virus quantity detected in bioassay c Bioassay outcome Donor fish Sham injection negative 0/2 (0/1) not applicable not applicable ISKNV injection 1 x 10 6 6/6 (3/3) not applicable not applicable Fresh inoculum Pool A injection 2.5 x 10 5 – 1.3 x 10 6 14/15 (3/3) 2.6 x 10 6 – 1.1 x 10 7 positive immersion 5.0 x 10 6 – 2.6 x 10 7 12/15 (3/3) 7.7 x 10 3 – 9.9 x 10 5 positive Pool B injection 7.4 x 10 3 – 1.0 x 10 5 15/15 (3/3) 2.6 x 10 5 – 1.3 x 10 7 positive immersion 1.5 x 10 5 – 2.0 x 10 6 3/15 (2/3) 1.2 x 10–3.1 x 10 positive Pool C injection 2.8 x 10 5 – 7.6 x 10 6 15/15 (3/3) 6.4 x 10 6 – 2 x 10 7 positive immersion 5.5 x 10 6 – 1.5 x 10 8 11/15 (3/3) 4.7 x 10 4 – 8.7 x 10 6 positive Sham Pool C injection negative 0/5 (0/1) negative negative Frozen inoculum Pool A injection 1.1 x 10 5 – 7 x 10 5 14/15 (3/3) 7.8 x 10 5 – 3.4 x 10 6 positive immersion 2.1 x 10 6 – 1.4 x 10 7 17/30 (5/6) 1.8 x 10 4 – 4.4 x 10 6 positive Pool B injection 5.7 x 10 3 – 4.6 x 10 4 14/15 (3/3) 4.0 x 10 6 – 1.3 x 10 7 positive immersion 2.2 x 10 5 – 9.2 x 10 5 10/30 (4/6) 6.4 x 10–3.4 x 10 5 positive Pool C injection 1.7 x 10 5 – 1.0 x 10 6 10/12 (3/3) 2.1 x 10 3 – 1.4 x 10 7 positive immersion 3.3 x 10 6 – 2.0 x 10 7 9/30 (5/6) 1.2 x 10–1.1 x 10 7 positive Sham Pool C injection negative 0/5 (0/1) negative negative a amount of virus in 50 µL injection dose or amount of virus per 1 L of immersion bath for replicate tanks b bracketed number is the proportion of positive replicate tanks in a treatment group c the mean quantity of virus detected in positive fish per tank represented as number of viral genome copies per qPCR reaction Table 2 Titration of ISKNV (isolate EFIV-2018) to determine the median infectious dose. Albino rainbow sharks ( Epalzeorhynchos frenatum ) were challenged by intraperitoneal (IP) injection of a cell culture derived inoculum quantified by qPCR. Data are pooled for each dose which included two replicate aquariums with five injected fish with five naïve fish in cohabitation. The outcome was determined by testing for ISKNV in tissues by qPCR at the time of observing clinical signs and morbidity or the end of the trial (28 days). Experiment Dose (genome copies/fish) Challenge method Proportion moribund Proportion positive by qPCR Quantity of ISKNV DNA (range) a 1 0 (Control) IP 0/9 0/9 negative 10 3 IP 10/10 10/10 1.12 x 10 5 – 3.95 x 10 7 Cohabitation 10/10 10/10 5.16 x 10 4 – 1.21 x 10 6 10 6 IP 10/10 10/10 7.73 x 10 4 – 1.8 x 10 6 Cohabitation 10/10 10/10 6.59 x 10 4 – 3.5 x 10 5 10 8.6 IP 10/10 10/10 3.35 x 10 4 – 4.12 x 10 6 Cohabitation 10/10 10/10 9.09 x 10 4 – 4.52 x 10 6 2 0 (Control) IP 0/10 0/10 Negative 10 1 IP 1/8 1/8 2.79 x 10 4 Cohabitation 0/10 0/10 negative 10 2 IP 7/10 8/10 1.09 x 10–3.15 x 10 8 Cohabitation 3/10 3/10 1.95 x 10 1 -4.84 x 10 3 10 6 IP 9/9 9/9 1.99 x 10 2 -4.61 x 10 8 Cohabitation b 4/6 6/6 9.21 x 10 4 − 3.56 x 10 7 a number of viral copies per qPCR reaction b some fish were lost due to misadventure Trial 1: Risk of ISKNV spread from frozen infected tissue On day 13 after injection, all six donor fish developed clinical signs and were qPCR positive for ISKNV. The quantity of ISKNV (per 50 µL injection), prepared from tissue pools of the donor fish for the injection challenge, ranged from a low of 5.7 x 10³ genome copies in Pool B (skin and muscle) to a high of 7.6 x 10⁶ in Pool C (viscera). The quantity of ISKNV per 1 L of immersion bath, also prepared from tissue pools of the donor fish for the immersion challenge, ranged from a low of 1.5 x 10⁵ genome copies in Pool B (skin and muscle) to a high of 1.5 x 10⁸ in Pool C (viscera) (Table 1 ). Positive bioassays were observed in all cases when fish were subjected to intraperitoneal (IP) or immersion challenge from freshly prepared or frozen tissue from all pools (Table 1 ). For the fresh inocula, all replicate tanks were positive for ISKNV infection except for one tank that received an immersion challenge with Pool B tissues (Table 1 ). All replicate tanks that received an IP challenge with frozen inocula (Pool A, B, and C) were positive. For the immersion baths with frozen inocua, one replicate tank for Pool A and Pool C, and two tanks for Pool B, did not result in ISKNV infection (Table 1 ). The lowest proportion of ISKNV-positive fish (13/45) was observed in tanks challenged with Pool B (skin and muscle) by immersion (fresh and frozen combined). A wide range of virus quantities were observed in moribund fish and those collected on day 14 (final sample day), from a low of 10 0 to a high of 10 7 genome copies per qPCR reaction (Table 1 ). There was no statistical association between the quantity of virus detected and being exposed to fresh or frozen inoculum (p = 0.126) or being sampled at the time of morbidity compared to the final day (p = 0.757) (Fig. 3 ). Histopathological changes consistent with ISKNV infection were observed in fish challenged with frozen tissues (Fig. 4 ). The most affected organ was the spleen, followed by the caudal kidney, cranial kidney, liver, intestines, stomach, heart, gills, and skin. These tissues exhibited large (20–50 µm in diameter), round to polygonal, basophilic to amphophilic hypertrophied cells (megalocytes), with large vesicular or small and densely basophilic nuclei. These cells replaced extensive areas, and in some cases nearly all, of the splenic parenchyma, which also exhibited multifocal areas of necrosis of lymphocytes and hematopoietic precursors. Multifocal to locally extensive areas of the renal interstitium and glomeruli were also expanded and effaced by megalocytes. Smaller numbers of the megalocytes were found in the hepatic parenchyma (occasionally accompanied by single cell necrosis), epicardium and myocardium, gastric and intestinal lamina propria, as well as in the soft tissues of the gills and skin. Trial 2: Median infectious dose (ID) of ISKNV Experiment 1 tested dose ranges from 1 x 10 3 to 1 x 10 8.6 genome copies per injection. This resulted in all fish becoming moribund and succumbing to ISKNV infection for both IP and cohabitation challenge methods (Table 2 ). Histopathological changes associated with ISKNV infection were observed in injected (Fig. 2 B and 2 F) and cohabiting fish (Fig. 2 D). Experiment 2 evaluated exposure to lower doses of ISKNV down to 10 copies per injection. From the 1x 10 1 IP group, one fish died on day 10 post-injection and was ISKNV positive by qPCR. All the cohabitating fish in the 1 x 10 1 group were negative for ISKNV (Table 2 ). The ID 50 for IP injection of ISKNV using this challenge model was 42 ISKNV genome equivalents/fish-injection (95% CI: 19–98) (Fig. 5 ). Discussion The ISKNV genotype of M. pagrus1 emerged in 1994 in the freshwater mandarin fish ( Siniperca chuatsi ) being farmed in China 37 . Known for a wide host range, ISKNV causes high-mortality disease outbreaks in marine and freshwater environments in tropical and temperate regions, negatively impacting both capture fisheries and aquaculture 17 . Tilapia and mandarin fish are fast-growing, affordable freshwater species easily adapted to many farming systems, including polyculture. These two species make up 10.8% (5.7 million tonnes) of global inland aquaculture 3 , 38 , making repeated ISKNV outbreaks a serious threat to food security in Asia, South America, and Africa, where these fish are essential protein sources. The first ISKNV outbreaks in Ghana in 2018–2019 resulted in a 47% reduction in tilapia production, and the virus has spread to nearly all farms in Lake Volta, limiting production 22 , 39 . Risks of transboundary spread for ISKNV continue to be very high with annual emergence in new areas and fish hosts (for recent examples see 40 – 43 ). ISKNV has recently emerged in Western Asia, causing significant mortality in the fourfinger threadfin ( Eleutheronema tetradactylum ) 43 . In November 2021, hatchlings were imported from Singapore, where ISKNV outbreaks are known, to support Kuwait’s developing aquaculture sector. It remains undetermined whether the fish were already infected upon arrival or became infected through local seawater. The recent revision to the WOAH notifiable disease listing 12 to be ‘infection with M. pagrus1 ’, now covering all genotypes, is intended to strengthen efforts to control and prevent the spread of these viruses. However, at the same time, import controls could limit trade from countries with documented ISKNV outbreaks, resulting in food security challenges. ISKNV spreads horizontally, with infectious water alone sufficient to cause disease 36 ; however, there is no evidence of vertical transmission 35 . Within a farm, fomites (e.g., nets and gloves) were identified as an important source of RSIV spread at sea cages in Japan 44 . The current study was the first to test the potential for ISKNV spread associated with frozen fish fillets, a commonly traded product on international markets. With the ISKNV genotype now listed by WOAH, countries can establish regulatory import controls to support freedom declarations and safeguard against introduction. Using the albino rainbow shark model and a natural transmission pathway of bath immersion, we demonstrated that ISKNV remains infectious in fish tissues that were frozen at -20°C for seven days. Positive bioassays were observed in fish challenged with tissues consisting of the anterior section of the fish, including the eye and brain (Pool A), as well as the posterior area of only the caudal skin and muscle (Pool B). Previous research by He et al. 35 showed that partially purified virus prepared from spleen and kidney tissues held at − 20°C for 18 months and injected into mandarin fish caused 100% mortality. Disease caused by ISKNV infection is systemic, with inclusion-bearing cells (megalocytes), considered a sign of active viral replication, commonly observed in the spleen and kidney, as well as the gill, intestine, brain, heart, eye, and fin 45 – 48 . The risk of transboundary spread of aquatic viruses is affected by the interactions of numerous factors relating to virus virulence and persistence within host(s), and stability in the environment 49 . The accuracy of epidemiological simulation models for assessing transmission risks for aquatic viruses requires accurate parameter estimates to support a country in imposing pre- and post-import measures to ensure sanitary trade of fish and fish products within the WOAH guidelines 49 . The present study was the first to estimate the ID 50 to be 42 ISKNV genome equivalents (95% CI: 19–98). This outcome is aligned with a previous study where ISKNV transmission was demonstrated in Murray cod ( Maccullochella peelii ) injected with an ISKNV dose that was below the level of quantification using the Rimmer et al. 50 qPCR assay (estimated to be around 100 copies per mg of fish tissue) 36 . Although the ID 50 for ISKNV was previously unknown, the value determined in this study reflects a non-natural route of exposure (e.g. intraperitoneal injection) using cell culture derived virus. Natural transmission, albeit with inherent challenges in being able to accurately quantify the dose, may result in a different ID 50 . The ID 50 estimated from this study will be used to inform quantitative risk assessments for the spread of M. pagrus1 (ISKNV) and introduction to countries where the virus is considered exotic (e.g. Australia). The three genotypes of M. pagrus1 are considered exotic to Australia and are of high biosecurity concern, with additional controls for imported ornamental fish and seafood products to meet Australia’s acceptable level of protection 51 . Given the potential impacts for large-scale environmental damage if an incursion of M. pagrus1 were to occur, the three genotypes are considered the highest risk on Australia’s national list of environmental pests and pathogens 52 . A previous study identified that imported uncooked seafood could be a potential risk pathway for M. pagrus1 into Australia 34 . ISKNV DNA was detected in uncooked barramundi and golden pomfret (assumed to be Trachinotus blochii ) imported as frozen whole and eviscerated (i.e., gills and viscera removed with head on, eyes and brain intact) seafood for human consumption 34 . Whole and eviscerated fish products are usually further processed in the importing country, generating liquid and solid waste that could be used for crab or lobster bait and fishing berley, posing a risk to fisheries and aquaculture industries 34 , 53 . For this study, to closely mimic post-import processing of frozen seafood, tissues from clinically affected fish were excised and stored at -20°C for one week before pathogenicity testing. The likelihood of the introduction and establishment of an exotic virus is related to the viral load in the traded product, the rate of viral decay or inactivation, and the likelihood that the introduced virus would have sufficient contact with a susceptible host 49 . A study assessing the transboundary spread of tilapia lake virus (TiLV) from frozen tilapia fillets identified that the risk was very low 54 . Freezing tilapia fillets infected with low quantities of TiLV (e.g., 100 and 1000 copies ca.) at -20°C for 14 days inactivated the virus compared to the fresh inocula that caused 30% mortality in a bioassay 54 . Subclinical infections are typical of ISKNV, with little apparent link between viral load and clinical signs, heightening the risk of inadvertently trading infected fish. From our previous studies, we have observed several species of ornamental fish with natural infections that were apparently healthy or moribund with overlapping viral loads from a low range (e.g., 10 2 copies per mg of fish tissue) to a high range (e.g., 10 7 to 10 8 ) 26 . In agreement with this, from the present study, an apparently healthy fish sampled on day 28 following an IP injection with 10 2 (Table 2 ) had an estimated quantity of 1 x 10 5.87 genome copies. A study by Joiner et al. 55 showed that subclinical rainbow trout infected with VHSV had large quantities of virus in the muscle (e.g. 4.17 × 10 7 TCID 50 g − 1 ), and fillet-only seafood processing generated wastewater with virus quantities exceeding 10 4 TCID 50 g − 1 . These virus quantities are equivalent to or higher than the minimum infectious dose (10 4.7 TCID 50 mL − 1 ) for VHSV 56 . Consequently, waste generated from processing imported rainbow trout carcasses with VHSV can carry considerable viral loads and was identified as a potential route of pathogen introduction for the UK 55 . With the revised listing of the ISKNV genotypes of M. pagrus1 as a WOAH-notifiable infection, countries free of the pathogen can put in place import measures following a risk assessment process. Outcomes of this study provide evidence to support an import risk analysis and show there is a strong need to test spread pathways for uncooked frozen fish with lower viral loads and from subclinical hosts. A hindrance to M. pagrus1 research has been the inability to easily culture the viruses in vitro with a limited number of cell lines, which are often hard to obtain 57 . As a result, bioassays remain a reliable, albeit costly and time-consuming model for studying ISKNV infectivity. Given that the median infectious dose for ISKNV is less than 100 genome equivalents per injection, the findings from this and other studies 35 , 36 , 44 suggest that both solid and liquid waste from fish processing could potentially facilitate spread by horizontal transmission of ISKNV. Evaluation of these waste streams as potential pathways for pathogen introduction should be the focus of future research. Methods Source of ISKNV and virus culture The isolate of ISKNV used in this study was EFIV-2018 58 and belongs to ISKNV clade 1 30 . It was obtained in 2018 from a single moribund albino rainbow shark collected from a group of conspecifics experiencing a disease outbreak confirmed to be caused by ISKNV. A frozen aliquot of EFIV-2018 stored in liquid nitrogen was thawed on ice. Then, 1 mL of the virus stock was inoculated into a 25 cm 2 culture flask containing a confluent monolayer of spotted knifejaw (SKF-9) cells 57 , with an incubation period of one hour at room temperature to allow viral attachment. Following the adsorption period, the inoculum was replaced with 5 mL of Eagle’s Minimal Essential Medium (EMEM; Gibco, USA) containing 2% foetal bovine serum (FBS; Gibco) with 1× antibiotic/antimycotic (AA; Gibco), resulting in a final concentration of 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. The cells were observed daily for cytopathic effects (CPE), and after CPE was observed in more than 90% of the cells, the infected cells and spent media were collected, clarified by centrifugation at 4°C at 3000 × g for 10 min, and filtered using a 0.45 µm sterile syringe filter. The clarified supernatant was used as inoculum after quantification by qPCR (as described below). Detection and quantification of ISKNV For the detection and quantification of ISKNV DNA, the partially validated qPCR assay described in Koda et al. (2023) 59 was used. Diagnostic sensitivity and specificity have been determined to be 92% (95% CI: 87.3–95.6) and 89.8% (95% CI: 83.5–94.8), respectively 59 . To determine viral copy number in the clarified supernatant, DNA was extracted by using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. The concentration of the DNA was determined fluorometrically with a Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific). The qPCR assay was performed in a 20 µL reaction mixture, which included 0.9 µM of each forward (SKNV104R-F; 5’-GGCCCTTCAGTTGTATGC-3’) and reverse (SKNV104R-R; 5’-TAGGGCACAGTCCAATGG-3’) primer, 0.25 µM of the probe (ISKNV104R-P; 5’-ACTGTGATTGAAGTCTTC-3’), 4 µL of nucleic acid template (50 ng of total DNA per reaction), 10 µL of universal qPCR mix (TaqMan® Fast Universal PCR Master Mix 2X, Applied Biosystems), and 3 µL of molecular grade water. The amplification was conducted on a QuantStudio 5 Real-Time PCR System (Applied Biosystems) using the following thermocycling conditions: 95°C for 20 s followed by 40 cycles at 95°C for 3 s and 60°C for 30 s. The viral copy number was calculated by comparing the threshold cycle (C t ) value obtained from the samples to a standard curve generated from the known concentrations using the QuantStudio™ Design & Analysis Software v1.4.1. To confirm ISKNV infection and determine the viral copy number in the fish used in the challenge studies, internal tissue samples were collected and pooled by individual fish (brain tissue was collected for testing in Trial 2: experiment 1; all others used kidney, liver, and spleen). Due to the large number of samples, DNA was extracted using the MagMAX DNA Multi-Sample Ultra 2.0 Kit on the KingFisher Flex purification platform, which is designed to efficiently process multiple samples simultaneously. The concentration of the extracted DNA was measured using a Qubit 4.0 Fluorometer, and qPCR was performed as described above. Fish and fish care The bioassay studies were conducted at the University of Florida from October 2023 to July 2024. The use of fish in research was approved by the University of Florida Institutional Animal Care and Use Committee (IACUC protocol #202300000667 and 202400000245). The study protocol adhered to the ARRIVE guidelines as well as the relevant national laws on the protection of animals. Two batches of albino rainbow sharks were obtained in October 2023 (n = 80) and June 2024 (n = 360) from two ornamental fish farms located in Florida, USA. In both instances, fish were collected from rearing ponds, administered with a salt bath at 5 ppt, placed in plastic bags with oxygen, and transported by road to the University of Florida (Gainesville, FL). Upon arrival, fish were equally distributed into two 560 L aquariums and acclimated for 5–7 days. The aquariums contained dechlorinated municipal water on a flow-through system set at four volume changes per hour with constant aeration, and the water temperature was 27 ± 1°C. The photoperiod was set at 12:12 day:night. Fish were fed twice daily to satiation with pelleted feed designed for ornamental fish. Prior to use in experiments, three and 10 fish were randomly selected from each batch, respectively, for a health assessment (examination for ectoparasites and gross pathology) and tissue collection for the detection of ISKNV. All procedures requiring anaesthesia and euthanasia were completed with 100 mg/L and 1000 mg/L of tricaine methanesulfonate (MS-222®, Argent Laboratories, Finquel®), respectively, buffered 1:1 with sodium bicarbonate. Challenge trials were undertaken in a biocontainment aquatic laboratory with chlorination of all effluent water. Trial 1: Risk of ISKNV spread from frozen infected tissue The aim of this trial was to evaluate whether freezing at temperatures used in commercial seafood trade (-20°C) would impact the infectivity of fish tissues containing ISKNV. A schematic overview of the trial design is presented in Fig. 1 . Generation of donor infected and control fish tissues : Six albino rainbow sharks (mean 9.4 g ± 1 SD) received an IP-injection of 50 µL containing 1 x 10 6 ISKNV genome copies. Referred to as donor fish, they were held under the same fish care conditions with daily observation as described above for 13 days. To serve as the negative control, two fish from the same batch received a sham injection of 50 µL containing cell culture medium without virus. On day 13 post-injection, each donor fish was separately euthanized, weighed, measured, and held chilled for dissection. All dissections and tissue collections occurred in a biological safety cabinet that was UV-irradiated and decontaminated with Oxivir Tb (Diversey Inc.) and Eliminase® (Decon Labs). To create each tissue pool (see Fig. 1 A), firstly, the head was severed just anterior to the operculum to include the eye and brain and to exclude the gills (Pool A). Next, the body was dissected posterior to the anus to include only the skin and muscle tissue from the caudal area (Pool B). Thirdly, the body cavity was opened and the visceral organs, including the heart, kidneys, spleen, and gastrointestinal tract, were removed (Pool C). Gills were not included in any tissue pool. A new sterile scalpel blade was used to collect each tissue pool. The tissue collected from each pool was divided approximately in half for either immediate use (referred to as the fresh treatment groups) or placed in a 50 mL Falcon® tube within a polystyrene foam container and held at -20°C for seven days (referred to as the frozen treatment groups). The two sham-injected fish were treated in the same manner, with only tissue Pool C dissected for bioassay testing. Preparation of inocula from tissues : To have sufficient volume of tissue of each pool for the fresh and frozen inoculum preparations, two ISKNV-donor fish were randomly selected and the tissues combined (see Fig. 1 A). There were three replicates for each tissue pool, consisting of combined tissues from two donor fish. During the processing of fresh and frozen inocula, tissues were held at 4°C or on ice. To thaw the frozen tissues to create frozen inoculum, the polystyrene foam container was held at 4°C for two hours prior to use. Briefly, a ratio of 100 mg of tissue was added to 900 µL of Eagle’s MEM (1:10 w/v dilution), homogenized using a disposable pestle, and then clarified by centrifugation at 4°C at 3000 × g for 10 minutes. The clarified supernatant was then filtered using a 0.45 µm sterile syringe filter, and the viral copy number was determined as described above. For IP injections, 50 µL of a 1/10 dilution of the filtered homogenate was administered, while the immersion dose consisted of 1 mL of filtered homogenate per 1 L of bath. Bioassay : For the fresh ISKNV inoculum, each replicate tissue pool was tested using five albino rainbow sharks in one tank using either IP-injection or immersion challenge methods (Fig. 1 B). Similar testing conditions were used for the frozen ISKNV inoculum with two tanks of five fish exposed via immersion and an additional tank containing five fish that were exposed using IP-injection for Pool A and B. Pool C was tested with groups of four fish in a tank for the frozen inoculum IP-injection and two tanks for immersion exposure (Fig. 1 B). The fresh and frozen control inocula for Pool C were treated in the same manner and tested using five fish in one tank (serving as the negative control). Fish were held as described for the general care conditions and observed 2–3 times daily for clinical signs and morbidity. Clinical signs included observation of cutaneous haemorrhages, erratic swimming, irregular operculum movements, anorexia, and loss of buoyancy 36 , 48 . Fish were sampled upon morbidity or on day 14 post-exposure. This endpoint was chosen based on prior knowledge to detect evidence of infection subsequent to ISKNV exposure 36 , 48 . Individually, fish were weighed and measured, and for most fish, the kidney, liver, and spleen were collected as a tissue pool into one tube and placed at -80°C until testing for the detection of ISKNV by qPCR (described above). As appropriate, a few moribund fish were collected for histopathological analysis for disease confirmation. A bioassay was declared positive if one or more replicate tanks had one or more moribund fish positive for ISKNV as detected by the qPCR assay. The bioassay was declared negative if all fish exposed to the treatment appeared healthy and were negative by qPCR. Note, the limit of detection for the qPCR assay is < 10 copies of template per qPCR reaction or 2.5 copies per µL of DNA extract 59 . Trial 2: Median infectious dose of ISKNV The aim of Trial 2 was to determine the median infective dose of ISKNV as assessed by IP injection with confirmation of infection through transmission to cohabitating naive fish. For this experiment, two trials were conducted beginning on 16 November 2023 and 21 June 2024. Fish were randomly selected from the holding aquariums, and treatment groups were randomly allocated to experimental tanks. Fish were held in 38 L tanks under similar conditions as the acclimation period (described above) at 27.5 ± 0.5°C with constant aeration and flow through set at two exchanges of volume per hour. Each treatment group was completed in duplicate and consisted of five IP-injected and five cohabitating unexposed fish. For the treatment groups, the IP-injected fish received a 50 µL injection of EFIV-2018 at a dose of 10 8.6 , 10 6 , and 10 3 genome copies per injection for the first trial, and 10 6 , 10 2 , and 10 1 for the second trial. The person (JB) conducting the injections was blinded to treatment doses and was the same person responsible for the daily care of the fish and determining signs of morbidity. The cohabitating fish were subjected to a small fin clip under anaesthesia for identification and were placed in the tank 24 hours after the IP-injected fish were added. For the negative control, two groups of albino rainbow sharks received an IP injection of 50 µL of Eagle’s MEM without the virus and were held in the same conditions as the exposed fish. Fish were observed 2–3 times daily for the appearance of clinical signs and morbidity for 28 days. Individually, collected fish were weighed and measured, and for most fish, the kidney, liver, and spleen were collected as a tissue pool into one tube and placed at -80°C until testing for the detection of ISKNV by qPCR. As appropriate, one or two moribund fish per treatment group were collected for histopathological analysis for disease confirmation. Histopathology Due to the small size of the fish, whole specimens were fixed in 10% neutral buffered formalin for a minimum of 24 hours. After fixation, the bodies were decalcified in EDTA (0.5 M, pH 8.0) for 48 hours. The tissues were then processed using standard procedures through a series of graded ethanol concentrations, embedded in paraffin wax, and sectioned at 5 µm for hematoxylin and eosin (H&E) staining. Stained sections were examined for general histopathology using light microscopy (Olympus BX41), and images were recorded with an automated upright microscope system (Olympus DP28). Samples were classified as positive or negative based on the presence or absence of megalocytes. Statistical analysis The ID 50 for infection of juvenile albino rainbow shark with ISKNV from tissue culture by IP injection was calculated by probit regression using MedCalc® Statistical Software version 23.2.1 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org ; 2025). Data were derived from two titration experiments (Table 2 ) where the dose was estimated by qPCR quantification of ISKNV DNA in the inoculum. Individual fish were considered to be infected when: they were from an aquarium in which transmission occurred by cohabitation AND they had clinical signs of disease AND ISKNV DNA was detected in tissues OR the quantity of ISKNV DNA detected at the time of disease, or the end of the experiment (28 days) exceeded the amount injected. For Trial 1, linear regression was used to assess the relationship between the quantity of virus detected and the dependant variables of being challenged with fresh or frozen inoculum material and clinical status at time of collection (e.g. moribund or alive on day 14) (Stata 19, StataCorp LLC, College Station, TX, USA). Declarations Competing interests statement The authors declare no conflict of interest. Funding J. Becker was awarded a fellowship from the OECD Co-operative Research Programme: Sustainable Agricultural and Food Systems to support her travel to complete the study. Additional funding was provided by The University of Sydney. Author Contribution JB: Conceptualization; Methodology / Study design; Formal analysis; Investigation; Resources; Data curation; Writing – original draft; Visualization; Project administration; Funding acquisition PH: Conceptualization; Methodology / Study design, Formal analysis DM: Methodology / Study design; InvestigationHS: Formal analysis; Data curation FP: Formal analysis; VisualizationSG: Formal analysis; Data curation KS: Conceptualization; Methodology / Study design; Formal analysis; Investigation; Resources; Data curation; Supervision; Project administration Acknowledgement J. Becker was awarded a fellowship from the OECD Co-operative Research Programme: Sustainable Agricultural and Food Systems to support her travel to complete the study. Additional funding was provided by The University of Sydney. The authors would like to acknowledge the technical contributions from Drs. P. Khrongsee, N. Falconnier, and U. Gottipati. The authors thank Dr. Y. 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Complete genome sequences of infectious spleen and kidney necrosis virus isolated from farmed albino rainbow sharks Epalzeorhynchos frenatum in the United States. Virus Genes . 57 , 448–452. https://doi.org/10.1007/s11262-021-01857-6 (2021). Koda, S. A. et al. Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of infectious spleen and kidney necrosis virus. PLoS One . 18 , e0281292. https://doi.org/10.1371/journal.pone.0281292 (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6816932","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469266788,"identity":"ef6efa12-aafc-459c-9f30-6c1413a6db59","order_by":0,"name":"Joy A. 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Replicates (R) with the same numeral represent tissues that originated from the same two fish. Fish were challenged by intraperitoneal (IP) injection and bath immersion (IMM) with freshly prepared tissues and those prepared after being frozen at -20°C for 7 days. The homogenizing medium (HM) was Eagle’s Minimal Essential Medium.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6816932/v1/3348c8d3eb759dc6c4482378.png"},{"id":84398484,"identity":"15c6f68d-d4c2-4d1f-8d4d-46e0d88aff3d","added_by":"auto","created_at":"2025-06-11 12:56:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":842600,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology of organs of albino rainbow sharks (\u003cem\u003eEpalzeorhynchos frenatum\u003c/em\u003e) infected with ISKNV (\u003cem\u003eMegalocytivirus pagrus1\u003c/em\u003e). (A) Section of exemplar spleen of a fish from the control group (H\u0026amp;E, 400X). (B) Section of spleen of an infected fish showing necrosis and depletion of lymphocytes and hematopoietic precursors, and numerous basophilic hypertrophied cells (megalocytes) scattered throughout the parenchyma (H\u0026amp;E, 400X). (C) Section of exemplar small intestine of a fish from the control group (H\u0026amp;E, 400X). (D) Section of small intestine of an infected fish showing large numbers of megalocytes in the lamina propria (H\u0026amp;E, 400X). (E) Section of exemplar kidney of a fish from the control group (H\u0026amp;E, 400X). (F) Section of kidney of an infected fish showing numerous megalocytes within the glomerulus and in the interstitium, with multifocal small areas of necrosis of hematopoietic cells (H\u0026amp;E, 400X).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6816932/v1/3d3971495d630c6afca33eb5.jpg"},{"id":84398487,"identity":"3d4a6bcd-4d46-4827-9c80-7fb73ea6650d","added_by":"auto","created_at":"2025-06-11 12:56:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69636,"visible":true,"origin":"","legend":"\u003cp\u003eQuantity of ISKNV (viral genome copies per qPCR reaction) for all positive albino rainbow sharks (\u003cem\u003eEpalzeorhynchos frenatum\u003c/em\u003e) that were sampled at the time of morbidity or sampled on day 14 from Trial 1. There was no statistical difference in the quantity of virus detected from dead/moribund fish compared to ones sampled on the final day (p = 0.757), and this was also independent of being exposed to fresh or frozen inocula (p = 0.126).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6816932/v1/031338fa5851c571dd341d8d.png"},{"id":84398485,"identity":"45e0cbb7-2d29-46ad-a6f5-ffea4bb11faa","added_by":"auto","created_at":"2025-06-11 12:56:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":888815,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathology of organs of albino rainbow sharks (\u003cem\u003eEpalzeorhynchos frenatum\u003c/em\u003e) infected with ISKNV following exposure from frozen tissue inocula. (A, B) Section of spleen of an infected fish showing necrosis and depletion of hematopoietic precursors and replacement of the parenchyma by a myriad of basophilic hypertrophied cells (megalocytes) (H\u0026amp;E, 200X and 400X). (C, D) Section of small intestine of an infected fish showing large numbers of megalocytes in the lamina propria. Observe occasional cells with amphophilic cytoplasm and pyknotic nuclei (H\u0026amp;E, 200x and 400X). (E, F) Section of liver of an infected fish showing occasional megalocytes scattered throughout the hepatic parenchyma. Observe the moderate amount of cytoplasm and the macronuclei and macronucleoli of the hypertrophied cells at higher magnification (H\u0026amp;E, 200 x and 400X).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6816932/v1/af38585a4e75311a181e70b6.jpg"},{"id":84398489,"identity":"dff04cb9-8741-416d-8fab-974e77b40237","added_by":"auto","created_at":"2025-06-11 12:56:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3610693,"visible":true,"origin":"","legend":"\u003cp\u003eDose response curve generated from probit regression of \u003cem\u003eMegalocytivirus pagrus1\u003c/em\u003e in albino rainbow sharks (\u003cem\u003eEpalzeorhynchos frenatum\u003c/em\u003e) following intraperitoneal injection with cell culture derived virus. Dashed lines and blue shading depict the 95% confidence interval for the dose corresponding to a particular probability (MedCalc).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6816932/v1/d3e3e646d3ebdf68cde7aec1.png"},{"id":86944085,"identity":"7e7607e2-ac23-4450-914d-8e73a0454e0d","added_by":"auto","created_at":"2025-07-17 12:31:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6454884,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6816932/v1/237400f2-2ed9-4a80-bd0c-14257a419842.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Risk of spread of Megalocytivirus pagrus1 (infectious spleen and kidney necrosis virus) from frozen fillets","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobally, aquatic foods from wild-harvest and farmed sources are one of the most heavily traded food commodities, with an estimated annual value of \u003cspan\u003e$\u003c/span\u003e190 billion\u003csup\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/sup\u003e. Generally, it is recognized that aquatic foods are vital to food and nutrition security by providing 15% of the global animal protein supply, and this proportion reaches over 50% in some Asian and African countries\u003csup\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e \u003c/sup\u003e. However, significant and enduring challenges to the global aquatic food supply are threatening food and nutrition security, which include the COVID-19 pandemic, the war in Ukraine, escalating energy costs, climate change, and disease outbreaks\u003csup\u003e \u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e \u003c/sup\u003e. A recent economic analysis of the impact of the COVID-19 pandemic showed that the global seafood trade markets have a lower resilience to trade disruptions compared to other sectors within the agri-food industry\u003csup\u003e \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e \u003c/sup\u003e. By 2030, economic forecasts have predicted that hundreds of millions of people living in South Asia and Africa will be unable to meet their aquatic food consumption needs due to expected population growth and the lingering effects of the COVID-19 pandemic\u003csup\u003e \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e \u003c/sup\u003e. The synergistic negative impacts of climate change on aquatic species distributions and abundance, and on the frequency and severity of aquatic disease outbreaks have long been recognized as a key threat to food security\u003csup\u003e \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e \u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInternationally relevant animal diseases are regulated through the World Organization for Animal Health (WOAH) to support the fair and safe trade of live animals and animal products and to protect animal health and welfare. Frozen seafood products for human consumption are generally considered to pose a substantially lower risk for pathogen transmission compared to live animal movements\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, evidence suggests that transboundary spread has occurred for WOAH-notifiable pathogens through this pathway. Infected viscera from imported rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) were the likely source of viral haemorrhagic septicaemia virus (VHSV) to the United Kingdom in 2006\u003csup\u003e9\u003c/sup\u003e. Imported uncooked prawn products for human consumption being used as fishing bait have been implicated in the introduction of white spot syndrome virus (WSSV) to Australia\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFirst identified in the early 1990s as the cause of high mortality in marine finfish and initially listed by WOAH as red seabream iridovirus (RSIV)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, megalocytiviruses have since been subject to biosecurity and regulatory measures due to their potential impact on aquaculture and wild fish populations\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Previously, the exemplar virus species was known as infectious spleen and kidney necrosis viru\u003cem\u003es\u003c/em\u003e (ISKNV) with three recognized genotypes \u0026mdash; ISKNV (Genotype II), RSIV (Genotype I), and turbot reddish body iridovirus (TRBIV; Genotype III)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e \u0026mdash; each of which is further divided into two clades\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. A recent taxonomic revision assigned the binomial name \u003cem\u003eMegalocytivirus pagrus1\u003c/em\u003e to infections caused by any of the genotypes\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, which enabled a revision to the Aquatic Animal Code to consider the three genotypes as being internationally notifiable to WOAH\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDisease associated with \u003cem\u003eM. pagrus1\u003c/em\u003e infections occurs most frequently in Asia \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e in numerous species of freshwater and marine fishes, often resulting in high mortality but subclinical phases can also develop (see recent reviews\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e). The three genotypes of \u003cem\u003eM. pagrus1\u003c/em\u003e have a broad and overlapping host range with non-specific clinical signs and significant disease outbreaks have commonly occurred in many important aquaculture species, such as barramundi (\u003cem\u003eLates calcarifer\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, red sea bream (\u003cem\u003ePagrus major\u003c/em\u003e), rock bream (\u003cem\u003eOplegnathus fasciatus\u003c/em\u003e), tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e)\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and grouper (\u003cem\u003eEpinephelus\u003c/em\u003e spp.)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The transboundary spread of the ISKNV genotype has been linked to the international trade of ornamental fish between several continents\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e A significant threat to global aquatic food security is the emergence of ISKNV, causing mass mortalities in tilapia aquaculture in Southeast Asia, USA, South America, and Africa\u003csup\u003e \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e \u003c/sup\u003e. Global tilapia aquaculture has grown 11% annually since 1990 to reach 5.2\u0026nbsp;million tonnes valued at \u003cspan\u003e$\u003c/span\u003e14\u0026nbsp;billion USD\u003csup\u003e \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e \u003c/sup\u003e. Fresh and frozen tilapia fillets are major commodities in international trade, but tariff disputes between the USA (the largest importer) and China (the leading producer and exporter) have reduced profit margins, creating opportunities for other countries such as Colombia, Brazil, and Mexico to expand their exports to the U.S. market\u003csup\u003e \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e \u003c/sup\u003e. The ISKNV DNA has been detected in frozen seafood imported to Australia, which is considered free of megalocytiviruses\u003csup\u003e \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e \u003c/sup\u003e, though it is untested if the virus in these products is infectious. As there are no effective vaccines or treatments for ISKNV, risk management and prevention are done through trade restrictions, pathogen surveillance, and biosecurity controls, such as effective disinfection\u003csup\u003e \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e \u003c/sup\u003e. Understanding ISKNV spread through frozen fish products and the live fish trade remains a critical knowledge gap in managing disease risks across local, national, and international levels. Therefore, the objectives of this study were to (1) evaluate spread pathways for \u003cem\u003eM. pagrus\u003c/em\u003e1 (ISKNV) through the trade in frozen seafood products and to (2) determine the median infectious dose (ID\u003csub\u003e50\u003c/sub\u003e) for ISKNV.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFor Trials 1 and 2, all albino rainbow sharks (\u003cem\u003eEpalzeorhynchos frenatum\u003c/em\u003e) sampled immediately prior to experimentation and negative control groups at the end of the observation periods were confirmed to be negative for ISKNV by qPCR (see Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for negative control results). Further, no ectoparasites were observed, and no histopathological changes that would indicate a prior ISKNV infection were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eE present exemplar sections from control fish).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantity of ISKNV in inocula and bioassay outcomes for Trial 1 to evaluate the spread risk of ISKNV from different infected tissue pools with and without freezing at -20\u0026deg;C for 7 days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003cp\u003egroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChallenge method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eISKNV challenge\u003c/p\u003e \u003cp\u003edose, range\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProportion of fish\u003c/p\u003e \u003cp\u003epositive from bioassay\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRange of mean virus quantity detected in bioassay\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBioassay outcome\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDonor fish\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/2 (0/1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enot applicable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enot applicable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISKNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6/6 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enot applicable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enot applicable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFresh inoculum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePool A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 1.3 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14/15 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.6 x 10\u003csup\u003e6\u003c/sup\u003e \u0026ndash; 1.1 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eimmersion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0 x 10\u003csup\u003e6\u003c/sup\u003e \u0026ndash; 2.6 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12/15 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.7 x 10\u003csup\u003e3\u003c/sup\u003e \u0026ndash; 9.9 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePool B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.4 x 10\u003csup\u003e3\u003c/sup\u003e \u0026ndash; 1.0 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15/15 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.6 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 1.3 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eimmersion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 2.0 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/15 (2/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.2 x 10\u0026ndash;3.1 x 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePool C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 7.6 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15/15 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.4 x 10\u003csup\u003e6\u003c/sup\u003e \u0026ndash; 2 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eimmersion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5 x 10\u003csup\u003e6\u003c/sup\u003e \u0026ndash; 1.5 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11/15 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.7 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 8.7 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSham Pool C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/5 (0/1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFrozen inoculum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePool A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 7 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14/15 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.8 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 3.4 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eimmersion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1 x 10\u003csup\u003e6\u003c/sup\u003e \u0026ndash; 1.4 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17/30 (5/6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.8 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 4.4 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePool B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.7 x 10\u003csup\u003e3\u003c/sup\u003e \u0026ndash; 4.6 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14/15 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.0 x 10\u003csup\u003e6\u003c/sup\u003e \u0026ndash; 1.3 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eimmersion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.2 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 9.2 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/30 (4/6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.4 x 10\u0026ndash;3.4 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePool C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 1.0 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/12 (3/3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.1 x 10\u003csup\u003e3\u003c/sup\u003e \u0026ndash; 1.4 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eimmersion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.3 x 10\u003csup\u003e6\u003c/sup\u003e \u0026ndash; 2.0 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9/30 (5/6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.2 x 10\u0026ndash;1.1 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSham Pool C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einjection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/5 (0/1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003e amount of virus in 50 \u0026micro;L injection dose or amount of virus per 1 L of immersion bath for replicate tanks\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003eb\u003c/sup\u003e bracketed number is the proportion of positive replicate tanks in a treatment group\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ec\u003c/sup\u003e the mean quantity of virus detected in positive fish per tank represented as number of viral genome copies per qPCR reaction\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTitration of ISKNV (isolate EFIV-2018) to determine the median infectious dose. Albino rainbow sharks (\u003cem\u003eEpalzeorhynchos frenatum\u003c/em\u003e) were challenged by intraperitoneal (IP) injection of a cell culture derived inoculum quantified by qPCR. Data are pooled for each dose which included two replicate aquariums with five injected fish with five na\u0026iuml;ve fish in cohabitation. The outcome was determined by testing for ISKNV in tissues by qPCR at the time of observing clinical signs and morbidity or the end of the trial (28 days).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperiment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDose\u003c/p\u003e \u003cp\u003e(genome copies/fish)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChallenge\u003c/p\u003e \u003cp\u003emethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProportion\u003c/p\u003e \u003cp\u003emoribund\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProportion positive by qPCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eQuantity of ISKNV DNA (range)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.12 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 3.95 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCohabitation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.16 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 1.21 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.73 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 1.8 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCohabitation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.59 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 3.5 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e8.6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.35 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 4.12 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCohabitation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.09 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 4.52 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.79 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCohabitation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.09 x 10\u0026ndash;3.15 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCohabitation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.95 x 10\u003csup\u003e1\u003c/sup\u003e -4.84 x 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.99 x 10\u003csup\u003e2\u003c/sup\u003e -4.61 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCohabitation \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4/6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6/6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.21 x 10\u003csup\u003e4\u003c/sup\u003e \u0026minus;\u0026thinsp;3.56 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003e number of viral copies per qPCR reaction\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003eb\u003c/sup\u003e some fish were lost due to misadventure\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTrial 1: Risk of ISKNV spread from frozen infected tissue\u003c/h2\u003e \u003cp\u003eOn day 13 after injection, all six donor fish developed clinical signs and were qPCR positive for ISKNV. The quantity of ISKNV (per 50 \u0026micro;L injection), prepared from tissue pools of the donor fish for the injection challenge, ranged from a low of 5.7 x 10\u0026sup3; genome copies in Pool B (skin and muscle) to a high of 7.6 x 10⁶ in Pool C (viscera). The quantity of ISKNV per 1 L of immersion bath, also prepared from tissue pools of the donor fish for the immersion challenge, ranged from a low of 1.5 x 10⁵ genome copies in Pool B (skin and muscle) to a high of 1.5 x 10⁸ in Pool C (viscera) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Positive bioassays were observed in all cases when fish were subjected to intraperitoneal (IP) or immersion challenge from freshly prepared or frozen tissue from all pools (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For the fresh inocula, all replicate tanks were positive for ISKNV infection except for one tank that received an immersion challenge with Pool B tissues (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All replicate tanks that received an IP challenge with frozen inocula (Pool A, B, and C) were positive. For the immersion baths with frozen inocua, one replicate tank for Pool A and Pool C, and two tanks for Pool B, did not result in ISKNV infection (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The lowest proportion of ISKNV-positive fish (13/45) was observed in tanks challenged with Pool B (skin and muscle) by immersion (fresh and frozen combined). A wide range of virus quantities were observed in moribund fish and those collected on day 14 (final sample day), from a low of 10\u003csup\u003e0\u003c/sup\u003e to a high of 10\u003csup\u003e7\u003c/sup\u003e genome copies per qPCR reaction (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was no statistical association between the quantity of virus detected and being exposed to fresh or frozen inoculum (p\u0026thinsp;=\u0026thinsp;0.126) or being sampled at the time of morbidity compared to the final day (p\u0026thinsp;=\u0026thinsp;0.757) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathological changes consistent with ISKNV infection were observed in fish challenged with frozen tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The most affected organ was the spleen, followed by the caudal kidney, cranial kidney, liver, intestines, stomach, heart, gills, and skin. These tissues exhibited large (20\u0026ndash;50 \u0026micro;m in diameter), round to polygonal, basophilic to amphophilic hypertrophied cells (megalocytes), with large vesicular or small and densely basophilic nuclei. These cells replaced extensive areas, and in some cases nearly all, of the splenic parenchyma, which also exhibited multifocal areas of necrosis of lymphocytes and hematopoietic precursors. Multifocal to locally extensive areas of the renal interstitium and glomeruli were also expanded and effaced by megalocytes. Smaller numbers of the megalocytes were found in the hepatic parenchyma (occasionally accompanied by single cell necrosis), epicardium and myocardium, gastric and intestinal lamina propria, as well as in the soft tissues of the gills and skin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTrial 2: Median infectious dose (ID) of ISKNV\u003c/h3\u003e\n\u003cp\u003eExperiment 1 tested dose ranges from 1 x 10\u003csup\u003e3\u003c/sup\u003e to 1 x 10\u003csup\u003e8.6\u003c/sup\u003e genome copies per injection. This resulted in all fish becoming moribund and succumbing to ISKNV infection for both IP and cohabitation challenge methods (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Histopathological changes associated with ISKNV infection were observed in injected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) and cohabiting fish (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Experiment 2 evaluated exposure to lower doses of ISKNV down to 10 copies per injection. From the 1x 10\u003csup\u003e1\u003c/sup\u003e IP group, one fish died on day 10 post-injection and was ISKNV positive by qPCR. All the cohabitating fish in the 1 x 10\u003csup\u003e1\u003c/sup\u003e group were negative for ISKNV (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The ID\u003csub\u003e50\u003c/sub\u003e for IP injection of ISKNV using this challenge model was 42 ISKNV genome equivalents/fish-injection (95% CI: 19\u0026ndash;98) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ISKNV genotype of \u003cem\u003eM. pagrus1\u003c/em\u003e emerged in 1994 in the freshwater mandarin fish (\u003cem\u003eSiniperca chuatsi\u003c/em\u003e) being farmed in China\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Known for a wide host range, ISKNV causes high-mortality disease outbreaks in marine and freshwater environments in tropical and temperate regions, negatively impacting both capture fisheries and aquaculture\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Tilapia and mandarin fish are fast-growing, affordable freshwater species easily adapted to many farming systems, including polyculture. These two species make up 10.8% (5.7\u0026nbsp;million tonnes) of global inland aquaculture\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, making repeated ISKNV outbreaks a serious threat to food security in Asia, South America, and Africa, where these fish are essential protein sources. The first ISKNV outbreaks in Ghana in 2018\u0026ndash;2019 resulted in a 47% reduction in tilapia production, and the virus has spread to nearly all farms in Lake Volta, limiting production\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Risks of transboundary spread for ISKNV continue to be very high with annual emergence in new areas and fish hosts (for recent examples see \u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e). ISKNV has recently emerged in Western Asia, causing significant mortality in the fourfinger threadfin (\u003cem\u003eEleutheronema tetradactylum\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In November 2021, hatchlings were imported from Singapore, where ISKNV outbreaks are known, to support Kuwait\u0026rsquo;s developing aquaculture sector. It remains undetermined whether the fish were already infected upon arrival or became infected through local seawater. The recent revision to the WOAH notifiable disease listing\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e to be \u0026lsquo;infection with \u003cem\u003eM. pagrus1\u003c/em\u003e\u0026rsquo;, now covering all genotypes, is intended to strengthen efforts to control and prevent the spread of these viruses. However, at the same time, import controls could limit trade from countries with documented ISKNV outbreaks, resulting in food security challenges.\u003c/p\u003e \u003cp\u003eISKNV spreads horizontally, with infectious water alone sufficient to cause disease\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e; however, there is no evidence of vertical transmission\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Within a farm, fomites (e.g., nets and gloves) were identified as an important source of RSIV spread at sea cages in Japan\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The current study was the first to test the potential for ISKNV spread associated with frozen fish fillets, a commonly traded product on international markets. With the ISKNV genotype now listed by WOAH, countries can establish regulatory import controls to support freedom declarations and safeguard against introduction. Using the albino rainbow shark model and a natural transmission pathway of bath immersion, we demonstrated that ISKNV remains infectious in fish tissues that were frozen at -20\u0026deg;C for seven days. Positive bioassays were observed in fish challenged with tissues consisting of the anterior section of the fish, including the eye and brain (Pool A), as well as the posterior area of only the caudal skin and muscle (Pool B). Previous research by He et al.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e showed that partially purified virus prepared from spleen and kidney tissues held at \u0026minus;\u0026thinsp;20\u0026deg;C for 18 months and injected into mandarin fish caused 100% mortality. Disease caused by ISKNV infection is systemic, with inclusion-bearing cells (megalocytes), considered a sign of active viral replication, commonly observed in the spleen and kidney, as well as the gill, intestine, brain, heart, eye, and fin\u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe risk of transboundary spread of aquatic viruses is affected by the interactions of numerous factors relating to virus virulence and persistence within host(s), and stability in the environment\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The accuracy of epidemiological simulation models for assessing transmission risks for aquatic viruses requires accurate parameter estimates to support a country in imposing pre- and post-import measures to ensure sanitary trade of fish and fish products within the WOAH guidelines\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The present study was the first to estimate the ID\u003csub\u003e50\u003c/sub\u003e to be 42 ISKNV genome equivalents (95% CI: 19\u0026ndash;98). This outcome is aligned with a previous study where ISKNV transmission was demonstrated in Murray cod (\u003cem\u003eMaccullochella peelii\u003c/em\u003e) injected with an ISKNV dose that was below the level of quantification using the Rimmer et al.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e qPCR assay (estimated to be around 100 copies per mg of fish tissue)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Although the ID\u003csub\u003e50\u003c/sub\u003e for ISKNV was previously unknown, the value determined in this study reflects a non-natural route of exposure (e.g. intraperitoneal injection) using cell culture derived virus. Natural transmission, albeit with inherent challenges in being able to accurately quantify the dose, may result in a different ID\u003csub\u003e50\u003c/sub\u003e. The ID\u003csub\u003e50\u003c/sub\u003e estimated from this study will be used to inform quantitative risk assessments for the spread of \u003cem\u003eM. pagrus1\u003c/em\u003e (ISKNV) and introduction to countries where the virus is considered exotic (e.g. Australia).\u003c/p\u003e \u003cp\u003eThe three genotypes of \u003cem\u003eM. pagrus1\u003c/em\u003e are considered exotic to Australia and are of high biosecurity concern, with additional controls for imported ornamental fish and seafood products to meet Australia\u0026rsquo;s acceptable level of protection\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Given the potential impacts for large-scale environmental damage if an incursion of \u003cem\u003eM. pagrus1\u003c/em\u003e were to occur, the three genotypes are considered the highest risk on Australia\u0026rsquo;s national list of environmental pests and pathogens\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. A previous study identified that imported uncooked seafood could be a potential risk pathway for \u003cem\u003eM. pagrus1\u003c/em\u003e into Australia\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. ISKNV DNA was detected in uncooked barramundi and golden pomfret (assumed to be \u003cem\u003eTrachinotus blochii\u003c/em\u003e) imported as frozen whole and eviscerated (i.e., gills and viscera removed with head on, eyes and brain intact) seafood for human consumption\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Whole and eviscerated fish products are usually further processed in the importing country, generating liquid and solid waste that could be used for crab or lobster bait and fishing berley, posing a risk to fisheries and aquaculture industries\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. For this study, to closely mimic post-import processing of frozen seafood, tissues from clinically affected fish were excised and stored at -20\u0026deg;C for one week before pathogenicity testing.\u003c/p\u003e \u003cp\u003eThe likelihood of the introduction and establishment of an exotic virus is related to the viral load in the traded product, the rate of viral decay or inactivation, and the likelihood that the introduced virus would have sufficient contact with a susceptible host\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. A study assessing the transboundary spread of tilapia lake virus (TiLV) from frozen tilapia fillets identified that the risk was very low\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Freezing tilapia fillets infected with low quantities of TiLV (e.g., 100 and 1000 copies ca.) at -20\u0026deg;C for 14 days inactivated the virus compared to the fresh inocula that caused 30% mortality in a bioassay\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Subclinical infections are typical of ISKNV, with little apparent link between viral load and clinical signs, heightening the risk of inadvertently trading infected fish. From our previous studies, we have observed several species of ornamental fish with natural infections that were apparently healthy or moribund with overlapping viral loads from a low range (e.g., 10\u003csup\u003e2\u003c/sup\u003e copies per mg of fish tissue) to a high range (e.g., 10\u003csup\u003e7\u003c/sup\u003e to 10\u003csup\u003e8\u003c/sup\u003e)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In agreement with this, from the present study, an apparently healthy fish sampled on day 28 following an IP injection with 10\u003csup\u003e2\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) had an estimated quantity of 1 x 10\u003csup\u003e5.87\u003c/sup\u003e genome copies. A study by Joiner et al.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e showed that subclinical rainbow trout infected with VHSV had large quantities of virus in the muscle (e.g. 4.17 \u0026times; 10 \u003csup\u003e7\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and fillet-only seafood processing generated wastewater with virus quantities exceeding 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These virus quantities are equivalent to or higher than the minimum infectious dose (10\u003csup\u003e4.7\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for VHSV\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Consequently, waste generated from processing imported rainbow trout carcasses with VHSV can carry considerable viral loads and was identified as a potential route of pathogen introduction for the UK\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWith the revised listing of the ISKNV genotypes of \u003cem\u003eM. pagrus1\u003c/em\u003e as a WOAH-notifiable infection, countries free of the pathogen can put in place import measures following a risk assessment process. Outcomes of this study provide evidence to support an import risk analysis and show there is a strong need to test spread pathways for uncooked frozen fish with lower viral loads and from subclinical hosts. A hindrance to \u003cem\u003eM. pagrus1\u003c/em\u003e research has been the inability to easily culture the viruses \u003cem\u003ein vitro\u003c/em\u003e with a limited number of cell lines, which are often hard to obtain\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. As a result, bioassays remain a reliable, albeit costly and time-consuming model for studying ISKNV infectivity. Given that the median infectious dose for ISKNV is less than 100 genome equivalents per injection, the findings from this and other studies\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e suggest that both solid and liquid waste from fish processing could potentially facilitate spread by horizontal transmission of ISKNV. Evaluation of these waste streams as potential pathways for pathogen introduction should be the focus of future research.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSource of ISKNV and virus culture\u003c/h2\u003e \u003cp\u003eThe isolate of ISKNV used in this study was EFIV-2018\u003csup\u003e58\u003c/sup\u003e and belongs to ISKNV clade 1\u003csup\u003e30\u003c/sup\u003e. It was obtained in 2018 from a single moribund albino rainbow shark collected from a group of conspecifics experiencing a disease outbreak confirmed to be caused by ISKNV. A frozen aliquot of EFIV-2018 stored in liquid nitrogen was thawed on ice. Then, 1 mL of the virus stock was inoculated into a 25 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e culture flask containing a confluent monolayer of spotted knifejaw (SKF-9) cells\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, with an incubation period of one hour at room temperature to allow viral attachment. Following the adsorption period, the inoculum was replaced with 5 mL of Eagle\u0026rsquo;s Minimal Essential Medium (EMEM; Gibco, USA) containing 2% foetal bovine serum (FBS; Gibco) with 1\u0026times; antibiotic/antimycotic (AA; Gibco), resulting in a final concentration of 100 units/mL penicillin, 100 \u0026micro;g/mL streptomycin, and 0.25 \u0026micro;g/mL amphotericin B. The cells were observed daily for cytopathic effects (CPE), and after CPE was observed in more than 90% of the cells, the infected cells and spent media were collected, clarified by centrifugation at 4\u0026deg;C at 3000 \u0026times; g for 10 min, and filtered using a 0.45 \u0026micro;m sterile syringe filter. The clarified supernatant was used as inoculum after quantification by qPCR (as described below).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetection and quantification of ISKNV\u003c/h2\u003e \u003cp\u003eFor the detection and quantification of ISKNV DNA, the partially validated qPCR assay described in Koda et al. (2023)\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e was used. Diagnostic sensitivity and specificity have been determined to be 92% (95% CI: 87.3\u0026ndash;95.6) and 89.8% (95% CI: 83.5\u0026ndash;94.8), respectively\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. To determine viral copy number in the clarified supernatant, DNA was extracted by using the DNeasy Blood \u0026amp; Tissue Kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions. The concentration of the DNA was determined fluorometrically with a Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific). The qPCR assay was performed in a 20 \u0026micro;L reaction mixture, which included 0.9 \u0026micro;M of each forward (SKNV104R-F; 5\u0026rsquo;-GGCCCTTCAGTTGTATGC-3\u0026rsquo;) and reverse (SKNV104R-R; 5\u0026rsquo;-TAGGGCACAGTCCAATGG-3\u0026rsquo;) primer, 0.25 \u0026micro;M of the probe (ISKNV104R-P; 5\u0026rsquo;-ACTGTGATTGAAGTCTTC-3\u0026rsquo;), 4 \u0026micro;L of nucleic acid template (50 ng of total DNA per reaction), 10 \u0026micro;L of universal qPCR mix (TaqMan\u0026reg; Fast Universal PCR Master Mix 2X, Applied Biosystems), and 3 \u0026micro;L of molecular grade water. The amplification was conducted on a QuantStudio 5 Real-Time PCR System (Applied Biosystems) using the following thermocycling conditions: 95\u0026deg;C for 20 s followed by 40 cycles at 95\u0026deg;C for 3 s and 60\u0026deg;C for 30 s. The viral copy number was calculated by comparing the threshold cycle (C\u003csub\u003et\u003c/sub\u003e) value obtained from the samples to a standard curve generated from the known concentrations using the QuantStudio\u0026trade; Design \u0026amp; Analysis Software v1.4.1. To confirm ISKNV infection and determine the viral copy number in the fish used in the challenge studies, internal tissue samples were collected and pooled by individual fish (brain tissue was collected for testing in Trial 2: experiment 1; all others used kidney, liver, and spleen). Due to the large number of samples, DNA was extracted using the MagMAX DNA Multi-Sample Ultra 2.0 Kit on the KingFisher Flex purification platform, which is designed to efficiently process multiple samples simultaneously. The concentration of the extracted DNA was measured using a Qubit 4.0 Fluorometer, and qPCR was performed as described above.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFish and fish care\u003c/h3\u003e\n\u003cp\u003eThe bioassay studies were conducted at the University of Florida from October 2023 to July 2024. The use of fish in research was approved by the University of Florida Institutional Animal Care and Use Committee (IACUC protocol #202300000667 and 202400000245). The study protocol adhered to the ARRIVE guidelines as well as the relevant national laws on the protection of animals. Two batches of albino rainbow sharks were obtained in October 2023 (n\u0026thinsp;=\u0026thinsp;80) and June 2024 (n\u0026thinsp;=\u0026thinsp;360) from two ornamental fish farms located in Florida, USA. In both instances, fish were collected from rearing ponds, administered with a salt bath at 5 ppt, placed in plastic bags with oxygen, and transported by road to the University of Florida (Gainesville, FL). Upon arrival, fish were equally distributed into two 560 L aquariums and acclimated for 5\u0026ndash;7 days. The aquariums contained dechlorinated municipal water on a flow-through system set at four volume changes per hour with constant aeration, and the water temperature was 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The photoperiod was set at 12:12 day:night. Fish were fed twice daily to satiation with pelleted feed designed for ornamental fish. Prior to use in experiments, three and 10 fish were randomly selected from each batch, respectively, for a health assessment (examination for ectoparasites and gross pathology) and tissue collection for the detection of ISKNV. All procedures requiring anaesthesia and euthanasia were completed with 100 mg/L and 1000 mg/L of tricaine methanesulfonate (MS-222\u0026reg;, Argent Laboratories, Finquel\u0026reg;), respectively, buffered 1:1 with sodium bicarbonate. Challenge trials were undertaken in a biocontainment aquatic laboratory with chlorination of all effluent water.\u003c/p\u003e\n\u003ch3\u003eTrial 1: Risk of ISKNV spread from frozen infected tissue\u003c/h3\u003e\n\u003cp\u003eThe aim of this trial was to evaluate whether freezing at temperatures used in commercial seafood trade (-20\u0026deg;C) would impact the infectivity of fish tissues containing ISKNV. A schematic overview of the trial design is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGeneration of donor infected and control fish tissues\u003c/span\u003e: Six albino rainbow sharks (mean 9.4 g\u0026thinsp;\u0026plusmn;\u0026thinsp;1 SD) received an IP-injection of 50 \u0026micro;L containing 1 x 10\u003csup\u003e6\u003c/sup\u003e ISKNV genome copies. Referred to as donor fish, they were held under the same fish care conditions with daily observation as described above for 13 days. To serve as the negative control, two fish from the same batch received a sham injection of 50 \u0026micro;L containing cell culture medium without virus. On day 13 post-injection, each donor fish was separately euthanized, weighed, measured, and held chilled for dissection. All dissections and tissue collections occurred in a biological safety cabinet that was UV-irradiated and decontaminated with Oxivir Tb (Diversey Inc.) and Eliminase\u0026reg; (Decon Labs). To create each tissue pool (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), firstly, the head was severed just anterior to the operculum to include the eye and brain and to exclude the gills (Pool A). Next, the body was dissected posterior to the anus to include only the skin and muscle tissue from the caudal area (Pool B). Thirdly, the body cavity was opened and the visceral organs, including the heart, kidneys, spleen, and gastrointestinal tract, were removed (Pool C). Gills were not included in any tissue pool. A new sterile scalpel blade was used to collect each tissue pool. The tissue collected from each pool was divided approximately in half for either immediate use (referred to as the fresh treatment groups) or placed in a 50 mL Falcon\u0026reg; tube within a polystyrene foam container and held at -20\u0026deg;C for seven days (referred to as the frozen treatment groups). The two sham-injected fish were treated in the same manner, with only tissue Pool C dissected for bioassay testing.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePreparation of inocula from tissues\u003c/span\u003e: To have sufficient volume of tissue of each pool for the fresh and frozen inoculum preparations, two ISKNV-donor fish were randomly selected and the tissues combined (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). There were three replicates for each tissue pool, consisting of combined tissues from two donor fish. During the processing of fresh and frozen inocula, tissues were held at 4\u0026deg;C or on ice. To thaw the frozen tissues to create frozen inoculum, the polystyrene foam container was held at 4\u0026deg;C for two hours prior to use. Briefly, a ratio of 100 mg of tissue was added to 900 \u0026micro;L of Eagle\u0026rsquo;s MEM (1:10 w/v dilution), homogenized using a disposable pestle, and then clarified by centrifugation at 4\u0026deg;C at 3000 \u0026times; g for 10 minutes. The clarified supernatant was then filtered using a 0.45 \u0026micro;m sterile syringe filter, and the viral copy number was determined as described above. For IP injections, 50 \u0026micro;L of a 1/10 dilution of the filtered homogenate was administered, while the immersion dose consisted of 1 mL of filtered homogenate per 1 L of bath.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBioassay\u003c/span\u003e: For the fresh ISKNV inoculum, each replicate tissue pool was tested using five albino rainbow sharks in one tank using either IP-injection or immersion challenge methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similar testing conditions were used for the frozen ISKNV inoculum with two tanks of five fish exposed via immersion and an additional tank containing five fish that were exposed using IP-injection for Pool A and B. Pool C was tested with groups of four fish in a tank for the frozen inoculum IP-injection and two tanks for immersion exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The fresh and frozen control inocula for Pool C were treated in the same manner and tested using five fish in one tank (serving as the negative control).\u003c/p\u003e \u003cp\u003eFish were held as described for the general care conditions and observed 2\u0026ndash;3 times daily for clinical signs and morbidity. Clinical signs included observation of cutaneous haemorrhages, erratic swimming, irregular operculum movements, anorexia, and loss of buoyancy\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Fish were sampled upon morbidity or on day 14 post-exposure. This endpoint was chosen based on prior knowledge to detect evidence of infection subsequent to ISKNV exposure\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Individually, fish were weighed and measured, and for most fish, the kidney, liver, and spleen were collected as a tissue pool into one tube and placed at -80\u0026deg;C until testing for the detection of ISKNV by qPCR (described above). As appropriate, a few moribund fish were collected for histopathological analysis for disease confirmation. A bioassay was declared positive if one or more replicate tanks had one or more moribund fish positive for ISKNV as detected by the qPCR assay. The bioassay was declared negative if all fish exposed to the treatment appeared healthy and were negative by qPCR. Note, the limit of detection for the qPCR assay is \u0026lt;\u0026thinsp;10 copies of template per qPCR reaction or 2.5 copies per \u0026micro;L of DNA extract\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTrial 2: Median infectious dose of ISKNV\u003c/h2\u003e \u003cp\u003eThe aim of Trial 2 was to determine the median infective dose of ISKNV as assessed by IP injection with confirmation of infection through transmission to cohabitating naive fish. For this experiment, two trials were conducted beginning on 16 November 2023 and 21 June 2024. Fish were randomly selected from the holding aquariums, and treatment groups were randomly allocated to experimental tanks. Fish were held in 38 L tanks under similar conditions as the acclimation period (described above) at 27.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C with constant aeration and flow through set at two exchanges of volume per hour. Each treatment group was completed in duplicate and consisted of five IP-injected and five cohabitating unexposed fish. For the treatment groups, the IP-injected fish received a 50 \u0026micro;L injection of EFIV-2018 at a dose of 10\u003csup\u003e8.6\u003c/sup\u003e, 10\u003csup\u003e6\u003c/sup\u003e, and 10\u003csup\u003e3\u003c/sup\u003e genome copies per injection for the first trial, and 10\u003csup\u003e6\u003c/sup\u003e, 10\u003csup\u003e2\u003c/sup\u003e, and 10\u003csup\u003e1\u003c/sup\u003e for the second trial. The person (JB) conducting the injections was blinded to treatment doses and was the same person responsible for the daily care of the fish and determining signs of morbidity. The cohabitating fish were subjected to a small fin clip under anaesthesia for identification and were placed in the tank 24 hours after the IP-injected fish were added. For the negative control, two groups of albino rainbow sharks received an IP injection of 50 \u0026micro;L of Eagle\u0026rsquo;s MEM without the virus and were held in the same conditions as the exposed fish.\u003c/p\u003e \u003cp\u003eFish were observed 2\u0026ndash;3 times daily for the appearance of clinical signs and morbidity for 28 days. Individually, collected fish were weighed and measured, and for most fish, the kidney, liver, and spleen were collected as a tissue pool into one tube and placed at -80\u0026deg;C until testing for the detection of ISKNV by qPCR. As appropriate, one or two moribund fish per treatment group were collected for histopathological analysis for disease confirmation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eDue to the small size of the fish, whole specimens were fixed in 10% neutral buffered formalin for a minimum of 24 hours. After fixation, the bodies were decalcified in EDTA (0.5 M, pH 8.0) for 48 hours. The tissues were then processed using standard procedures through a series of graded ethanol concentrations, embedded in paraffin wax, and sectioned at 5 \u0026micro;m for hematoxylin and eosin (H\u0026amp;E) staining. Stained sections were examined for general histopathology using light microscopy (Olympus BX41), and images were recorded with an automated upright microscope system (Olympus DP28). Samples were classified as positive or negative based on the presence or absence of megalocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe ID\u003csub\u003e50\u003c/sub\u003e for infection of juvenile albino rainbow shark with ISKNV from tissue culture by IP injection was calculated by probit regression using MedCalc\u0026reg; Statistical Software version 23.2.1 (MedCalc Software Ltd, Ostend, Belgium; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.medcalc.org\u003c/span\u003e\u003cspan address=\"https://www.medcalc.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; 2025). Data were derived from two titration experiments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) where the dose was estimated by qPCR quantification of ISKNV DNA in the inoculum. Individual fish were considered to be infected when: they were from an aquarium in which transmission occurred by cohabitation AND they had clinical signs of disease AND ISKNV DNA was detected in tissues OR the quantity of ISKNV DNA detected at the time of disease, or the end of the experiment (28 days) exceeded the amount injected. For Trial 1, linear regression was used to assess the relationship between the quantity of virus detected and the dependant variables of being challenged with fresh or frozen inoculum material and clinical status at time of collection (e.g. moribund or alive on day 14) (Stata 19, StataCorp LLC, College Station, TX, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests statement\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eJ. Becker was awarded a fellowship from the OECD Co-operative Research Programme: Sustainable Agricultural and Food Systems to support her travel to complete the study. Additional funding was provided by The University of Sydney.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJB: Conceptualization; Methodology / Study design; Formal analysis; Investigation; Resources; Data curation; Writing \u0026ndash; original draft; Visualization; Project administration; Funding acquisition\u003c/p\u003e\n\u003cp\u003ePH: Conceptualization; Methodology / Study design, Formal analysis\u003c/p\u003e\n\u003cp\u003eDM: Methodology / Study design; InvestigationHS: Formal analysis; Data curation\u003c/p\u003e\n\u003cp\u003eFP: Formal analysis; VisualizationSG: Formal analysis; Data curation\u003c/p\u003e\n\u003cp\u003eKS: Conceptualization; Methodology / Study design; Formal analysis; Investigation; Resources; Data curation; Supervision; Project administration\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eJ. Becker was awarded a fellowship from the OECD Co-operative Research Programme: Sustainable Agricultural and Food Systems to support her travel to complete the study. Additional funding was provided by The University of Sydney. The authors would like to acknowledge the technical contributions from Drs. P. Khrongsee, N. Falconnier, and U. Gottipati. The authors thank Dr. Y. Kawato (Fisheries Technology Institute, Japan Fisheries Research and Education Agency) for providing spotted knifejaw (SKF-9) cells.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLem, A., Castro de Souza, M. \u0026amp; Griffin, W. \u003cem\u003eThe Importance of International Trade for Fisheries and Aquaculture Products\u003c/em\u003e (FAO, 2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026eacute;n\u0026eacute;, C. et al. 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Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of infectious spleen and kidney necrosis virus. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e18\u003c/b\u003e, e0281292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0281292\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0281292\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RSIV, transboundary disease risk, Iridoviridae, frozen seafood, ornamental fish, epidemiology, ID50, food security","lastPublishedDoi":"10.21203/rs.3.rs-6816932/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6816932/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe emergence of infectious spleen and kidney necrosis virus (ISKNV) is a significant threat to global aquatic food security by causing large scale mortality in the aquaculture of tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) and mandarin fish (\u003cem\u003eSiniperca chuatsi\u003c/em\u003e). ISKNV (Genotype II) is a genogroup of \u003cem\u003eMegalocytivirus pagrus1\u003c/em\u003e, along with RSIV (Genotype I) and TRBIV (Genotype III). Their recent listing as WOAH-notifiable diseases highlights the need to assess ISKNV spread pathways to support quantitative risk assessments to prevent exotic pathogen incursions. The objectives were to evaluate the risk of ISKNV introduction from the trade in frozen seafood products by determining viability after freezing and the median infectious dose (ID\u003csub\u003e50\u003c/sub\u003e). An albino rainbow shark (\u003cem\u003eEpalzeorhynchos frenatum\u003c/em\u003e) challenge model was used with juvenile fish held at 27°C in an aerated freshwater flow-through aquaculture system. Six donor fish were injected with ISKNV with tissues collected after clinical signs appeared and used immediately or stored at –20 °C for seven days. Tissue pools (challenge inocula) were prepared consisting of snout, eyes, and brain (Pool A), caudal skin and muscle (Pool B), or peritoneal viscera (Pool C). Naïve albino rainbow sharks were challenged by intraperitoneal (IP) injection with a clarified tissue homogenate or by bath immersion for one hour. Each treatment group was completed in triplicate, with the frozen immersion bath treatment repeated six times. Fish were sampled for the detection of ISKNV by qPCR at the time of morbidity/death or on day 14. Negative control fish all survived without detection of ISKNV. All tissue pools caused infection and disease via IP injection or immersion, whether used fresh or frozen, showing ISKNV remains infectious after seven days at −20 °C. From dose titration experiments by IP injection, the ID\u003csub\u003e50\u003c/sub\u003e was 42 ISKNV genome equivalents (95% CI: 19-98) estimated by probit regression. This study is the first to investigate the potential for ISKNV spread via frozen fish fillets, a commodity frequently traded in international markets. The findings provide evidence to inform import risk assessments and highlight the need for further investigation into spread pathways involving uncooked, frozen fish products.\u003c/p\u003e","manuscriptTitle":"Risk of spread of Megalocytivirus pagrus1 (infectious spleen and kidney necrosis virus) from frozen fillets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 12:56:36","doi":"10.21203/rs.3.rs-6816932/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f1fbff2f-a14c-476d-9ea9-83173b2f1558","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49822945,"name":"Biological sciences/Microbiology/Virology"},{"id":49822946,"name":"Biological sciences/Zoology/Ichthyology"}],"tags":[],"updatedAt":"2025-07-17T12:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 12:56:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6816932","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6816932","identity":"rs-6816932","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}