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Protective immunity against rock bream iridovirus (RBIV) infection and TLR3-mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus) following poly (I:C) administration
Accepted Manuscript Protective immunity against rock bream iridovirus (RBIV) infection and TLR3mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus) following poly (I:C) administration Myung-Hwa Jung, Sung-Ju Jung PII:
S1050-4648(17)30349-2
DOI:
10.1016/j.fsi.2017.06.026
Reference:
YFSIM 4647
To appear in:
Fish and Shellfish Immunology
Received Date: 22 February 2017 Revised Date:
27 April 2017
Accepted Date: 6 June 2017
Please cite this article as: Jung M-H, Jung S-J, Protective immunity against rock bream iridovirus (RBIV) infection and TLR3-mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus) following poly (I:C) administration, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.06.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Protective immunity against rock bream iridovirus (RBIV) infection and TLR3-
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mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus)
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following poly (I:C) administration
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Myung-Hwa Jung *, Sung-Ju Jung
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Department of Aqualife Medicine, Chonnam National University, Republic of Korea.
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National University, San96-1 Dunduck Dong, Yeosu, Chonnam 550-749, Republic of Korea.
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Tel.: +82-61-659-7175
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Fax: +82-61-659-7179
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E-mail address: [email protected] (M.-H. Jung).
Corresponding author contact information: Department of Aqualife Medicine, Chonnam
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ACCEPTED MANUSCRIPT Abstract
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In this study, we evaluated the potential of poly (I:C) to induce antiviral status for protecting
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rock bream from RBIV infection. Rock bream injected with poly (I:C) at 2 days before
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infection (1.1 × 104) at 20 °C had significantly higher protection with RPS 13.4% and 33.4%
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at 100 and 200 µg/fish, respectively, through 100 days post infection (dpi). The addition of
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boost immunization with poly (I:C) at before/post infection at 20 °C clearly enhanced the
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level of protection showing 33.4% and 60.0% at 100 and 200 µg/fish, respectively. To
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investigate the development of a protective immune response, rock bream were re-infected
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with RBIV (1.1 × 107) at 200 dpi. While 100% of the previously unexposed fish died, 100%
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of the previously infected fish survived. Poly (I:C) induced TLR3 and Mx responses were
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observed at several sampling time points in the spleen, kidney and blood. Moreover,
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significantly high expression levels of IRF3 (2.9- and 3.1-fold at 1 d and 2 days post
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administration (dpa), respectively), ISG15 and PKR expression (5.4- and 10.2-fold at 2 dpa,
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respectively) were observed in the blood, but the expression levels were low in the spleen and
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kidney after poly (I:C) administration. Our results showed the induction of antiviral immune
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responses and indicate the possibility of developing long term preventive measures against
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RBIV using poly (I:C).
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Key word: rock bream, rock bream iridovirus, poly (I:C), protection, TLR3, Mx 2
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1. Introduction Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus [1], causes
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mass mortality in rock bream (Oplegnathus fasciatus) [2]. Since first outbreak, high mortality
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rates have been occurred in rock bream annually due to RBIV. Prophylactic measures to
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control RBIV remain unsolved because of the high susceptibility of rock bream to RBIV [3-
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5]; thus, it has been difficult to develop an effective vaccine [1]. Vaccination is an important
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disease control measure and essential for good farming practices [6-9]. Therefore,
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understanding of host-RBIV interactions will be useful for the future strategies for controlling
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RBIV disease in cultured fish. Hence the development of control and preventive measures
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against RBIV is required to sustain rock bream aquaculture industry.
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The innate immune system is the first line of host defense against pathogens. Toll-like
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receptors (TLRs) are well known innate immune-recognition receptors for microbial
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pathogens [10-11]. TLR3 was identified as a receptor for double-stranded RNA (dsRNA).
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Polyinosinic:polycytidylic acid (poly (I:C)) is a commercially available synthetic analogue
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of dsRNA. Upon binding to TLR3, dsRNA or its synthetic analogue poly (I:C) leads to the
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induction of type I interferon (IFN) and inflammatory cytokine productions [12-14] and it is
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also important for activation of adaptive immunity. Poly (I:C) has been extensively studied
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in fish systems, where its induction of antiviral activities have been shown [15-22].
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Additionally, poly (I:C) has been shown to induce disease resistance against fish viruses
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such as infectious haematopoietic necrosis virus (IHNV) [23], nervous necrosis virus (NNV)
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[24-26] and viral haemorrhagic septicaemia virus (VHSV) [27,28]. However, the
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mechanism by which poly (I:C) functions in fish is unclear, and very little is known about
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the relationship between the poly (I:C) induced antiviral effect and TLR3-mediated type I
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IFN signaling. Moreover, no published reports are available on the systemic protective
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effects of poly (I:C) against RBIV infection in rock bream.
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ACCEPTED MANUSCRIPT Water temperature is known to affect the immune system and susceptibility of fish to virus
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infection [29,30]. In Korea, RBIV outbreaks typically occur in cultured rock bream in the
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summer season, when water temperature range between 23-27 °C [2]. Recently, we reported
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that rock bream mortality due to RBIV infection was dependent on susceptible water
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temperature [4]; fish mortality reaches 100% at water temperature of 29 °C to 20 °C, but
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delayed mortality at the low water temperatures. Furthermore, rock bream infected at
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different concentrations of RBIV (6.7 × 105 – 7.5 × 107 /100 µl) exhibited 100% mortality at
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26 °C with delayed mortality at the low virus concentrations. This findings indicate that the
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water temperature and RBIV infection dose are an important factor to rock bream.
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In the present study, poly (I:C) was evaluated for its antiviral potentials in rock bream under
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different virus replication levels at 26, 23 or 20 °C. The experiment involved the controlled
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variation of water temperatures, different RBIV infection dose, different doses of poly (I:C)
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and also the suitable time of poly (I:C) administration (before or post virus infection).
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Furthermore, the gene expression levels of TLR3, IL1β, IL8, TNFα, IRF3, Mx, ISG15 and
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PKR were assessed by poly (I:C) at different time points in rock bream for molecular
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understanding of enhanced immune protection against RBIV by poly (I:C).
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2. Materials and methods
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2.1. Experimental fish
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Rock bream were obtained from a local farm and reared at the Fisheries Science Institute at
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Chonnam National University. Approximately 1000 fish were maintained in large tanks (10
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ton) with a continuous sea water supply with aeration. The required numbers of fish for the
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experiments were transferred to the experimental facility.
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2.2. Isolation of RBIV 4
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2010 [31], were thawed for the experiment. The MCP gene copies of the original RBIV in
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the supernatant preparations, quantified by using quantitative real time polymerase chain
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reaction (qRT-PCR) was 7.5 × 107/100 µl and at 10, 100, 1000, 10000 and 100000 fold
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serially diluted virus solutions containing 1.1 × 107, 1.2 × 106, 6.7 × 105, 5.6 × 104 and 1.1 ×
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104/100 µl MCP gene copies, respectively.
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2.3. Effect of poly (I:C) on RBIV infection at 26 °C (Experiment I)
Since, optimum RBIV replication water temperature in fish body is 26 °C, this water
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temperature was selected at first to examine the antiviral property of poly (I:C) on RBIV
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infection. Rock bream (10.2 ± 1.4 cm, 17.2 ± 3.0 g) were administered intraperioneally (i.p.)
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with each of the poly (I:C) (Sigma) at 100 and 200 µg/fish or phosphate-buffered saline
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(PBS) as a virus injected control. At 2 days post administration (dpa) of poly (I:C) or PBS,
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the fish were injected with RBIV (100 µl/fish) containing 1.1 × 107 MCP gene copies. Each
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group of 20 fish were maintained at 26 °C for 15 days in the aquaria containing 30 L of UV-
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treated seawater.
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2.4. Analysis of the antiviral effect of poly (I:C) in relation to dose, time and water
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temperature (Experiment II)
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Rock bream are very susceptible to high RBIV dose (1.1 × 107 MCP gene copies) with a
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water temperature at 26 °C. Alterations in experimental conditions were made to determine
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whether a protective immune response could be induced in the surviving fish. To obtain
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survivors, experimental conditions were adjusted as follows; water temperature (3 °C or
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6 °C) and RBIV injection dose (1000-fold) were lower than the experiment I. To examine the
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effect of water temperature and time on the antiviral property of poly (I:C), 15 fish in each 5
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23 °C (100 µg and 200 µg/fish) and two groups at 20 °C (100 µg and 200 µg/fish) water
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temperature were administered i.p. with poly (I:C) and the other two groups were
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administered PBS as virus injected controls at the respective water temperatures. At 2 days
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post administration, the fish were injected with RBIV (100 µl/fish) containing 1.1 × 104 MCP
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gene copies. This group of fish were regarded as the 2 days before infection group.
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Rock bream (9.8 ± 2.2 cm, 16.8 ± 2.5 g) were divided randomly into four groups (15 fish
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per group); two groups at 23 °C and the other two groups at 20 °C were injected with RBIV
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(100 µl/fish) containing 1.1 × 104 MCP gene copies. At 2 days post infection (dpi), the four
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groups were administered i.p. with poly (I:C) at 100 and 200 µg/fish, respectively, these
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groups were regarded 2 days post infection group.
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15 fish in each group (10.4 ± 2.4 cm, 16.9 ± 2.4 g) were divided randomly into four groups
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and administered i.p. with poly (I:C) at the concentration of 100 µg (23 and 20 °C) and 200
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µg/fish (23 and 20 °C). At 2 days post poly (I:C) administration, the fish groups were injected
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with RBIV (100 µl/fish) containing 1.1 × 104 MCP gene copies and then at 2 days post
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infection (4 days post immunization), the fish were boost immunized with a same
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concentration of poly (I:C) as that for the primary immunization protocol; these groups were
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regarded as the boost immunization group. Fish were maintained at 23 and 20 °C for 200
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days in the aquaria containing 30 L of UV-treated seawater. Table 1 summarizes
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experimental conditions.
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2.5. Re-challenge of survivors from Experiment II with RBIV
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The fish that survived from Experiment II at 200 dpi were challenged for second time by an
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injection of 100 µl/fish of RBIV (1.1 × 107 MCP gene copies). Fifteen naive fish (11.2 ± 1.2
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cm, 30.2 ± 1.4 g) from virus injected control group (not previously exposed to the virus) were 6
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injected with RBIV in the same manner as the survivors. All the fish were kept for an
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additional 50 days in the aquaria containing 30 L of UV-treated seawater. Table 1
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summarizes experimental conditions.
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2.6. Effect of poly (I:C) administration on the gene expression
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The immune response effect of poly (I:C) administered rock bream and non-administered
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rock bream was evaluated as follows: Each of 25 fish (10.1 ± 2.2 cm, 17.7 ± 2.0 g) were
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maintained at 20 °C in the aquarium containing 250 L of UV-treated seawater. The fish
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were administered i.p. with poly (I:C) (100 µg/fish) or PBS (100 µl/fish) as control. The
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spleen, kidney and blood were collected from five fish each group at 0 h (before poly (I:C)
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administration), 12 hours, and 1, 2 and 4 days post poly (I:C) administration and flash
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frozen in liquid nitrogen. Samples were stored at –80 °C after being flash frozen in liquid
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nitrogen.
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2.7. Quantitative expression of immune genes and viral copy numbers Real-time PCR was carried out in an Exicycler 96 Real-Time Quantitative Thermal Block
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(Bioneer, Korea) using an AccuPre®2x Greenstar qPCR Master mix (Bioneer, Korea) as
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described previously [31]. The spleen, kidney and blood were obtained from poly (I:C)
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administered fish and PBS injected fish at several sampling points. Each assay was
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performed in duplicate with β-actin RNA as control. The primers used to amplify the
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immune-related genes are listed in Table 2. The quantitation of the mRNA was determined
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by the 2−∆∆Ct method [32].
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For the analysis of the RBIV copy number, genomic DNA was isolated from the whole
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spleen (35–130 mg) of each fish. A standard curve was generated to determine the RBIV
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MCP gene copy number by qRT-PCR as described previously [31]. As MCP-F 5′ 7
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determine the RBIV MCP copy numbers. The virus copy number was determined from 1 µl
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of DNA from 100 µl of total DNA that was extracted from a whole spleen. The detection
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limit of RBIV MCP copy number was 1.0 × 101/µl.
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2.8. Statistical analysis
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The statistical analysis was performed by one-way analysis of variance (ANOVA) using
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GraphPad Prism software version 5.0 for Windows (GraphPad Software, USA) and the
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comparison of gene expressions at different time were obtained by Dunnett’s multiple
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comparison test. A value of p<0.05 was considered to indicate the statistical significance. All
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the data were represented as the means ± the standard error.
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2.9. Relative percent survival (RPS)
The RPS was calculated according to the method described by Amend [33]. RPS = [1-(% mortality of poly (I:C) administered group/ % mortality in control] × 100
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3. Results
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3.1. Effect of poly (I:C) on RBIV infection at 26 °C (Experiment I)
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Rock bream were administered poly (I:C) at 100 and 200 µg/fish, injected with RBIV at 2
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days post poly (I:C) administration, and held 26 °C showed 100% cumulative mortality.
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However, the fish were 3 days to 4 days delayed final mortality as compared to the virus
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injected control fish (Fig. 1). Similar results were obtained in a repeat experiment.
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3.2. Effect of poly (I:C) on RBIV infection at 23 °C (Experiment II)
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days post poly (I:C) administration showed 100% cumulative mortality at 23 °C. However,
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there was a considerable delay in the final mortality compared to the virus injected control
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fish at 6 days (100 µg and 200 µg/fish, respectively) (Fig. 2) (Table 1).
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The fish groups maintained for 2 days at 23 °C with RBIV injection and then administered
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with poly (I:C) at 100 and 200 µg/fish showed 100% cumulative mortality, there was not a
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considerable delay in the final mortality compared to the virus injected control fish (Fig. 2)
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(Table 1).
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The boost immunization group showed 100% cumulative mortality at 23 °C. However,
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there was a considerable delay in the final mortality compared to the virus injected control
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fish at 2 days (100 µg/fish) and 7 days (200 µg/fish) (Fig. 2) (Table 1). Similar results were
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obtained in a repeat experiment.
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3.3. Protection efficacy of poly (I:C) at 20 °C (Experiment II) This experiment was designed to evaluate the efficacy and duration of protection provided
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by administration of the poly (I:C) to rock bream at 100 and 200 µg/fish at 20 °C. At 2 days
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post poly (I:C) administration were exposed to RBIV. The RPS obtained after
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administration was 13.4% (100 µg/fish) and 33.4% (200 µg/fish) for the respective
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administration dose and time points at the end of 200 dpi (Fig. 3) (Table 1).
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The fish groups maintained for 2 days at 20 °C with RBIV injection and then administered
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with poly (I:C) at 100 and 200 µg/fish showed 100% cumulative mortality, and there was a
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considerable delay in the final mortality compared to the virus injected control fish at 4 days
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(100 µg/fish) and 5 days (200 µg/fish) (Fig. 3) (Table 1).
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In boost immunization group; the RPS obtained after administration was 33.4% (100
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µg/fish) and 60.0% (200 µg/fish) for the respective administration dose and time points at the
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the end of 200 dpi (Fig. 3) (Table 1).
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3.4. RBIV re-challenge of survivors
Survivors from 2 days before infection and boost immunization group of Experiment II at
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20 °C, following the re-infection at 200 dpi showed no clinical signs or mortality through 250
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dpi (Fig. 3A and 3C), when they were determined to have low virus copy numbers (average
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1.2 × 101/µl). Specific mortality was observed after 16 days in the virus infected control fish
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(naive fish, not previously exposed to virus), with characteristic clinical signs of the RBIV
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disease, and the mortality reached 100% at 24 dpi. The dead fish demonstrated high MCP
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gene copy numbers in the spleen (greater than 7.1 × 107/µl). RPS values obtained for each
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groups are listed in Table 1.
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3.5. Effect of poly (I:C) on immune gene expression
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3.5.1. TLR3
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In the spleen, TLR3 showed a significantly increased expression level compared with the
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control (3.3-, 6.9-, 3.0- and 3.4-fold) at 12 h, 1 d, 2 d and 4 dpa, respectively (Fig. 4A).
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Significantly upregulation of TLR3 expression was observed at 1 dpa (4.1-fold) (p<0.01) in
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the kidney when compared with the control (Fig. 4A). In the blood, TLR3 expression was
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observed at 1 dpa (2.1-fold) and were significantly higher (p<0.001) than that of the control
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fish; increased TLR3 expression was observed at 2 d and 4 dpa (9.6- and 5.3-fold,
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respectively) compared with the control group (Fig. 4A).
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3.5.2. Inflammatory cytokines (IL1β, IL8 and TNFα) 10
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The expression levels of IL1β, IL8 and TNFα ranged between 0.1 ~ 2.1-fold (12 h to 4
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dpa) in the spleen, kidney and blood, and none of these expression levels showed significant
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differences when compared with those of controls (Fig. 4B, 4C and 4D).
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3.5.3. IRF3, Mx, ISG15 and PKR
A significant increases in IRF3 expression level were observed in the blood at 1 d (2.9fold) (p<0.05) and 2 dpa (3.1-fold) (p<0.01) (Fig. 4E).
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Similar gene expression trends were observed for Mx in the spleen, kidney and blood. Mx
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expression was significantly increased at 12 h (6.6-, 9.5- and 7.3-fold, respectively) and 1
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dpa (5.9-, 12.4- and 8.6-fold, respectively) in the spleen, kidney and blood, respectively (Fig.
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4F).
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ISG15 and PKR expression was significantly increased in the blood at 2 dpa (5.4- and
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10.2-fold, respectively) (p<0.001) (Fig. 4G and 4H). However, ISG15 and PKR expression
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levels in the spleen and kidney were not significant different from those of the control group
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in this experiment (Fig. 4G and 4H).
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4. Discussion
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Poly (I:C) is believed to activate an antiviral state of sufficient duration for inhibition of
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virus replication to protect fish from virus infection [23-28]. However, there is no report on
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the efficacy of poly (I:C) to provide protection against RBIV infection in rock bream.
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Recently, our previous studies have demonstrated that the virus replication speed with the
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water temperature is likely the more critical factor for rock bream mortality [4]. Hence, in
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the present study, poly (I:C) was evaluated for its antiviral potentials in rock bream under
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various experimental conditions such as different water temperature, different RBIV
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infection dose and the suitable time of poly (I:C) administration.
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104 MCP gene copies/100 µl) in the 100 µg and 200 µg with poly (I:C) administration groups
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showed no protection. A similar observation was reported by Oh et al. 2015 [34]. They
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observed no significantly protective effect (97% mortality) in red seabream iridovirus (RSIV)
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infected rock bream when 200 µg of poly (I:C) was administered at 28 °C. Fish injected with
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RBIV (1.1 × 104 MCP gene copies/100 µl) at 20 °C had a virus concentration that was lower
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than the concentration in the other experiments, showing the highest protection, while the
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virus injected control fish exhibited 100% mortality. This indicates that the poly (I:C) can
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induce better antiviral immune response in administered fish at 20 °C (lower virus replication
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speed) than in administered fish at 23 and 26 °C (higher virus replication speed) against
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RBIV infection.
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Furthermore, it has been reported that the immune effects of fish administered with
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immunostimulants are time dependent [24,35,36]. The before-injected fish with poly (I:C)
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followed by RGNNV challenge exhibited higher survival rates than poly (I:C) administration
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at post infection [24], suggesting that it required several days post poly (I:C) administration
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for the full development of poly (I:C) induced immune response. In agreement with these
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results, we found that poly (I:C) administered before infection showed the highest protective
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effect, while poly (I:C) administration at post infection showed 100% mortality. However,
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poly (I:C) administration at post infection delayed final mortality more than that observed in
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the virus injected control fish in this study. This indicates that although the poly (I:C)
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administration at post infection does not lead to the full development of poly (I:C) induced
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immune response, it could still induce an immune response against RBIV infection. It was
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evident by this study that the poly (I:C) administered two times at before/post infection
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provided 20~30% higher protection rates than poly (I:C) administered once before infection.
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This indicates that survival rates of immunized fish were clearly enhanced by the boost
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administration. To obtain survivors, considering the immunostimulant effects on fish
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mortality and water temperature, the suitable time of immunostimulant administration and
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virus replication speed are likely the critical factor in rock bream mortality because rock
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bream is more susceptible to RBIV infection. Finally, re-infection was tried to evaluate
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immune protection was developed in survived fish from poly (I:C) administration. Survivals
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at 200 dpi showed 100% survival rates at the end of experiment. This observation indicates
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that the immune defense system of survived fish can induced protection in rock bream
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against re-infection RBIV.
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Poly (I:C) exerts its biological effect by interacting with the TLR3. The signaling pathways
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activated by these TLR3 are broadly classified as adaptor protein TIR-domain-containing
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adapter inducing IFN-β (TRIF)-dependent. This activates the transcription factors such as
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interferon regulatory factor (IRF) IRF3, nuclear factor-κB (NF-κB) and activator protein 1
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(AP-1) and induce an antiviral state through secretion of pro-inflammatory cytokines and
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type I IFN [12-14]. We examined the two complementary TLR3-mediated gene expression,
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namely the inflammatory cytokine-mediated immune response and the IFN-mediated
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immune response in poly (I:C) administered rock bream.
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In this study, significantly higher level of TLR3 expression were observed at certain times
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in the spleen, kidney and blood after poly (I:C) administration. These results indicate a vital
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contribution of the TLR3 gene expression to the antiviral activity of poly (I:C) in rock
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bream. However, the expression levels of inflammatory cytokines (IL1β, IL8 and TNFα)
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were low in the spleen, kidney and blood, suggesting that pro-inflammatory gene expression
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was not activated when the rock bream were administered with poly (I:C). A similar
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observation was reported by Hong et al. 2016 [37]. They observed that the poly (I:C) did not
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significantly activated IL1β expression in the spleen and down regulation of IL8 and TNFα in
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head kidney and spleen of rock bream. Mx proteins are particularly induced by poly (I:C), a synthetic double-stranded RNA that is
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a known indicator of IFN production or antiviral activity in a number of fish species [38-43].
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In agreement with these results, Mx expression profiles of rock bream were regulated by poly
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(I:C). Similar significantly high expression patterns of Mx were observed in the spleen,
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kidney and blood in the early administration period at 12 h and 1 dpa, and the expression
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levels then decreased in the late administration period at 2 d to 4 dpa. Similar to our results,
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sevenband grouper (Epinephelus septemfasciatus) with poly (I:C) injection exhibited a high
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level of Mx expression at 3 hpi in head kidney and decreased levels at 24 hpi [22].
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Furthermore, a significantly high expression level of IRF3, ISG15 and PKR was observed
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only in the blood; there was no significantly upregulation of these interferon-related genes in
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the spleen and kidney. These observations highlight the importance of interferon-related gene
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measurement in blood and indicate the need for more detailed studies to obtain a better
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understanding of blood interferon-related gene levels to identify blood-interferon-related
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immune response interaction in rock bream.
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In conclusion, the present study demonstrated for the first time the possibility of developing
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long-term preventive measures against RBIV using poly (I:C). The protection against RBIV
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might have occurred through administration of poly (I:C) due to the activation of immune
336
response-related genes such as TLR3, IRF3, Mx, ISG15 and PKR in rock bream.
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337
338
Acknowledgements
14
ACCEPTED MANUSCRIPT 339
This research was supported by Basic Science Research Program through the National
340
Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future
341
Planning (2015R1C1A1A01053685).
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References
348
[1] Kurita J, Nakajima K. Megalocytiviruses. Viruses 2012;4:521–38.
349
[2] Jung SJ, Oh MJ. Iridovirus-like infection associated with high mortalities of striped
350
beakperch, Oplegnathus fasciatus (Temminck et Schlegel), in southern coastal areas of
351
the Korean peninsula. J Fish Dis 2000;23:223–6.
TE D
M AN U
347
[3] Jun LJ, Jeong JB, Kim JH, Nam JH, Shin KW, Kim J.K, et al. Influence of temperature
353
shifts on the onset and development of red sea bream iridoviral disease in rock bream
354
Oplegnathus fasciatus. Dis Aquat Org 2009;84: 201–8.
EP
352
[4] Jung MH, Jung SJ, Vinay TN, Nikapitiya C, Kim JO, Lee JH, et al. Effects of water
356
temperature on mortality in Megalocytivirus-infected rock bream Oplegnathus fasciatus
357
(Temminck et Schlegel) and development of protective immunity. J Fish Dis
358
2015;38:729–37.
AC C
355
359
[5] Jung MH, Lee J, Jung SJ. Low pathogenicity of FLIV (flounder iridovirus) and the
360
absence of cross-protection between FLIV and RBIV (rock bream iridovirus). J Fish Dis
361
2016;39:1325–33.
15
ACCEPTED MANUSCRIPT
363 364 365
[6] Secombes C. Will advances in fish immunology change vaccination strategies. Fish Shellfish Immunol 2008;25:409–16. [7] Ragnar T, Powell DB. Effects of disease risk, vaccine efficacy, and market priceon the economics of fish vaccination. Aquaculture 2006;256:42–9.
RI PT
362
366
[8] Vinay TN, Kim YJ, Jung MH, Kim WS, Kim DH, Jung SJ. Inactivated vaccine against
367
viral hemorrhagic septicemia (VHS) emulsified with squalene and aluminum hydroxide
368
adjuvant
369
olivaceus). Vaccine 2013;31:4603–10.
long
term
protection
in
olive
flounder
(Paralichthys
SC
provides
[9] Tafalla C, Bøgwald J, Dalmo RA. Adjuvants and immunostimulants in fish vaccines:
371
current knowledge and future perspectives. Fish Shellfish Immunol 2013;35:1740–50.
M AN U
370
372
[10] Akira S. Toll-like receptors and innate immunity. Adv Immunol 2001;78:1–56.
373
[11] Medzhitov
Janeway
C.
Innate
immune
recognition:
mechanisms
and
pathways. Immunol Rev 2000;173:89–97.
TE D
374
R,
[12] Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T. TICAM-1, an adaptor
376
molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat
377
Immunol 2003;4:161–7.
EP
375
[13] Lee KG, Xu S, Kang ZH, Huo J, Huang M, Liu D, et al. Bruton's tyrosine kinase
379
phosphorylates Toll-like receptor 3 to initiate antiviral response. P Natl Acad Sci USA
380
2012;109:5791–96.
AC C
378
381
[14] Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, et al. Role of adaptor
382
TRIF in the MyD88-independent toll-like receptor signaling pathway. Science
383
2003;301:640–3.
384 385
[15] Eaton WD. Antiviral activity in 4 species of salmonids following exposure to poly inosinic-cytidylic acid. Dis Aquat Org 1990;9:193–8.
16
ACCEPTED MANUSCRIPT 386
[16] Nygaard R, Husgard S, Sommer AI, Leong JAC, Robertsen B. Induction of Mx protein
387
by interferon and double-stranded RNA in salmonid cells. Fish Shellfish Immunol
388
2000;10:435–50.
390
[17] Lockhart K, Bowden TJ, Ellis AE. Poly I:C-induced Mx responses in Atlantic salmon
RI PT
389
parr, post-smolts and growers. Fish Shellfish Immunol 2004;17:245–54.
[18] Acosta F, Lockhart K, Gahlwat SK, Real F, Ellis AE. Mx expression in Atlantic salmon
392
(Salmo salar L.) parr in response to Listonella anguillarum bacterin, lipopolysaccharide
393
and chromsomal DNA. Fish Shellfish Immunol 2004;17:255–63.
SC
391
[19] Jensen I, Albuerque A, Sommer AI, Robertsen B. Effect of poly I:C on the expression of
395
Mx proteins and resistance against infection by infectious salmon anaemia virus in
396
Atlantic salmon. Fish Shellfish Immunol 2002;13:311–26.
M AN U
394
[20] Plant KP, Harbottle H, Thune RL. Poly I:C induces an antiviral state against Ictalurid
398
Herpesvirus 1 and Mx1 transcription in the channel catfish (Ictalurus punctatus). Dev
399
Comp Immunol 2005;29:627–35.
TE D
397
[21] Zhou ZX, Zhang BC, Sun L. Poly (I: C) induces antiviral immune responses in Japanese
401
flounder (Paralichthys olivaceus) that require TLR3 and MDA5 and is negatively
402
regulated by Myd88. PloS one 2014;9:e112918.
EP
400
[22] Thanasaksiri K, Sakai N, Yamashita H, Hirono I, Kondo H. Influence of temperature on
404
Mx gene expression profiles and the protection of sevenband grouper, Epinephelus
405
septemfasciatus, against red-spotted grouper nervous necrosis virus (RGNNV) infection
406
after poly (I:C) injection. Fish Shellfish Immunol 2014;40:441–5.
AC C
403
407
[23] Kim HJ, Oseko N, Nishizawa T, Yoshimizu M. Protection of rainbow trout from
408
infectious hematopoietic necrosis (IHN) by injection of infectious pancreatic necrosis
409
virus (IPNV) or Poly (I:C). Dis Aquat Org 2009;83:105–13.
17
ACCEPTED MANUSCRIPT 410
[24] Nishizawa T, Takami I, Kokawa Y, Yoshimizu M. Fish immunization using a synthetic
411
double-stranded RNA Poly (I:C), an interferon inducer, offers protection against
412
RGNNV, a fish nodavirus. Dis Aquat Org 2009;83:115–22. [25] Nishizawa T, Takami I, Yoshimizu M, Oh MJ. Required dose of fish nervous necrosis
414
virus (NNV) for Poly (I:C) immunization of sevenband grouper Epinephelus
415
septemfasciatus. Aquaculture 2011;311:100–4.
RI PT
413
[26] Oh MJ, Takami I, Nishizawa T, Kim WS, Kim CS, Kim SR, et al. Field tests of Poly
417
(I:C) immunization with nervous necrosis virus (NNV) in sevenband grouper,
418
Epinephelus septemfasciatus (Thunberg). J Fish Dis 2012;35:187–91.
M AN U
SC
416
419
[27] Takami I, Kwon SR, Nishizawa T, Yoshimizu M. Protection of Japanese flounder
420
Paralichthys olivaceus from viral hemorrhagic septicemia (VHS) by Poly (I:C)
421
immunization. Dis Aquat Org 2010;89:109–15.
[28] Nishizawa T, Takami I, Yang M, Oh MJ. Live vaccine of viral hemorrhagic septicemia
423
virus (VHSV) for Japanese flounder at fish rearing temperature of 21 °C instead of Poly
424
(I:C) administration. Vaccine 2011;29: 8397–404.
TE D
422
[29] Alcorn SW, Murray AL, Pascho RJ. Effects of rearing temperature on immune functions
426
in sockeye salmon (Oncorhynchus nerka). Fish Shellfish Immunol 2002;12:303–34.
427
[30] Bly JE, Clem LW. Temperature and teleost immune functions. Fish Shellfish Immunol
AC C
428
EP
425
1992;2:159–71.
429
[31] Jung MH, Nikapitiya C, Song JY, Lee JH, Lee JH, Oh MJ, et al. Gene expression of pro-
430
and anti-apoptotic proteins in rock bream (Oplegnathus fasciatus) infected with
431
Megalocytivirus (family Iridoviridae). Fish Shellfish Immunol 2014;37:122–30.
432 433
[32] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–8.
18
ACCEPTED MANUSCRIPT 434
[33] Amend DF. Potency testing of fish vaccines. In: Anderson DP, Hennessen W, editors.
435
Developments in Biological Standardization, vol. 49. Basel: Fish bio-logics.
436
Serodiagnostics and Vaccines; 1981. p. 447–54. [34] Oh SY, Kim WS, Oh MJ, Nishizawa T. Quantitative change of red seabream iridovirus
438
(RSIV)
439
administration. Aquacult Int 2015;23:93–8.
441
rock
bream
Oplegnathus
fasciatus,
following
Poly
(I:C)
[35] Tassakka ACMA, Sakai M. The in vitro effect of CpG-ODNs on the innate immune
SC
440
in
RI PT
437
response of common carp, Cyprinus carpio L. Aquaculture 2003;220:27–36. [36] Nakatani M, Iwasaki T, Watarai S, Kodama H. In vitro enhancement by CpG ODN of
443
phagocytic activity of rainbow trout head kidney phagocytes against fish pathogen Vibrio
444
ordalii, but not against polystyrene particles. J Vet Med Sci 2007;69:1287–90.
M AN U
442
[37] Hong S, Jin JW, Park JH, Kim JK, Jeong HD. Analysis of proinflammatory gene
446
expression by RBIV infection in rock bream, Oplegnathus faciatus. Fish Shellfish
447
Immunol 2016;50:317–26.
TE D
445
[38] Fernandez-Trujillo A, Ferro P, Garcia-Rosado E, Infante C, Alonso MC, Bejar J, et al.
449
Poly I:C induces Mx transcription and promotes an antiviral state against sole
450
aquabirnavirus in the flatfish Senegalese sole (Solea senegalensis Kaup). Fish Shellfish
451
Immunol 2008;24:279–85.
AC C
EP
448
452
[39] Fernández-Trujillo MA, Novel P, Manchado M, Sepulcre MP, Mulero V, Borrego JJ, et
453
al. Three Mx genes with differential response to VNNV infection have been identified in
454
Gilthead seabream (Sparus aurata). Mol Immunol 2011;48:1216–23.
455
[40] Chen YM, Su YL, Lin JHY, Yang HL, Chen TY. Cloning of an orange-spotted grouper
456
(Epinephelus coioides) Mx cDNA and characterisation of its expression in response to
457
nodavirus. Fish Shellfish Immunol 2006;20:58–71.
19
ACCEPTED MANUSCRIPT 458
[41] Chen YM, Su YL, Shie PS, Huang SL, Yang HL, Chen TY. Grouper Mx confers
459
resistance to nodavirus and interacts with coat protein. Dev Comp Immunol
460
2008;32:825–36. [42] Trobridge GD, Chiou PP, Kim CH, Leong JC. Induction of the Mx protein of rainbow
462
trout Oncorhynchus mykiss in vitro and in vivo with poly I:C dsRNA and infectious
463
hematopoietic necrosis virus. Dis Aquat Org 1997;30:91–8.
RI PT
461
[43] Ohta T, Ueda Y, Ito K, Miura C, Yamashita H, Miura T, et al. Anti-viral effects of
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interferon administration on sevenband grouper, Epinephelus septemfasciatus. Fish
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Shellfish Immunol 2011;30:1064–1071.
M AN U
SC
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Figure legend
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Figure 1. The mortality patterns of rock bream in the poly (I:C) treated groups (100 and 200
469
ug/fish) and virus injected control group following an i.p. injected at 26 °C with RBIV (1.1 ×
470
107) at 2 days post poly (I:C) administration.
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Figure 2. The effect of poly (I:C) on RBIV (1.1 × 104) at 23 °C. Fifteen fish in each group
473
were intraperitoneally (i.p) injected with poly (I:C) at 100 µg/fish. A) poly (I:C)
474
administration at 2 days before infection. B) poly (I:C) administration at 2 days post infection.
475
C) poly (I:C) administration at 2 days before/post infection (boost immunization).
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Figure 3. Protection efficacy of poly (I:C) at 20 °C. Fifteen fish in each group were
478
administered i.p. with poly (I:C) (100 µg/fish) at 2 days before infection (A), 2 days post
479
infection (B) and 2 days before/post infection (boost immunization) (C). To establish mild
480
infection to reduce virus replication speed, fish were injected with RBIV (1.1 × 104) and
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ACCEPTED MANUSCRIPT 481
maintained at 20 °C in the aquaria. Surviving fish were infected a second time (period of 200
482
to 250 dpi with 1.1 × 107 MCP copies/fish).
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Figure 4. Relative expression analysis of immune response-related genes in the spleen,
485
kidney and blood of poly (I:C) administered rock bream. The expression level of β-actin was
486
used as an internal control for a real-time PCR-based relative expression study. Each
487
experiment was performed in duplicate. The bars represent the standard error (SE) of the
488
mean for 5 individuals. A- TLR3; B- IL1β; C- IL8; D- TNFα; E- IRF3; F- Mx; G- ISG15; H-
489
PKR. The relative expression fold-change at 0 h post injection (before injection) was used as
490
the basal line. Differences between the poly (I:C) administered rock bream and non-
491
administered rock bream were compared using a one-way analysis of variance followed by
492
the Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01 and ***p < 0.001 were
493
considered significant.
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Table 1 Summary of poly (I:C) efficacy test.
Boost immunization (2 days before/post infection)
First infection Control Second infection
RBIV infection dose
Day of final mortality
Mortality% at 100 dpi
RPS
RBIV re-infection for survivors
RBIV infection dose
Days observed
Mortality%
100 µg
23 °C
15
1.1 × 104/fish
29 day
100%
-
-
-
-
-
200 µg
23 °C
15
1.1 × 104/fish
29 day
100%
-
-
-
-
-
100 µg
20 °C
15
1.1 × 104/fish
36 day
86.6%
13.4%
200 dpi
1.1 × 107/fish
50 days
0%
200 µg
20 °C
15
1.1 × 104/fish
32 day
66.6%
33.4%
200 dpi
1.1 × 107/fish
50 days
0%
100 µg
23 °C
15
1.1 × 104/fish
23 day
100%
-
-
-
-
-
200 µg
23 °C
15
1.1 × 104/fish
23 day
100%
-
-
-
-
-
100 µg
20 °C
15
1.1 × 104/fish
38 day
100%
-
-
-
-
-
200 µg
20 °C
15
1.1 × 104/fish
39 day
100%
-
-
-
-
-
100 µg
23 °C
15
1.1 × 104/fish
25 day
100%
-
-
-
-
-
200 µg
23 °C
15
1.1 × 104/fish
30 day
100%
-
-
-
-
-
100 µg
20 °C
15
1.1 × 104/fish
37 day
66.6%
33.4%
200 dpi
1.1 × 107/fish
50 days-
0%
200 µg
20 °C
15
1.1 × 104/fish
35 day
39.9%
60.0%
200 dpi
1.1 × 107/fish
50 days
0%
-
23 °C
15
1.1 × 104/fish
23 day
100%
-
-
-
-
-
-
20 °C
15
1.1 × 104/fish
34 day
100%
-
-
-
-
-
-
20 °C
15
-
-
-
-
200 dpi
1.1 × 107/fish
24 days
100%
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No. of fish
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2 days post infection
RBIV infection at
EP
2 days before infection
Poly (I:C) administration
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Group (time of poly (I:C) administration)
ACCEPTED MANUSCRIPT
Table 2 Primers used in this study.
Sequence
Accession number
RI PT
Name β-actin
F CAGGGAGAAGATGACCCAGA R CATAGATGGGCACTGTGTGG
MCP
F TGCACAATCTAGTTGAGGAGGTG R AGGCGTTCCAAAAGTCAAGG
TLR3
F TAAAGCCCATCAGGCACTTCAC R AGGAAGAGTGAGCGAGACAACC
KY613955
IL1β
F ATCTGGAGACGGTGGACAAC R GCTGATGTACCAGTCGCTGA
KC522967
IL8
F CCCTCCTGACCATCAGTGAA R TGATCTCAGTCTCCTCGCAGT
KC522965
TNFα
F GCAATCAGGCCAAACAGAAGCACT R TTGGCTTTGCTGCTGATACGCTTC
FJ623187
IRF3
F GTGTCTAAAGGGACTGAACATCG R CCCTCAAACGTTACTGGATACTG
KF267453.1
Mx
F GATCGCCTCTCCTGATGTTC R ACCACCAAGCTGATGGTTTC
FJ155359.1
ISG15
F CTTTAAGACCAAGGTCCAATGC R GCCTCCACATTGTAGTCTGAAAG
BAJ16365.1
F GCTCAACAAATAGTCAGTGGAGT R CTCTGGTGACCAGACCAAAGTCC
FJ179396.1
SC
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TE D
EP
AC C
PKR
FJ975145
AY849394
ACCEPTED MANUSCRIPT
Poly (I:C) administration at 26 °C at 2 days before infection 100 200 µg
80
RI PT
Virus injected control 60
SC
40 20
5
10
15
TE D
Days post infection
EP
0
M AN U
0
AC C
Cumulative mortality%
100 µg
Figure 1
ACCEPTED MANUSCRIPT
Poly (I:C) administration at 23 °C at 2 days before infection
A
80 60
RI PT
100 µg 200 µg Virus injected control
40
SC
Cumulative mortality%
100
20
0
10
M AN U
0 20
30
Poly (I:C) administration at 23 °C at 2 days post infection
B
80
EP
100 µg
60
200 µg 40
Virus injected control
20
AC C
Cumulative mortality%
100
TE D
Days post infection
0 0
5
10
15
20
25
Days post infection
Figure 2 is continued to the next page
ACCEPTED MANUSCRIPT
Poly (I:C) administration at 23 °C at 2 days before/post infection
C
80
RI PT
100 µg (boost)
60
200 µg (boost) 40
SC
Virus injected control
20
10
15
20
25
Days post infection
30
TE D
5
EP
0
M AN U
0
AC C
Cumulative mortality%
100
Figure 2
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Poly (I:C) administration at 20 °C at 2 days before RBIV infection
A
60 100 µg 40
200 µg 1st virus injected control
20
Second infection
2nd virus injected control 0
B
10
20
30
40
50 60 200 Days post infection
100 µg
240
250
Virus injected control
20
AC C
200 µg 40
230
EP
80 60
220
TE D
Poly (I:C )administration at 2 days post infection at 20 °C
100
210
M AN U
0
Cumulative mortality%
RI PT
80
SC
Cumulative mortality%
100
0 0
5
10
15
20
25
30
35
40
Days post infection
Figure 3 is continued to the next page
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80
200 µg (boost) 1st virus injected control 2nd virus injected control
60 40
Second infection
20 0 30
40
50 60 200 Days post infection
210
220
M AN U
20
230
240
250
TE D
10
EP
0
SC
100 µg (boost)
AC C
Cumulative mortality%
100
RI PT
Poly (I:C) administration at 20 °C at 2 days before/post infection
C
Figure 3
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TLR3 1d
15
*** 10
*** **
** * *
*
1d
12 h
4d
2d
8
6
4
2
0
Spleen
Kidney
Blood
IL8
C
12 h
1d
2d
AC C
8
6
4
2
0
Spleen
10
Kidney
Blood
Organs
TNFα
D
4d
EP
0h
TE D
Organs
10
0h
M AN U
5
0
Relative expression to β.actin
10
4d
2d
RI PT
12 h
SC
0h
Relative expression to β.actin
Relative expression to β.actin
20
IL1β
B
Relative expression to β.actin
A
0h
12 h
1d
4d
2d
8
6
4
2
0 Spleen
Kidney Organs
Blood
Spleen
Kidney
Blood
Figure 4 is continued to the next page Organs
ACCEPTED MANUSCRIPT
IRF3 1d
12 h
Relative expression to β.actin
8
6
*
4
**
10
**
1d
12 h
*
4d
2d
** ** *
*
0
Spleen
Kidney
Blood
ISG15
G 0h
12 h
1d
2d
TE D
Organs
***
AC C
8
6
4
2
0
Spleen
20
Kidney
Blood
Organs
PKR
H
4d
EP
10
20
0h
M AN U
2
0
Relative expression to β.actin
30
4d
2d
RI PT
0h
Relative expression to β.actin
Relative expression to β.actin
10
Mx
F
SC
E
0h
12 h
1d
15
4d
2d
***
10
5
0 Spleen
Kidney Organs
Blood
Spleen
Kidney Organs
Blood
Figure 4
ACCEPTED MANUSCRIPT Highlights: Treatment with poly (I:C) at before/post infection had significantly higher protection. Poly (I:C) provided significant protection for 200 days. TLR3 and Mx were highly expressed in poly (I:C) administered rock bream.
AC C
EP
TE D
M AN U
SC
RI PT
IRF3, ISG15 and PKR induced immune responses were high activated in blood.
S1050-4648(17)30349-2
DOI:
10.1016/j.fsi.2017.06.026
Reference:
YFSIM 4647
To appear in:
Fish and Shellfish Immunology
Received Date: 22 February 2017 Revised Date:
27 April 2017
Accepted Date: 6 June 2017
Please cite this article as: Jung M-H, Jung S-J, Protective immunity against rock bream iridovirus (RBIV) infection and TLR3-mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus) following poly (I:C) administration, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.06.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Protective immunity against rock bream iridovirus (RBIV) infection and TLR3-
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mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus)
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following poly (I:C) administration
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Myung-Hwa Jung *, Sung-Ju Jung
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Department of Aqualife Medicine, Chonnam National University, Republic of Korea.
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*
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National University, San96-1 Dunduck Dong, Yeosu, Chonnam 550-749, Republic of Korea.
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Tel.: +82-61-659-7175
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Fax: +82-61-659-7179
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E-mail address: [email protected] (M.-H. Jung).
Corresponding author contact information: Department of Aqualife Medicine, Chonnam
20 1
ACCEPTED MANUSCRIPT Abstract
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In this study, we evaluated the potential of poly (I:C) to induce antiviral status for protecting
23
rock bream from RBIV infection. Rock bream injected with poly (I:C) at 2 days before
24
infection (1.1 × 104) at 20 °C had significantly higher protection with RPS 13.4% and 33.4%
25
at 100 and 200 µg/fish, respectively, through 100 days post infection (dpi). The addition of
26
boost immunization with poly (I:C) at before/post infection at 20 °C clearly enhanced the
27
level of protection showing 33.4% and 60.0% at 100 and 200 µg/fish, respectively. To
28
investigate the development of a protective immune response, rock bream were re-infected
29
with RBIV (1.1 × 107) at 200 dpi. While 100% of the previously unexposed fish died, 100%
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of the previously infected fish survived. Poly (I:C) induced TLR3 and Mx responses were
31
observed at several sampling time points in the spleen, kidney and blood. Moreover,
32
significantly high expression levels of IRF3 (2.9- and 3.1-fold at 1 d and 2 days post
33
administration (dpa), respectively), ISG15 and PKR expression (5.4- and 10.2-fold at 2 dpa,
34
respectively) were observed in the blood, but the expression levels were low in the spleen and
35
kidney after poly (I:C) administration. Our results showed the induction of antiviral immune
36
responses and indicate the possibility of developing long term preventive measures against
37
RBIV using poly (I:C).
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Key word: rock bream, rock bream iridovirus, poly (I:C), protection, TLR3, Mx 2
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1. Introduction Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus [1], causes
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mass mortality in rock bream (Oplegnathus fasciatus) [2]. Since first outbreak, high mortality
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rates have been occurred in rock bream annually due to RBIV. Prophylactic measures to
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control RBIV remain unsolved because of the high susceptibility of rock bream to RBIV [3-
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5]; thus, it has been difficult to develop an effective vaccine [1]. Vaccination is an important
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disease control measure and essential for good farming practices [6-9]. Therefore,
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understanding of host-RBIV interactions will be useful for the future strategies for controlling
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RBIV disease in cultured fish. Hence the development of control and preventive measures
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against RBIV is required to sustain rock bream aquaculture industry.
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The innate immune system is the first line of host defense against pathogens. Toll-like
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receptors (TLRs) are well known innate immune-recognition receptors for microbial
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pathogens [10-11]. TLR3 was identified as a receptor for double-stranded RNA (dsRNA).
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Polyinosinic:polycytidylic acid (poly (I:C)) is a commercially available synthetic analogue
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of dsRNA. Upon binding to TLR3, dsRNA or its synthetic analogue poly (I:C) leads to the
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induction of type I interferon (IFN) and inflammatory cytokine productions [12-14] and it is
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also important for activation of adaptive immunity. Poly (I:C) has been extensively studied
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in fish systems, where its induction of antiviral activities have been shown [15-22].
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Additionally, poly (I:C) has been shown to induce disease resistance against fish viruses
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such as infectious haematopoietic necrosis virus (IHNV) [23], nervous necrosis virus (NNV)
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[24-26] and viral haemorrhagic septicaemia virus (VHSV) [27,28]. However, the
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mechanism by which poly (I:C) functions in fish is unclear, and very little is known about
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the relationship between the poly (I:C) induced antiviral effect and TLR3-mediated type I
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IFN signaling. Moreover, no published reports are available on the systemic protective
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effects of poly (I:C) against RBIV infection in rock bream.
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infection [29,30]. In Korea, RBIV outbreaks typically occur in cultured rock bream in the
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summer season, when water temperature range between 23-27 °C [2]. Recently, we reported
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that rock bream mortality due to RBIV infection was dependent on susceptible water
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temperature [4]; fish mortality reaches 100% at water temperature of 29 °C to 20 °C, but
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delayed mortality at the low water temperatures. Furthermore, rock bream infected at
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different concentrations of RBIV (6.7 × 105 – 7.5 × 107 /100 µl) exhibited 100% mortality at
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26 °C with delayed mortality at the low virus concentrations. This findings indicate that the
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water temperature and RBIV infection dose are an important factor to rock bream.
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In the present study, poly (I:C) was evaluated for its antiviral potentials in rock bream under
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different virus replication levels at 26, 23 or 20 °C. The experiment involved the controlled
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variation of water temperatures, different RBIV infection dose, different doses of poly (I:C)
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and also the suitable time of poly (I:C) administration (before or post virus infection).
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Furthermore, the gene expression levels of TLR3, IL1β, IL8, TNFα, IRF3, Mx, ISG15 and
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PKR were assessed by poly (I:C) at different time points in rock bream for molecular
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understanding of enhanced immune protection against RBIV by poly (I:C).
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2. Materials and methods
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2.1. Experimental fish
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Rock bream were obtained from a local farm and reared at the Fisheries Science Institute at
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Chonnam National University. Approximately 1000 fish were maintained in large tanks (10
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ton) with a continuous sea water supply with aeration. The required numbers of fish for the
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experiments were transferred to the experimental facility.
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2.2. Isolation of RBIV 4
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2010 [31], were thawed for the experiment. The MCP gene copies of the original RBIV in
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the supernatant preparations, quantified by using quantitative real time polymerase chain
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reaction (qRT-PCR) was 7.5 × 107/100 µl and at 10, 100, 1000, 10000 and 100000 fold
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serially diluted virus solutions containing 1.1 × 107, 1.2 × 106, 6.7 × 105, 5.6 × 104 and 1.1 ×
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104/100 µl MCP gene copies, respectively.
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2.3. Effect of poly (I:C) on RBIV infection at 26 °C (Experiment I)
Since, optimum RBIV replication water temperature in fish body is 26 °C, this water
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temperature was selected at first to examine the antiviral property of poly (I:C) on RBIV
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infection. Rock bream (10.2 ± 1.4 cm, 17.2 ± 3.0 g) were administered intraperioneally (i.p.)
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with each of the poly (I:C) (Sigma) at 100 and 200 µg/fish or phosphate-buffered saline
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(PBS) as a virus injected control. At 2 days post administration (dpa) of poly (I:C) or PBS,
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the fish were injected with RBIV (100 µl/fish) containing 1.1 × 107 MCP gene copies. Each
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group of 20 fish were maintained at 26 °C for 15 days in the aquaria containing 30 L of UV-
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treated seawater.
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2.4. Analysis of the antiviral effect of poly (I:C) in relation to dose, time and water
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temperature (Experiment II)
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Rock bream are very susceptible to high RBIV dose (1.1 × 107 MCP gene copies) with a
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water temperature at 26 °C. Alterations in experimental conditions were made to determine
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whether a protective immune response could be induced in the surviving fish. To obtain
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survivors, experimental conditions were adjusted as follows; water temperature (3 °C or
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6 °C) and RBIV injection dose (1000-fold) were lower than the experiment I. To examine the
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effect of water temperature and time on the antiviral property of poly (I:C), 15 fish in each 5
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23 °C (100 µg and 200 µg/fish) and two groups at 20 °C (100 µg and 200 µg/fish) water
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temperature were administered i.p. with poly (I:C) and the other two groups were
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administered PBS as virus injected controls at the respective water temperatures. At 2 days
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post administration, the fish were injected with RBIV (100 µl/fish) containing 1.1 × 104 MCP
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gene copies. This group of fish were regarded as the 2 days before infection group.
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Rock bream (9.8 ± 2.2 cm, 16.8 ± 2.5 g) were divided randomly into four groups (15 fish
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per group); two groups at 23 °C and the other two groups at 20 °C were injected with RBIV
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(100 µl/fish) containing 1.1 × 104 MCP gene copies. At 2 days post infection (dpi), the four
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groups were administered i.p. with poly (I:C) at 100 and 200 µg/fish, respectively, these
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groups were regarded 2 days post infection group.
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15 fish in each group (10.4 ± 2.4 cm, 16.9 ± 2.4 g) were divided randomly into four groups
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and administered i.p. with poly (I:C) at the concentration of 100 µg (23 and 20 °C) and 200
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µg/fish (23 and 20 °C). At 2 days post poly (I:C) administration, the fish groups were injected
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with RBIV (100 µl/fish) containing 1.1 × 104 MCP gene copies and then at 2 days post
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infection (4 days post immunization), the fish were boost immunized with a same
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concentration of poly (I:C) as that for the primary immunization protocol; these groups were
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regarded as the boost immunization group. Fish were maintained at 23 and 20 °C for 200
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days in the aquaria containing 30 L of UV-treated seawater. Table 1 summarizes
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experimental conditions.
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2.5. Re-challenge of survivors from Experiment II with RBIV
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The fish that survived from Experiment II at 200 dpi were challenged for second time by an
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injection of 100 µl/fish of RBIV (1.1 × 107 MCP gene copies). Fifteen naive fish (11.2 ± 1.2
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cm, 30.2 ± 1.4 g) from virus injected control group (not previously exposed to the virus) were 6
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injected with RBIV in the same manner as the survivors. All the fish were kept for an
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additional 50 days in the aquaria containing 30 L of UV-treated seawater. Table 1
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summarizes experimental conditions.
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2.6. Effect of poly (I:C) administration on the gene expression
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The immune response effect of poly (I:C) administered rock bream and non-administered
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rock bream was evaluated as follows: Each of 25 fish (10.1 ± 2.2 cm, 17.7 ± 2.0 g) were
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maintained at 20 °C in the aquarium containing 250 L of UV-treated seawater. The fish
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were administered i.p. with poly (I:C) (100 µg/fish) or PBS (100 µl/fish) as control. The
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spleen, kidney and blood were collected from five fish each group at 0 h (before poly (I:C)
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administration), 12 hours, and 1, 2 and 4 days post poly (I:C) administration and flash
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frozen in liquid nitrogen. Samples were stored at –80 °C after being flash frozen in liquid
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nitrogen.
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2.7. Quantitative expression of immune genes and viral copy numbers Real-time PCR was carried out in an Exicycler 96 Real-Time Quantitative Thermal Block
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(Bioneer, Korea) using an AccuPre®2x Greenstar qPCR Master mix (Bioneer, Korea) as
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described previously [31]. The spleen, kidney and blood were obtained from poly (I:C)
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administered fish and PBS injected fish at several sampling points. Each assay was
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performed in duplicate with β-actin RNA as control. The primers used to amplify the
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immune-related genes are listed in Table 2. The quantitation of the mRNA was determined
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by the 2−∆∆Ct method [32].
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For the analysis of the RBIV copy number, genomic DNA was isolated from the whole
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spleen (35–130 mg) of each fish. A standard curve was generated to determine the RBIV
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MCP gene copy number by qRT-PCR as described previously [31]. As MCP-F 5′ 7
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determine the RBIV MCP copy numbers. The virus copy number was determined from 1 µl
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of DNA from 100 µl of total DNA that was extracted from a whole spleen. The detection
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limit of RBIV MCP copy number was 1.0 × 101/µl.
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2.8. Statistical analysis
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The statistical analysis was performed by one-way analysis of variance (ANOVA) using
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GraphPad Prism software version 5.0 for Windows (GraphPad Software, USA) and the
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comparison of gene expressions at different time were obtained by Dunnett’s multiple
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comparison test. A value of p<0.05 was considered to indicate the statistical significance. All
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the data were represented as the means ± the standard error.
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2.9. Relative percent survival (RPS)
The RPS was calculated according to the method described by Amend [33]. RPS = [1-(% mortality of poly (I:C) administered group/ % mortality in control] × 100
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3. Results
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3.1. Effect of poly (I:C) on RBIV infection at 26 °C (Experiment I)
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Rock bream were administered poly (I:C) at 100 and 200 µg/fish, injected with RBIV at 2
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days post poly (I:C) administration, and held 26 °C showed 100% cumulative mortality.
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However, the fish were 3 days to 4 days delayed final mortality as compared to the virus
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injected control fish (Fig. 1). Similar results were obtained in a repeat experiment.
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3.2. Effect of poly (I:C) on RBIV infection at 23 °C (Experiment II)
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days post poly (I:C) administration showed 100% cumulative mortality at 23 °C. However,
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there was a considerable delay in the final mortality compared to the virus injected control
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fish at 6 days (100 µg and 200 µg/fish, respectively) (Fig. 2) (Table 1).
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The fish groups maintained for 2 days at 23 °C with RBIV injection and then administered
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with poly (I:C) at 100 and 200 µg/fish showed 100% cumulative mortality, there was not a
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considerable delay in the final mortality compared to the virus injected control fish (Fig. 2)
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(Table 1).
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The boost immunization group showed 100% cumulative mortality at 23 °C. However,
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there was a considerable delay in the final mortality compared to the virus injected control
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fish at 2 days (100 µg/fish) and 7 days (200 µg/fish) (Fig. 2) (Table 1). Similar results were
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obtained in a repeat experiment.
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3.3. Protection efficacy of poly (I:C) at 20 °C (Experiment II) This experiment was designed to evaluate the efficacy and duration of protection provided
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by administration of the poly (I:C) to rock bream at 100 and 200 µg/fish at 20 °C. At 2 days
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post poly (I:C) administration were exposed to RBIV. The RPS obtained after
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administration was 13.4% (100 µg/fish) and 33.4% (200 µg/fish) for the respective
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administration dose and time points at the end of 200 dpi (Fig. 3) (Table 1).
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The fish groups maintained for 2 days at 20 °C with RBIV injection and then administered
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with poly (I:C) at 100 and 200 µg/fish showed 100% cumulative mortality, and there was a
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considerable delay in the final mortality compared to the virus injected control fish at 4 days
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(100 µg/fish) and 5 days (200 µg/fish) (Fig. 3) (Table 1).
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In boost immunization group; the RPS obtained after administration was 33.4% (100
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µg/fish) and 60.0% (200 µg/fish) for the respective administration dose and time points at the
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the end of 200 dpi (Fig. 3) (Table 1).
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3.4. RBIV re-challenge of survivors
Survivors from 2 days before infection and boost immunization group of Experiment II at
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20 °C, following the re-infection at 200 dpi showed no clinical signs or mortality through 250
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dpi (Fig. 3A and 3C), when they were determined to have low virus copy numbers (average
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1.2 × 101/µl). Specific mortality was observed after 16 days in the virus infected control fish
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(naive fish, not previously exposed to virus), with characteristic clinical signs of the RBIV
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disease, and the mortality reached 100% at 24 dpi. The dead fish demonstrated high MCP
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gene copy numbers in the spleen (greater than 7.1 × 107/µl). RPS values obtained for each
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groups are listed in Table 1.
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3.5. Effect of poly (I:C) on immune gene expression
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3.5.1. TLR3
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In the spleen, TLR3 showed a significantly increased expression level compared with the
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control (3.3-, 6.9-, 3.0- and 3.4-fold) at 12 h, 1 d, 2 d and 4 dpa, respectively (Fig. 4A).
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Significantly upregulation of TLR3 expression was observed at 1 dpa (4.1-fold) (p<0.01) in
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the kidney when compared with the control (Fig. 4A). In the blood, TLR3 expression was
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observed at 1 dpa (2.1-fold) and were significantly higher (p<0.001) than that of the control
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fish; increased TLR3 expression was observed at 2 d and 4 dpa (9.6- and 5.3-fold,
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respectively) compared with the control group (Fig. 4A).
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3.5.2. Inflammatory cytokines (IL1β, IL8 and TNFα) 10
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The expression levels of IL1β, IL8 and TNFα ranged between 0.1 ~ 2.1-fold (12 h to 4
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dpa) in the spleen, kidney and blood, and none of these expression levels showed significant
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differences when compared with those of controls (Fig. 4B, 4C and 4D).
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3.5.3. IRF3, Mx, ISG15 and PKR
A significant increases in IRF3 expression level were observed in the blood at 1 d (2.9fold) (p<0.05) and 2 dpa (3.1-fold) (p<0.01) (Fig. 4E).
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Similar gene expression trends were observed for Mx in the spleen, kidney and blood. Mx
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expression was significantly increased at 12 h (6.6-, 9.5- and 7.3-fold, respectively) and 1
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dpa (5.9-, 12.4- and 8.6-fold, respectively) in the spleen, kidney and blood, respectively (Fig.
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ISG15 and PKR expression was significantly increased in the blood at 2 dpa (5.4- and
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10.2-fold, respectively) (p<0.001) (Fig. 4G and 4H). However, ISG15 and PKR expression
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levels in the spleen and kidney were not significant different from those of the control group
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in this experiment (Fig. 4G and 4H).
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4. Discussion
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Poly (I:C) is believed to activate an antiviral state of sufficient duration for inhibition of
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virus replication to protect fish from virus infection [23-28]. However, there is no report on
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the efficacy of poly (I:C) to provide protection against RBIV infection in rock bream.
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Recently, our previous studies have demonstrated that the virus replication speed with the
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water temperature is likely the more critical factor for rock bream mortality [4]. Hence, in
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the present study, poly (I:C) was evaluated for its antiviral potentials in rock bream under
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various experimental conditions such as different water temperature, different RBIV
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infection dose and the suitable time of poly (I:C) administration.
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104 MCP gene copies/100 µl) in the 100 µg and 200 µg with poly (I:C) administration groups
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showed no protection. A similar observation was reported by Oh et al. 2015 [34]. They
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observed no significantly protective effect (97% mortality) in red seabream iridovirus (RSIV)
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infected rock bream when 200 µg of poly (I:C) was administered at 28 °C. Fish injected with
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RBIV (1.1 × 104 MCP gene copies/100 µl) at 20 °C had a virus concentration that was lower
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than the concentration in the other experiments, showing the highest protection, while the
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virus injected control fish exhibited 100% mortality. This indicates that the poly (I:C) can
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induce better antiviral immune response in administered fish at 20 °C (lower virus replication
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speed) than in administered fish at 23 and 26 °C (higher virus replication speed) against
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RBIV infection.
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Furthermore, it has been reported that the immune effects of fish administered with
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immunostimulants are time dependent [24,35,36]. The before-injected fish with poly (I:C)
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followed by RGNNV challenge exhibited higher survival rates than poly (I:C) administration
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at post infection [24], suggesting that it required several days post poly (I:C) administration
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for the full development of poly (I:C) induced immune response. In agreement with these
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results, we found that poly (I:C) administered before infection showed the highest protective
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effect, while poly (I:C) administration at post infection showed 100% mortality. However,
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poly (I:C) administration at post infection delayed final mortality more than that observed in
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the virus injected control fish in this study. This indicates that although the poly (I:C)
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administration at post infection does not lead to the full development of poly (I:C) induced
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immune response, it could still induce an immune response against RBIV infection. It was
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evident by this study that the poly (I:C) administered two times at before/post infection
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provided 20~30% higher protection rates than poly (I:C) administered once before infection.
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This indicates that survival rates of immunized fish were clearly enhanced by the boost
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administration. To obtain survivors, considering the immunostimulant effects on fish
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mortality and water temperature, the suitable time of immunostimulant administration and
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virus replication speed are likely the critical factor in rock bream mortality because rock
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bream is more susceptible to RBIV infection. Finally, re-infection was tried to evaluate
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immune protection was developed in survived fish from poly (I:C) administration. Survivals
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at 200 dpi showed 100% survival rates at the end of experiment. This observation indicates
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that the immune defense system of survived fish can induced protection in rock bream
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against re-infection RBIV.
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Poly (I:C) exerts its biological effect by interacting with the TLR3. The signaling pathways
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activated by these TLR3 are broadly classified as adaptor protein TIR-domain-containing
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adapter inducing IFN-β (TRIF)-dependent. This activates the transcription factors such as
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interferon regulatory factor (IRF) IRF3, nuclear factor-κB (NF-κB) and activator protein 1
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(AP-1) and induce an antiviral state through secretion of pro-inflammatory cytokines and
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type I IFN [12-14]. We examined the two complementary TLR3-mediated gene expression,
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namely the inflammatory cytokine-mediated immune response and the IFN-mediated
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immune response in poly (I:C) administered rock bream.
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In this study, significantly higher level of TLR3 expression were observed at certain times
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in the spleen, kidney and blood after poly (I:C) administration. These results indicate a vital
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contribution of the TLR3 gene expression to the antiviral activity of poly (I:C) in rock
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bream. However, the expression levels of inflammatory cytokines (IL1β, IL8 and TNFα)
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were low in the spleen, kidney and blood, suggesting that pro-inflammatory gene expression
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was not activated when the rock bream were administered with poly (I:C). A similar
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observation was reported by Hong et al. 2016 [37]. They observed that the poly (I:C) did not
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significantly activated IL1β expression in the spleen and down regulation of IL8 and TNFα in
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head kidney and spleen of rock bream. Mx proteins are particularly induced by poly (I:C), a synthetic double-stranded RNA that is
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a known indicator of IFN production or antiviral activity in a number of fish species [38-43].
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In agreement with these results, Mx expression profiles of rock bream were regulated by poly
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(I:C). Similar significantly high expression patterns of Mx were observed in the spleen,
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kidney and blood in the early administration period at 12 h and 1 dpa, and the expression
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levels then decreased in the late administration period at 2 d to 4 dpa. Similar to our results,
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sevenband grouper (Epinephelus septemfasciatus) with poly (I:C) injection exhibited a high
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level of Mx expression at 3 hpi in head kidney and decreased levels at 24 hpi [22].
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Furthermore, a significantly high expression level of IRF3, ISG15 and PKR was observed
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only in the blood; there was no significantly upregulation of these interferon-related genes in
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the spleen and kidney. These observations highlight the importance of interferon-related gene
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measurement in blood and indicate the need for more detailed studies to obtain a better
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understanding of blood interferon-related gene levels to identify blood-interferon-related
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immune response interaction in rock bream.
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In conclusion, the present study demonstrated for the first time the possibility of developing
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long-term preventive measures against RBIV using poly (I:C). The protection against RBIV
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might have occurred through administration of poly (I:C) due to the activation of immune
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response-related genes such as TLR3, IRF3, Mx, ISG15 and PKR in rock bream.
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Acknowledgements
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This research was supported by Basic Science Research Program through the National
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Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future
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Planning (2015R1C1A1A01053685).
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References
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[1] Kurita J, Nakajima K. Megalocytiviruses. Viruses 2012;4:521–38.
349
[2] Jung SJ, Oh MJ. Iridovirus-like infection associated with high mortalities of striped
350
beakperch, Oplegnathus fasciatus (Temminck et Schlegel), in southern coastal areas of
351
the Korean peninsula. J Fish Dis 2000;23:223–6.
TE D
M AN U
347
[3] Jun LJ, Jeong JB, Kim JH, Nam JH, Shin KW, Kim J.K, et al. Influence of temperature
353
shifts on the onset and development of red sea bream iridoviral disease in rock bream
354
Oplegnathus fasciatus. Dis Aquat Org 2009;84: 201–8.
EP
352
[4] Jung MH, Jung SJ, Vinay TN, Nikapitiya C, Kim JO, Lee JH, et al. Effects of water
356
temperature on mortality in Megalocytivirus-infected rock bream Oplegnathus fasciatus
357
(Temminck et Schlegel) and development of protective immunity. J Fish Dis
358
2015;38:729–37.
AC C
355
359
[5] Jung MH, Lee J, Jung SJ. Low pathogenicity of FLIV (flounder iridovirus) and the
360
absence of cross-protection between FLIV and RBIV (rock bream iridovirus). J Fish Dis
361
2016;39:1325–33.
15
ACCEPTED MANUSCRIPT
363 364 365
[6] Secombes C. Will advances in fish immunology change vaccination strategies. Fish Shellfish Immunol 2008;25:409–16. [7] Ragnar T, Powell DB. Effects of disease risk, vaccine efficacy, and market priceon the economics of fish vaccination. Aquaculture 2006;256:42–9.
RI PT
362
366
[8] Vinay TN, Kim YJ, Jung MH, Kim WS, Kim DH, Jung SJ. Inactivated vaccine against
367
viral hemorrhagic septicemia (VHS) emulsified with squalene and aluminum hydroxide
368
adjuvant
369
olivaceus). Vaccine 2013;31:4603–10.
long
term
protection
in
olive
flounder
(Paralichthys
SC
provides
[9] Tafalla C, Bøgwald J, Dalmo RA. Adjuvants and immunostimulants in fish vaccines:
371
current knowledge and future perspectives. Fish Shellfish Immunol 2013;35:1740–50.
M AN U
370
372
[10] Akira S. Toll-like receptors and innate immunity. Adv Immunol 2001;78:1–56.
373
[11] Medzhitov
Janeway
C.
Innate
immune
recognition:
mechanisms
and
pathways. Immunol Rev 2000;173:89–97.
TE D
374
R,
[12] Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T. TICAM-1, an adaptor
376
molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat
377
Immunol 2003;4:161–7.
EP
375
[13] Lee KG, Xu S, Kang ZH, Huo J, Huang M, Liu D, et al. Bruton's tyrosine kinase
379
phosphorylates Toll-like receptor 3 to initiate antiviral response. P Natl Acad Sci USA
380
2012;109:5791–96.
AC C
378
381
[14] Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, et al. Role of adaptor
382
TRIF in the MyD88-independent toll-like receptor signaling pathway. Science
383
2003;301:640–3.
384 385
[15] Eaton WD. Antiviral activity in 4 species of salmonids following exposure to poly inosinic-cytidylic acid. Dis Aquat Org 1990;9:193–8.
16
ACCEPTED MANUSCRIPT 386
[16] Nygaard R, Husgard S, Sommer AI, Leong JAC, Robertsen B. Induction of Mx protein
387
by interferon and double-stranded RNA in salmonid cells. Fish Shellfish Immunol
388
2000;10:435–50.
390
[17] Lockhart K, Bowden TJ, Ellis AE. Poly I:C-induced Mx responses in Atlantic salmon
RI PT
389
parr, post-smolts and growers. Fish Shellfish Immunol 2004;17:245–54.
[18] Acosta F, Lockhart K, Gahlwat SK, Real F, Ellis AE. Mx expression in Atlantic salmon
392
(Salmo salar L.) parr in response to Listonella anguillarum bacterin, lipopolysaccharide
393
and chromsomal DNA. Fish Shellfish Immunol 2004;17:255–63.
SC
391
[19] Jensen I, Albuerque A, Sommer AI, Robertsen B. Effect of poly I:C on the expression of
395
Mx proteins and resistance against infection by infectious salmon anaemia virus in
396
Atlantic salmon. Fish Shellfish Immunol 2002;13:311–26.
M AN U
394
[20] Plant KP, Harbottle H, Thune RL. Poly I:C induces an antiviral state against Ictalurid
398
Herpesvirus 1 and Mx1 transcription in the channel catfish (Ictalurus punctatus). Dev
399
Comp Immunol 2005;29:627–35.
TE D
397
[21] Zhou ZX, Zhang BC, Sun L. Poly (I: C) induces antiviral immune responses in Japanese
401
flounder (Paralichthys olivaceus) that require TLR3 and MDA5 and is negatively
402
regulated by Myd88. PloS one 2014;9:e112918.
EP
400
[22] Thanasaksiri K, Sakai N, Yamashita H, Hirono I, Kondo H. Influence of temperature on
404
Mx gene expression profiles and the protection of sevenband grouper, Epinephelus
405
septemfasciatus, against red-spotted grouper nervous necrosis virus (RGNNV) infection
406
after poly (I:C) injection. Fish Shellfish Immunol 2014;40:441–5.
AC C
403
407
[23] Kim HJ, Oseko N, Nishizawa T, Yoshimizu M. Protection of rainbow trout from
408
infectious hematopoietic necrosis (IHN) by injection of infectious pancreatic necrosis
409
virus (IPNV) or Poly (I:C). Dis Aquat Org 2009;83:105–13.
17
ACCEPTED MANUSCRIPT 410
[24] Nishizawa T, Takami I, Kokawa Y, Yoshimizu M. Fish immunization using a synthetic
411
double-stranded RNA Poly (I:C), an interferon inducer, offers protection against
412
RGNNV, a fish nodavirus. Dis Aquat Org 2009;83:115–22. [25] Nishizawa T, Takami I, Yoshimizu M, Oh MJ. Required dose of fish nervous necrosis
414
virus (NNV) for Poly (I:C) immunization of sevenband grouper Epinephelus
415
septemfasciatus. Aquaculture 2011;311:100–4.
RI PT
413
[26] Oh MJ, Takami I, Nishizawa T, Kim WS, Kim CS, Kim SR, et al. Field tests of Poly
417
(I:C) immunization with nervous necrosis virus (NNV) in sevenband grouper,
418
Epinephelus septemfasciatus (Thunberg). J Fish Dis 2012;35:187–91.
M AN U
SC
416
419
[27] Takami I, Kwon SR, Nishizawa T, Yoshimizu M. Protection of Japanese flounder
420
Paralichthys olivaceus from viral hemorrhagic septicemia (VHS) by Poly (I:C)
421
immunization. Dis Aquat Org 2010;89:109–15.
[28] Nishizawa T, Takami I, Yang M, Oh MJ. Live vaccine of viral hemorrhagic septicemia
423
virus (VHSV) for Japanese flounder at fish rearing temperature of 21 °C instead of Poly
424
(I:C) administration. Vaccine 2011;29: 8397–404.
TE D
422
[29] Alcorn SW, Murray AL, Pascho RJ. Effects of rearing temperature on immune functions
426
in sockeye salmon (Oncorhynchus nerka). Fish Shellfish Immunol 2002;12:303–34.
427
[30] Bly JE, Clem LW. Temperature and teleost immune functions. Fish Shellfish Immunol
AC C
428
EP
425
1992;2:159–71.
429
[31] Jung MH, Nikapitiya C, Song JY, Lee JH, Lee JH, Oh MJ, et al. Gene expression of pro-
430
and anti-apoptotic proteins in rock bream (Oplegnathus fasciatus) infected with
431
Megalocytivirus (family Iridoviridae). Fish Shellfish Immunol 2014;37:122–30.
432 433
[32] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–8.
18
ACCEPTED MANUSCRIPT 434
[33] Amend DF. Potency testing of fish vaccines. In: Anderson DP, Hennessen W, editors.
435
Developments in Biological Standardization, vol. 49. Basel: Fish bio-logics.
436
Serodiagnostics and Vaccines; 1981. p. 447–54. [34] Oh SY, Kim WS, Oh MJ, Nishizawa T. Quantitative change of red seabream iridovirus
438
(RSIV)
439
administration. Aquacult Int 2015;23:93–8.
441
rock
bream
Oplegnathus
fasciatus,
following
Poly
(I:C)
[35] Tassakka ACMA, Sakai M. The in vitro effect of CpG-ODNs on the innate immune
SC
440
in
RI PT
437
response of common carp, Cyprinus carpio L. Aquaculture 2003;220:27–36. [36] Nakatani M, Iwasaki T, Watarai S, Kodama H. In vitro enhancement by CpG ODN of
443
phagocytic activity of rainbow trout head kidney phagocytes against fish pathogen Vibrio
444
ordalii, but not against polystyrene particles. J Vet Med Sci 2007;69:1287–90.
M AN U
442
[37] Hong S, Jin JW, Park JH, Kim JK, Jeong HD. Analysis of proinflammatory gene
446
expression by RBIV infection in rock bream, Oplegnathus faciatus. Fish Shellfish
447
Immunol 2016;50:317–26.
TE D
445
[38] Fernandez-Trujillo A, Ferro P, Garcia-Rosado E, Infante C, Alonso MC, Bejar J, et al.
449
Poly I:C induces Mx transcription and promotes an antiviral state against sole
450
aquabirnavirus in the flatfish Senegalese sole (Solea senegalensis Kaup). Fish Shellfish
451
Immunol 2008;24:279–85.
AC C
EP
448
452
[39] Fernández-Trujillo MA, Novel P, Manchado M, Sepulcre MP, Mulero V, Borrego JJ, et
453
al. Three Mx genes with differential response to VNNV infection have been identified in
454
Gilthead seabream (Sparus aurata). Mol Immunol 2011;48:1216–23.
455
[40] Chen YM, Su YL, Lin JHY, Yang HL, Chen TY. Cloning of an orange-spotted grouper
456
(Epinephelus coioides) Mx cDNA and characterisation of its expression in response to
457
nodavirus. Fish Shellfish Immunol 2006;20:58–71.
19
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[41] Chen YM, Su YL, Shie PS, Huang SL, Yang HL, Chen TY. Grouper Mx confers
459
resistance to nodavirus and interacts with coat protein. Dev Comp Immunol
460
2008;32:825–36. [42] Trobridge GD, Chiou PP, Kim CH, Leong JC. Induction of the Mx protein of rainbow
462
trout Oncorhynchus mykiss in vitro and in vivo with poly I:C dsRNA and infectious
463
hematopoietic necrosis virus. Dis Aquat Org 1997;30:91–8.
RI PT
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[43] Ohta T, Ueda Y, Ito K, Miura C, Yamashita H, Miura T, et al. Anti-viral effects of
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interferon administration on sevenband grouper, Epinephelus septemfasciatus. Fish
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Shellfish Immunol 2011;30:1064–1071.
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SC
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Figure legend
468
Figure 1. The mortality patterns of rock bream in the poly (I:C) treated groups (100 and 200
469
ug/fish) and virus injected control group following an i.p. injected at 26 °C with RBIV (1.1 ×
470
107) at 2 days post poly (I:C) administration.
471
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Figure 2. The effect of poly (I:C) on RBIV (1.1 × 104) at 23 °C. Fifteen fish in each group
473
were intraperitoneally (i.p) injected with poly (I:C) at 100 µg/fish. A) poly (I:C)
474
administration at 2 days before infection. B) poly (I:C) administration at 2 days post infection.
475
C) poly (I:C) administration at 2 days before/post infection (boost immunization).
AC C
476
EP
472
477
Figure 3. Protection efficacy of poly (I:C) at 20 °C. Fifteen fish in each group were
478
administered i.p. with poly (I:C) (100 µg/fish) at 2 days before infection (A), 2 days post
479
infection (B) and 2 days before/post infection (boost immunization) (C). To establish mild
480
infection to reduce virus replication speed, fish were injected with RBIV (1.1 × 104) and
20
ACCEPTED MANUSCRIPT 481
maintained at 20 °C in the aquaria. Surviving fish were infected a second time (period of 200
482
to 250 dpi with 1.1 × 107 MCP copies/fish).
RI PT
483
Figure 4. Relative expression analysis of immune response-related genes in the spleen,
485
kidney and blood of poly (I:C) administered rock bream. The expression level of β-actin was
486
used as an internal control for a real-time PCR-based relative expression study. Each
487
experiment was performed in duplicate. The bars represent the standard error (SE) of the
488
mean for 5 individuals. A- TLR3; B- IL1β; C- IL8; D- TNFα; E- IRF3; F- Mx; G- ISG15; H-
489
PKR. The relative expression fold-change at 0 h post injection (before injection) was used as
490
the basal line. Differences between the poly (I:C) administered rock bream and non-
491
administered rock bream were compared using a one-way analysis of variance followed by
492
the Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01 and ***p < 0.001 were
493
considered significant.
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EP
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21
ACCEPTED MANUSCRIPT
Table 1 Summary of poly (I:C) efficacy test.
Boost immunization (2 days before/post infection)
First infection Control Second infection
RBIV infection dose
Day of final mortality
Mortality% at 100 dpi
RPS
RBIV re-infection for survivors
RBIV infection dose
Days observed
Mortality%
100 µg
23 °C
15
1.1 × 104/fish
29 day
100%
-
-
-
-
-
200 µg
23 °C
15
1.1 × 104/fish
29 day
100%
-
-
-
-
-
100 µg
20 °C
15
1.1 × 104/fish
36 day
86.6%
13.4%
200 dpi
1.1 × 107/fish
50 days
0%
200 µg
20 °C
15
1.1 × 104/fish
32 day
66.6%
33.4%
200 dpi
1.1 × 107/fish
50 days
0%
100 µg
23 °C
15
1.1 × 104/fish
23 day
100%
-
-
-
-
-
200 µg
23 °C
15
1.1 × 104/fish
23 day
100%
-
-
-
-
-
100 µg
20 °C
15
1.1 × 104/fish
38 day
100%
-
-
-
-
-
200 µg
20 °C
15
1.1 × 104/fish
39 day
100%
-
-
-
-
-
100 µg
23 °C
15
1.1 × 104/fish
25 day
100%
-
-
-
-
-
200 µg
23 °C
15
1.1 × 104/fish
30 day
100%
-
-
-
-
-
100 µg
20 °C
15
1.1 × 104/fish
37 day
66.6%
33.4%
200 dpi
1.1 × 107/fish
50 days-
0%
200 µg
20 °C
15
1.1 × 104/fish
35 day
39.9%
60.0%
200 dpi
1.1 × 107/fish
50 days
0%
-
23 °C
15
1.1 × 104/fish
23 day
100%
-
-
-
-
-
-
20 °C
15
1.1 × 104/fish
34 day
100%
-
-
-
-
-
-
20 °C
15
-
-
-
-
200 dpi
1.1 × 107/fish
24 days
100%
M AN U
SC
RI PT
No. of fish
TE D
2 days post infection
RBIV infection at
EP
2 days before infection
Poly (I:C) administration
AC C
Group (time of poly (I:C) administration)
ACCEPTED MANUSCRIPT
Table 2 Primers used in this study.
Sequence
Accession number
RI PT
Name β-actin
F CAGGGAGAAGATGACCCAGA R CATAGATGGGCACTGTGTGG
MCP
F TGCACAATCTAGTTGAGGAGGTG R AGGCGTTCCAAAAGTCAAGG
TLR3
F TAAAGCCCATCAGGCACTTCAC R AGGAAGAGTGAGCGAGACAACC
KY613955
IL1β
F ATCTGGAGACGGTGGACAAC R GCTGATGTACCAGTCGCTGA
KC522967
IL8
F CCCTCCTGACCATCAGTGAA R TGATCTCAGTCTCCTCGCAGT
KC522965
TNFα
F GCAATCAGGCCAAACAGAAGCACT R TTGGCTTTGCTGCTGATACGCTTC
FJ623187
IRF3
F GTGTCTAAAGGGACTGAACATCG R CCCTCAAACGTTACTGGATACTG
KF267453.1
Mx
F GATCGCCTCTCCTGATGTTC R ACCACCAAGCTGATGGTTTC
FJ155359.1
ISG15
F CTTTAAGACCAAGGTCCAATGC R GCCTCCACATTGTAGTCTGAAAG
BAJ16365.1
F GCTCAACAAATAGTCAGTGGAGT R CTCTGGTGACCAGACCAAAGTCC
FJ179396.1
SC
M AN U
TE D
EP
AC C
PKR
FJ975145
AY849394
ACCEPTED MANUSCRIPT
Poly (I:C) administration at 26 °C at 2 days before infection 100 200 µg
80
RI PT
Virus injected control 60
SC
40 20
5
10
15
TE D
Days post infection
EP
0
M AN U
0
AC C
Cumulative mortality%
100 µg
Figure 1
ACCEPTED MANUSCRIPT
Poly (I:C) administration at 23 °C at 2 days before infection
A
80 60
RI PT
100 µg 200 µg Virus injected control
40
SC
Cumulative mortality%
100
20
0
10
M AN U
0 20
30
Poly (I:C) administration at 23 °C at 2 days post infection
B
80
EP
100 µg
60
200 µg 40
Virus injected control
20
AC C
Cumulative mortality%
100
TE D
Days post infection
0 0
5
10
15
20
25
Days post infection
Figure 2 is continued to the next page
ACCEPTED MANUSCRIPT
Poly (I:C) administration at 23 °C at 2 days before/post infection
C
80
RI PT
100 µg (boost)
60
200 µg (boost) 40
SC
Virus injected control
20
10
15
20
25
Days post infection
30
TE D
5
EP
0
M AN U
0
AC C
Cumulative mortality%
100
Figure 2
ACCEPTED MANUSCRIPT
Poly (I:C) administration at 20 °C at 2 days before RBIV infection
A
60 100 µg 40
200 µg 1st virus injected control
20
Second infection
2nd virus injected control 0
B
10
20
30
40
50 60 200 Days post infection
100 µg
240
250
Virus injected control
20
AC C
200 µg 40
230
EP
80 60
220
TE D
Poly (I:C )administration at 2 days post infection at 20 °C
100
210
M AN U
0
Cumulative mortality%
RI PT
80
SC
Cumulative mortality%
100
0 0
5
10
15
20
25
30
35
40
Days post infection
Figure 3 is continued to the next page
ACCEPTED MANUSCRIPT
80
200 µg (boost) 1st virus injected control 2nd virus injected control
60 40
Second infection
20 0 30
40
50 60 200 Days post infection
210
220
M AN U
20
230
240
250
TE D
10
EP
0
SC
100 µg (boost)
AC C
Cumulative mortality%
100
RI PT
Poly (I:C) administration at 20 °C at 2 days before/post infection
C
Figure 3
ACCEPTED MANUSCRIPT
TLR3 1d
15
*** 10
*** **
** * *
*
1d
12 h
4d
2d
8
6
4
2
0
Spleen
Kidney
Blood
IL8
C
12 h
1d
2d
AC C
8
6
4
2
0
Spleen
10
Kidney
Blood
Organs
TNFα
D
4d
EP
0h
TE D
Organs
10
0h
M AN U
5
0
Relative expression to β.actin
10
4d
2d
RI PT
12 h
SC
0h
Relative expression to β.actin
Relative expression to β.actin
20
IL1β
B
Relative expression to β.actin
A
0h
12 h
1d
4d
2d
8
6
4
2
0 Spleen
Kidney Organs
Blood
Spleen
Kidney
Blood
Figure 4 is continued to the next page Organs
ACCEPTED MANUSCRIPT
IRF3 1d
12 h
Relative expression to β.actin
8
6
*
4
**
10
**
1d
12 h
*
4d
2d
** ** *
*
0
Spleen
Kidney
Blood
ISG15
G 0h
12 h
1d
2d
TE D
Organs
***
AC C
8
6
4
2
0
Spleen
20
Kidney
Blood
Organs
PKR
H
4d
EP
10
20
0h
M AN U
2
0
Relative expression to β.actin
30
4d
2d
RI PT
0h
Relative expression to β.actin
Relative expression to β.actin
10
Mx
F
SC
E
0h
12 h
1d
15
4d
2d
***
10
5
0 Spleen
Kidney Organs
Blood
Spleen
Kidney Organs
Blood
Figure 4
ACCEPTED MANUSCRIPT Highlights: Treatment with poly (I:C) at before/post infection had significantly higher protection. Poly (I:C) provided significant protection for 200 days. TLR3 and Mx were highly expressed in poly (I:C) administered rock bream.
AC C
EP
TE D
M AN U
SC
RI PT
IRF3, ISG15 and PKR induced immune responses were high activated in blood.