Accepted Manuscript

Cyprinid viral diseases and vaccine development

Hang Su, Jianguo Su

PII: S1050-4648(18)30539-4
DOI: 10.1016/j.fsi.2018.09.003
Reference: YFSIM 5524

To appear in: Fish and Shellfish Immunology

Received Date: 15 July 2018

Revised Date: 31 August 2018
Accepted Date: 5 September 2018

Please cite this article as: Su H, Su J, Cyprinid viral diseases and vaccine development, Fish and
Shellfish Immunology (2018), doi: 10.1016/j.fsi.2018.09.003.

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2 Cyprinid Viral Diseases and Vaccine Development

4 Hang Sua, Jianguo Sua,b,*

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5

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6 Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University,

7 Wuhan 430070, China

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8 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine

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9 Science and Technology (Qingdao), Qingdao 266237, China
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15 Keywords: Cyprinid; Viral disease; Vaccine

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19 * Corresponding author: Tel/Fax: 86-27-87282227

20 E-mail address: sujianguo@mail.hzau.edu.cn (Jianguo Su).

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22 Highlights

23 1. Reviewed the six most popular cyprinid viral diseases.

24 2. Summarized the development of aquaculture vaccine.

25 3. Characterized the vaccines specifically against KHV, GCRV and SVCV in detail.

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27 Abstract

28 In the past decades, global freshwater fish production has been rapidly growing,

29 while cyprinid takes the largest portion. Along with the rapid rise of novel forms of

30 intensive aquaculture, increased global aquatic animal movement and various

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31 anthropogenic stress to aquatic ecosystems during the past century, freshwater fish

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32 farming industry encounter the emergence and breakout of many diseases, especially

33 viral diseases. Because of the ability to safely and effectively prevent aquaculture

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34 diseases, vaccines have become the mainstream technology for prevention and control

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35 of aquatic diseases in the world. In this review, authors summarized six major
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36 cyprinid viral diseases, including koi herpesvirus disease (KHVD), spring viraemia of

37 carp (SVC), grass carp hemorrhagic disease (GCHD), koi sleepy disease (KSD), carp
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38 pox disease (CPD) and herpesviral haematopoietic necrosis (HPHN). The present
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39 review described the characteristics of these diseases from epidemiology, pathology,

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40 etiology and diagnostics. Furthermore, the development of specific vaccines

41 respective to these diseases is stated according to preparation methods and

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42 immunization approaches. It is hoped that the review could contribute to aquaculture

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43 in prevention and controlling of cyprinid viral diseases, and serve the healthy and
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44 sustainable development of aquaculture industry.

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46 Keywords

47 Cyprinid; Viral disease; Vaccine

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49 1. Introduction

50 Aquaculture is an ancient practice and it is believed to date back to China at least

51 4000 years ago. There are also references of fish ponds in The Old Testament and in

52 Egyptian hieroglyphics of the Middle Kingdom. Fish farms were common in Europe

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53 in Roman times and a recent study of land forms in the Bolivian Amazon has revealed

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54 a complex array of fish weirs that pre-date the Hispanic era [1, 2]. Despite its ancient

55 origin, aquaculture remained a low-level farming activity until the mid-20th century

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56 when experimental husbandry practices for salmon, trout and a mass of tropical fish

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57 and shrimp species were developed. Nowadays, aquaculture is commended to be a
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58 major global industry, with a total annual production exceeding 80 million ton and

59 estimated value of almost 230 billion US dollar (Food and Agriculture Organization
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60 of the United Nations, FAO, 2016). In the past three decades, aquaculture industry has
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61 made impressive progress and constitutes high quality protein for world population
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62 accounting for nearly 50% of the global food fish supply in 2016. In addition to being

63 an important form of nutrition, aquaculture is also a critical form of employment

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64 (FAO, 2018).
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65 Aquaculture is a highly dynamic production and is characterized by enormous

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66 diversity in both the farmed species range and the production systems feature. Over

67 350 different species of aquatic animals are cultured in global in freshwater,

68 brackishwater and marine environments. In 2016, the yield of freshwater fish

69 production reached 46.6 million ton (FAO, 2016). Carps (Cyprinidae) make up almost

70 65% of the global farmed freshwater fish production and are an important source of
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71 food in China and India where 72.8% and 13.8%, respectively, of farmed cyprinid are

72 produced (FAO, 2016). Economic aquaculture species belonging to cyprinid

73 principally include grass carp (Ctenopharyngodon idella), common carp (Cyprinus

74 carpio), crucian carp (Carassius auratus), black carp (Mylopharyngodon piceus),

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75 silver carp (Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis) and

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76 blunt snout bream (Megalobrama amblycephala), etc. Meanwhile, in the international

77 ornamental fish trade, more than 90 % of the freshwater fishes are farm bred, and

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78 about 4000 species of freshwater ornamental fishes are traded every year [3]. Goldfish

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79 (Carassius auratus auratus) and koi (Cyprinus carpio haematopterus) are the most
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80 extensively traded fish species [4].

81 Importantly, the global fish trade relocates large quantities of live fish species
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82 between countries, and therefore it can be a potential source for spread of exotic
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83 pathogens, particularly viral pathogens, which have been associated with high
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84 morbidity and mortality of farmed fishes [5, 6]. In the past century, the rise of new

85 forms of intensive aquaculture, the increasing global movement of aquatic animals

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86 and their products, and various artificial pressures to aquatic ecosystems have led to
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87 the emergence and outbreak of numbers of diseases in fishes. The global expansion of
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88 fish farming industry and the consequent improvement in fish health monitoring have

89 led to discovery of several new scientific viruses, which are almost endemic in local

90 population and overflow opportunistically to infect fishes in aquaculture facilities,

91 while other well-known fish viruses also cause significant losses in aquaculture.

92 Diseases caused by various fish viruses, bacteria, parasites and other pathogens
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93 pose great hazards to aquaculture industry and threat to food safety. As people pay

94 more and more attention to the quality and safety of aquatic products and

95 environmental pollution, the utilization of various chemical drugs to prevent diseases

96 in aquaculture animals has been increasingly questioned. At present, prevention and

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97 control of aquaculture diseases mainly include methods of drug control and immune

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98 prevention. The potential risks of drug resistance, allergic reactions and poisoning

99 reactions caused by drug residues have a serious impact on the environment, farmed

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100 animals and consumption of cultured products. Immunoprevention can not only

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101 improve the specific immunity of fish, but also can enhance the ability to resist stress,
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102 meeting the requirements of no environment pollution and no residues in aquatic

103 products. Therefore, due to its ability to safely and effectively prevent aquaculture
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104 diseases, vaccines have become the mainstream technology for prevention and control
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105 of aquatic diseases in the world. Vaccination has also become the normative
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106 production standard for international modern aquaculture. In the late 1990s, the

107 commercialization of fish vaccines developed rapidly. According to incomplete

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108 statistics, there are 38 global approvals in 2003, over 100 in 2006, and over 140 in
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109 2012 [7].

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110 In the following sections, authors summarized the major emerging viral diseases

111 that cause major losses in cyprinid fish farming, expanding in host or geographic

112 scope due to the risk of spreading through the commercial trade of finfish. The

113 respective pathogen-targeted vaccines were described subsequently. It is hoped that

114 the information provided in this review could contribute to cyprinid farming industry
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115 decreasing the economic loss caused by viral disease and improving the quality of

116 aquatic products.

117 2. Cyprinid viral diseases

118 Six cyprinid viral diseases are summarized in Table 1.

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119 2.1. Koi herpesvirus disease

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120 Koi herpesvirus disease (KHVD) caused by koi herpesvirus (KHV) [8] is a listed

121 notifiable disease to the International Office of Epizootics (OIE, 2018), spreading to

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122 most regions around the world due to the global fish trade and international

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123 ornamental koi shows [9, 10] (FAO, 2018). First detected in the late 1990s, KHVD
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124 has been found in different continents (Europa, Asia, North America and Africa),

125 leading to serious worldwide financial losses in common carp and koi culture
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126 industries. Once fish are infected, the virus persists for some period of time in a latent
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127 or carrier state without obvious clinical signs [11]. Molecular analysis shows that little
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128 variation among isolates is found, as it might be expected for a virus that is being

129 rapidly disseminated by the global movement of infected fish [12].

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130 KHVD is relatively host-specific， while only common carp and its ornamental
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131 subspecies, koi [13], are involved in the explosive losses reported globally [8]. In
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132 addition to its negative economical and societal impacts, KHVD has also a negative

133 environmental impact by affecting wild populations of carp. In addition, hybrids of

134 common carp and goldfish show partly susceptibility to KHV infection, however the

135 mortality rate is observed to be rather limited [14]. Cohabitation experiments indicate

136 that some carp species such as goldfish, common bream (Abramis brama), silver carp
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137 and grass carp, can carry KHV asymptomatically and transmit it to wild carp [15-19].

138 KHVD is seasonal, occurring when water temperature is between 18 oC and 28 oC. It

139 is highly contagious and extremely virulent with mortality rate up to 80% to 100%.

140 Fish infected with KHV and kept at 23-28 °C die between 5 and 22 dpi (days post

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141 infection) with a peak of mortality between 8 and 12 dpi [8, 20, 21]. Carp of all ages,

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142 from juveniles upwards, are affected by KHV, but younger fish (1-3 months, 2.5-6 g)

143 seem to be more susceptible to infection than mature fish (1 year old, nearly 230 g)

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144 [22].

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145 Loss of osmoregulation of the gill, gut and kidney contributes to mortality during
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146 acute infection with KHV [23]. After infection, the initial clinical signs appear at 2-3

147 dpi, when fish become lethargic, lose appetite and lie at the bottom of the tank with
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148 the dorsal. In ponds, infected fish usually gasp at the surface of water gathering close
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149 to the water inlet or sides of the pond. Gill necrosis coupled with extensive
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150 discoloration and increased mucus secretion appear as early as 3 dpi. The skin

151 exhibits different clinical signs depending on the stage of the infection. Hyperemia
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152 particularly at the base of the fins and on the abdomen and pale, irregular patches on
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153 the skin associated with mucus hyper-secretion often appear at the beginning of
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154 infection. Then, dead epithelium peels away lacking of mucus cover. Appearance of

155 epidermis surface with a sandpaper-like texture and herpetic lesions are observed at

156 the following infection condition [24]. In the later stages of infection, fin erosion and

157 bilateral enophthalmia are observed. Some fish show neurologic signs, getting

158 disoriented and losing equilibrium in the final stage of disease [8, 22, 25-28].
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159 The enveloped virion of KHV is formally classified as the species Cyprnid

160 herpesvirus 3 (CyHV-3). CyHV-3 is a member of genus Cyprinivirus, family

161 Alloherpesviridae, order Herpesvirales [29]. The Alloherpesviridae is a newly

162 designated family which regroups herpesviruses infecting fish and amphibians [30],

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163 being divided into four genera: Cyprinivirus, Ictalurivirus, Salmonivirus and

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164 Batrachovirus [29]. The genus Cyprinivirus contains viruses that infect common carp

165 (Cyprinid herpesvirus, CyHV-1 and CyHV-3), goldfish (Cyprinid herpesvirus 2,

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166 CyHV-2) and freshwater eel (Anguillid herpesvirus 1, AngHV-1) (International

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167 Committee on Taxonomy of Viruses, ICTV, 2017). The genome of CyHV-3 is a
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168 typical herpesvirus containing a 295 kb, linear, double stranded DNA molecule

169 consisting of a large central portion flanked by two 22 kb repeat regions [31]. So far,
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170 it is the largest genome among all sequenced herpesviruses [32].

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171 Various CyHV-3 diagnostic methods have been developed based on detection of
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172 infectious particles, viral DNA, transcripts or antigens.. A series of molecular

173 techniques for detection of viral DNA fragments has been developed, such as DNA
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174 hybridization, PCR, nested PCR, one-tube semi-nested PCR, semi-quantitative PCR,
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175 real-time TaqMan PCR, and loop-mediated isothermal amplification [33]. CyHV-3
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176 can be detected in environmental water, infected fish tissues and cell cultures by

177 real-time PCR after viral concentration [34, 35], in carp serum by ELISA aiming at

178 the specific anti-CyHV-3 antibodies [33], in tissues and touch imprints of organs from

179 infected fish by immunohistochemistry, immunofluorescence assays [36] and

180 monoclonal antibodies against CyHV-3 ORF68 without cross-reaction against

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181 CyHV-1 and CyHV-2 [37]. A CyHV-3-detection kit (The FASTest® Koi HV kit)

182 allows detection of CyHV-3 in gill swabs in just 15 min [38].

183 CyHV-3 also attracts originally fundamental researches. It is phylogenetically

184 distant from the vast majority of studied herpesviruses, thereby providing an original

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185 field of research. As the genome sequence published, it is revealed to be a fascinating

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186 virus with unique properties in the Herpesvirales, such as an extremely large genome,

187 a high number of genes which are not homologous to known viral sequences and

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188 genes that are normally found exclusively in the Poxviridae [31]. Meanwhile,

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189 CyHV-3 genome analysis also reveals several genes encoding proteins potentially
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190 involved in immune evasion mechanisms [31].

191 Immune system of vertebrate recognizes virus particles by pattern recognition

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192 receptors (PRRs), which senses particular protein ligand and virus nucleic acids [39].
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193 PRRs detect viral pathogen associated molecular patterns (PAMPs) to trigger
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194 intracellular innate immune pathways and subsequent adaptive immune responses by

195 producing cytokines, chemokines, T-cell stimulatory factors, etc [40]. Mammalian
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196 cells have the capacity to detect viral proteins using a series of receptors, such as cell
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197 surface toll-like receptor 2 (TLR2) [41] and a multiple set of cytoplasm receptors to
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198 viral DNA including a DNA dependent activator of interferon regulatory factors (DAI)

199 [42], the interferon-inducible protein 16 (IFI16) [43] and RNA polymerase III [44]. In

200 teleost, endosome TLR9 has been identified in numbers of carp species [45-47], but

201 further information on receptors for viral DNA remains unclear and the recognition

202 mechanism of CyHV-3 by cyprinid cells need further research. The recognition of
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203 viral molecular patterns leads to the production of type I interferon (IFN-I), which is

204 crucial in antiviral response to many virus infections [48-50]. IFNs bind to specific

205 cell-surface receptors, then trigger the JAK/STAT signal transduction pathway and

206 subsequently activate interferon stimulated genes (ISGs), which encode for a wide

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207 array of proteins with antiviral properties [51]. In carp cells, CyHV-3 can be interfered

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208 by the activation of IFN-I. poly(I:C) stimulation in fibroblastic carp cells induces the

209 expression of IFN-I, limiting CyHV-3 infectivity and replication, delaying the

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210 spreading of CyHV-3 in vitro [52]. In carp infected by CyHV-3, IFN-I response is

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211 detected in skin at 24 h post infection [53] and subsequently in spleen [52]. IFN-I
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212 system triggers the expression of a quantity of genes encoding antiviral proteins,

213 including grass carp reovirus-induced genes 1 (GIG-1) protein, ubiquitin like protein
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214 (ISG15) and ubiquitin specific protease 18 (USP18), inhibiting viral replication
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215 directly [52]. Furthermore, in CyHV-3 infected carp, genes encoding chemotactic
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216 cytokines of CC family are up-regulated [54]. Additional observation indicated that

217 the induced inflammation may derive from the rapid prominent leukocytosis in the
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218 blood cycle of carps during the initial two days after CyHV-3 infection. Complement
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219 system also participate in the reaction against CyHV-3 displayed as pathway
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220 triggering and complement hemolytic activity increasing in blood plasma [52]. The

221 activation of the complement system leads to neutralisation of viruses, phagocytosis

222 of coated virus particles or elimination of virus infected cells by lysis. In conclusion,

223 innate immune system including IFN-I, inflammation and complement are induced by

224 CyHV-3 infection, while the importance for resisting the infection still needs to be
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225 addressed in greater detail.

226 2.2. Spring viraermia of carp

227 Spring viraemia of carp (SVC) is one of ten piscine viruses listed by the OIE

228 (OIE, 2018) as a notable animal disease. It is caused by a fish rhabdovirus, spring

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229 viraemia of carp virus (SVCV). However, unlike infectious haematopoietic necrosis

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230 virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), SVCV is related to

231 genus Vesiculovirus of the family Rhabdoviridae, order Mononegavirales [55]. It

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232 exhibits an enveloped, bullet-shaped virion and measures ~80-180 nm in length and

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233 60-90 nm in diameter [55, 56]. It has a negative-sense, ssRNA genome of ~11 kb,
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234 containing five ORFs encoding five structural proteins: nucleoprotein (N),

235 phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA dependent RNA
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236 polymerase (L). The five viral genes are organized in the order typical of
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237 rhabdoviruses: 3’-N–P–M–G–L-5’ [57-59]. Genotypes of SVCV isolates from various

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238 locations form four major genetic clades [60]. SVCV isolates of the recent emergence

239 and geographic range expansion link to the spread of the virus within China where
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240 common carp are reared in large numbers for food and koi are reared for export [61].
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241 It is responsible for the highly contagious SVC, especially in common carp [55, 62,
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242 63].

243 SVCV was first detected in Yugoslavia in 1971 [64]. It was initially believed to

244 be endemic among common carp in Eastern and Western Europe, and subsequently

245 reported in the Americas [61, 65] and Asia [55, 58, 66]. SVCV infection has now

246 emerged in many European countries, including the UK, Denmark, Germany, the
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247 Netherlands, Austria, Spain, France, the Czech Republic, Georgia, Belarus, Moldova,

248 Ukraine and Russia associated with numerous financial losses in common carp and

249 koi [60, 61, 67-70]. These outbreaks occur in both farmed and wild fishes suggesting

250 the expansion of influenced range. The emergence of SVC in region where is free of

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251 SVC, such as North America, Asia and in portions of Europe, appears to be the

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252 consequence of both improved surveillance and the large volume of ornamental fish

253 global shipment.

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254 Outbreaks of SVC usually occur in spring, which is why the disease is called so

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255 [71]. Studies in carp have shown that few adult fish are infected when the water
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256 temperature is over 17 oC, but juveniles can be infected even at 22 oC -23 oC [71].

257 SVCV infection is highly lethal in young fish, with mortality rate up to 90% [63].
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258 Other risk factors associated with morbidity and mortality include fish density,
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259 geographical location, fish species and the immune status of susceptible fish [71].
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260 SVCV infection can spread by fomites and parasitic in vertebrates. Natural SVCV

261 infections have been reported in other cyprinid fish, including goldfish, koi, silver
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262 carp, crucian carp, bighead carp, grass carp, tench (Tinca tinca) and orfe (Leuciscus
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263 idus) [55, 62, 72]. Experimental infections have been reported in zebrafish (Danio
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264 rerio), golden shiner (Notemigonus crysoleucas), roach (Rutilus rutilus), guppy

265 (Lebistes reticulatus), pumpkinseed (Lepsomis gibbosus), northern pike (Esox lucius),

266 fathead minnow (Pimephales promelas), emerald shiner (Notropis atherinoides) and

267 white sucker (Catostomus commersonii) [55, 73-75].

268 SVCV infection is generally associated with non-specific symptoms, such as

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269 exophthalmia, abdominal distension and oedema of the vent region [55]. Petechial

270 hemorrhages can be seen on the skin, gills, eyes and internal organs, particularly on

271 the walls of the swim bladder [55, 76]. Other lesions may include degenerated gill

272 lamellae, oedematous internal organs, swollen and coarse-textured spleen, hepatic

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273 necrosis, enteritis, and pericarditis [75, 77]. The histological changes associated with

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274 hepatopancreas may range from perivasculitis to panvasculitis with a higher degree of

275 oedematization and loss of structure of blood vessels walls. The hepatopancreas

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276 parenchyma show hyperaemia, multifocal necrosis and adipose degeneration [77],

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277 whilst the spleen is often hyperaemic, showing a co-significant hyperplasia of the
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278 reticuloendothelium [55]. Multifocal necrosis and non-purulent inflammation often

279 occur in the pancreas of affected fish, and the heart shows pericarditis and
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280 discontinuous myodegeneration [75]. The visceral and parietal serosa of the
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281 peritoneum show peritonitis. In the intestine, perivasculitis with subsequent atrophy of
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282 the villi is often observed [55, 75]. The epithelial layer of the swim bladder changes

283 into a discontinuous multilayer and hemorrhages are commonly observed in the
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284 submucosa [55].

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285 As SVCV has a significant impact on carp aquaculture, its rapid detection and
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286 identification are crucial for effective control of the disease. Conventional serological

287 techniques include the virus neutralization test, immunoperoxidase assay, indirect

288 immunofluorescence assay and ELISA. However, these techniques are laborious and

289 time consuming [78-81]. Moreover, the indirect immunofluorescence assay and

290 ELISA appear to cross-react with other rhabdoviruses [80], leading to possible
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291 false-positive diagnoses. mAbs are also useful tools for detecting SVCV and studying

292 the function of virus-specific proteins [82, 83]. Recently, a single-chain fragment

293 variable antibody against SVCV has been developed using phage display technology

294 and employed for rapid detection of SVCV [84]. This antibody reacts specifically

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295 with SVCV, not to cross-react with other viruses. Thus, this approach provides the

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296 basis for establishing simple and cost-effective way for the development of

297 immunological detection assays for SVCV. Various PCR-based assays have also been

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298 used to detect SVCV owing to their high sensitivity. These include reverse

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299 transcription (RT)-PCR combined with nested PCR [85], multiplex real-time
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300 quantitative RT-PCR [86] and one-step TaqMan real-time quantitative RT-PCR [87].

301 These assays have clearly improved the specificity and sensitivity of detection [88].
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302 Although PCR is generally considered impractical for routine diagnostics in the field
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303 owing to the need for specialized instruments, skilled operators and isolation of RNA
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304 extracts, a recent report described an improved RT-PCR assay that is able to

305 accurately detect SVCV directly from fish tissues, indicating the potential application
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306 of this technology for SVCV detection in infected fish in the field [89]. A
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307 loop-mediated isothermal amplification (LAMP) assay has been increasingly used for
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308 the detection of viruses [90-93]. RT-LAMP is a simple and effective technique to

309 rapidly amplify specific nucleic acid sequences under isothermal conditions.

310 Moreover, it requires uncomplicated and inexpensive equipment easily manipulated at

311 fish farms. This assay has been successfully applied for disease control in aquaculture

312 [94, 95]. Two studies have used RT-LAMP to detect SVCV based on nucleotide
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313 sequences of the G and M genes [96, 97].

314 2.3. Grass carp hemorrhagic disease

315 In 1950s, Chinese experts found that sick grass carp suffer from bleeding

316 symptoms, suspecting that the hemorrhage disease was caused by virus [98]. Grass

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317 carp hemorrhagic disease caused by grass carp reovirus (GCRV) leads to billions of

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318 RMB yuan of economic loss every year [99]. The virus is identified as a viral etiology

319 in 1978 and is the first fish viral disease studied in China [100]. The mortality rate of

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320 grass carp can reach more than 90% at the fingerling stage, and it can also infect other

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321 species, such as black carp, making these fish die of bleeding symptoms [101].
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322 The prevalence of GCRV has a typical seasonality. The high incidence period of

323 GCRV is mainly in summer when water temperature is 25 oC-30 oC. The main reason
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324 is that the optimum temperature of the virus polymerase is 28 °C. In many high
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325 density grass carp pools, when the temperature of the water exceeds 24 °C, the virus
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326 rapidly proliferates and may result in serious dysfunction of the fish and death. When

327 the water temperature is lower than 20 °C, the virus proliferation will be inhibited,
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328 and even lose their infectivity.

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329 The clinical signs are organ hemorrhages showing spots or plate forms, in
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330 combination of some or all of the following signs: exophthalmia, body darkening,

331 hemorrhage of the mouth cavity, hemorrhagic or pale gills, and hemorrhage at the

332 base of fins and branchiostegites. Internal hemorrhage may occur throughout the

333 musculature, hepatopancreas, spleen, kidney, and intestines. Present researches

334 classify the disease into three types according to clinical signs: red muscles, enteritis,
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335 or red fin and branchiostegite [102].

336 GCRV belongs to group C, Aquareovirus genus in the Spinareovirinae subfamily,

337 Reoviridae family. It is the first fish virus isolated in mainland China and is also

338 known as the most virulent virus in Aquareovirus [103]. The genome of GCRV

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339 consists of 11 segments of dsRNA, encoding 12 proteins, including seven structural

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340 proteins and five non-structural proteins [104, 105]. To date, more than thirty GCRV

341 strains have been isolated from infected grass carp in global. Based on the difference

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342 in VP6 genome constitution, GCRV could be mainly clustered into three types, and

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343 the representative strains of three types are GCRV-873 (type I), GD108 (type II), and
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344 GCRV104 (type III), respectively [104-106]. As a consequence of the fast evolution,

345 identities of amino acid sequences among types are less than 30% [104-106]. Among
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346 the three types, GCRV type II is considered to be the most pathogenic and prevalent
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347 type currently in China and may be closer to Orthoreovirus than any other known
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348 species of Aquareovirus showed by phylogenetic analysis [105, 107].

349 The virus identification by RT-PCR are the main method of surveillance and
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350 diagnosis of GCRV infection. Primary diagnosis in fish farms is based on typical
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351 external and internal clinical signs, especially the “red muscles” that appears in some
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352 sick fish. Epidemiological investigations are usually carried out in summer when it is

353 easy to detect the virus from the carrier or sick fish. The virus can be identified by

354 ELISA or RT-PCR.

355 At present, the antiviral immune responses against GCRV in grass carp are

356 broadly researched. As a dsRNA virus, GCRV activates the similar PRR-PAMP
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357 recognition mechanism of innate immune system, being sensed by different receptors.

358 Cytoplasm viral infection is primarily detected by RIG-I-like receptors (RLRs), a

359 PRR family consisting of retinoic acid-inducible gene I (RIG-I), melanoma

360 differentiation-associated gene 5 (MDA5), and laboratory of genetic and physiology 2

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361 (LGP2) [108]. RIG-I preferentially binds to relatively short 5’-phosphorylated dsRNA,

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362 while MDA5 binds to long dsRNA [109, 110]. By GCRV or poly(I:C) challenge,

363 mRNA level of RIG-I is up-regulated in CIK cells [111]. CPE assay and viral titter

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364 reveal the significant antiviral activity of full-length RIG-I in response to GCRV

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365 infection. Meanwhile, the expression levels of these three genes are significantly
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366 induced in spleen and hepatopancreas tissues after GCRV infection [111-114]. LGP2

367 is found as a negative regulator for both RIG-I and MDA5 in resting state or early
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368 stage of GCRV infection. TLR signaling pathways also participate in the immune
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369 responses to GCRV infection. Up-regulations of TLR3, TLR7, TLR8, TLR19, TLR22
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370 and other TLRs are observed post poly(I:C) or GCRV challenge [115-119]. The

371 enhancement of NOD1 and NOD2 also occur in grass carp post GCRV infection [120,
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372 121]. All the members of HMGB family can respond to poly(I:C) and GCRV
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373 challenge in grass carp, delaying the CPE in CIK cells, meanwhile modulating RLR
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374 and TLR pathway genes [122-126]. Similar to mammals, fish IFN antiviral responses

375 are initiated through the pattern recognition of virus component by TLRs and RLRs

376 [127]. Although antiviral immune researches in teleost have achieved great progresses

377 and several fish specific immune genes have been identified, studies on the regulation
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378 mechanisms of fish antiviral immune signaling pathways remain far behind those in

379 mammals.

380 2.4. Koi sleepy disease

381 Koi sleepy disease (KSD) is an emerging problem harmful to koi. It was first

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382 detected in Japan in 1974 [128, 129]. After its initial detection in koi populations in

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383 Japan, the disease was limited to Japan until 2013 when it was reported in both koi

384 and common carp in numerous European countries, including Germany, England,

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385 Austria, Poland, France and the Netherlands [130-133]. In addition, it also spread to

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386 Brazil, the United States, China and India [134-136]. The disease is exclusively
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387 described in common carp and koi breeding facilities.

388 KSD shows clinical symptoms when the water temperature is 15 oC-25 oC [129,
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389 137]. Clinical signs are seen in extreme association with stressful farm conditions,
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390 often occurring when fish are transferred from earthen to concrete ponds, causing a
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391 mortality of up to 80-100% and prevalence to 87.5%, especially in juvenile koi [137].

392 The typical appearance, lying on the bottom of the ponds, leads to the name KSD.
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393 Clinical signs are lethargy, skin hemorrhages with oedema in the underlying tissues,
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394 enophthalmos and pale swollen gills [5, 129, 137]. In addition, gill hyperplasia and
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395 necrosis as well as lamellae clubbing, leading to dyspnoea and hypoxia are also seen

396 in infected fish [5, 128, 137].

397 Carp edema virus (CEV), a double-stranded DNA virus, belonging to the family

398 Poxviridae, is the causative agent of KSD, based on transmission electron microscopy

399 [129, 137]. This mulberry-like enveloped member of the pox virus group has been
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400 identified in gill cells from diseased fish, and further morphological characterization

401 is provided by electron microscopy [5, 128, 137]. Sequencing the known DNA

402 fragment encoding the core protein P4a from infected fish in different locations in

403 Europe and Asia shows a 6%-10% of genetic diversity. It is classified into three

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404 genetic genogroups, called I, IIa and IIb [138, 139]. Viruses from genogroup I usually

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405 present in farmed carp, while CEV from genogroup IIa mainly in diseased koi [133,

406 138]. Infection experiments with both genogroups in koi and farmed carp confirmed

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407 biological differences between virus isolates [140]. The genetic diversity and the

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408 observed differences in biology reveal that the virus has been present in the European
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409 carp population for a prolonged period and infections are diagnosed until recently.

410 Similarities of pathognomonic signs of viruses affecting carp in spring, such as

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411 CEV, KHV and SVCV [6, 130], highlight the necessity for exploiting specific
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412 diagnostic procedures. Although the disease has been successfully transmitted, using
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413 filtrated suspensions of gill homogenates obtained from sick fish [5], the virus has yet

414 to be isolated successfully and grown in cell culture. KSD diagnosis relies on
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415 detection of viral DNA by PCR [129, 133, 141]. In the present reports, the diagnosis
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416 relied on clinical findings is confirmed by molecular detection of an undefined

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417 segment of the viral genome. A nested PCR protocol is initially designed in Tokyo

418 University of Fisheries [129], recently is modified in CEFAS-Weymouth Laboratory,

419 England, to improve reliability of detection. The latter method of detection is also

420 used for an extensive phylogenetic analysis to reveal the presence of two potential

421 main viral lineages.

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422 2.5. Carp pox disease

423 Carp pox disease caused by CyHV-1 is probably the earliest recognized viral

424 disease of fish, dating back to 1563 and had broken out in Europe, North Korea and

425 Japan [1]. It has also been found in Hubei, Henan, Hebei and many other provinces in

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426 China. CyHV-1 mainly infects common carp over 1 year old and also has some effect

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427 on crucian carp, hybrids of common carp and goldfish, having no harm to black carp,

428 grass carp, silver carp, bream [2].

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429 The proper temperature of carp pox is 9 oC-16 oC. Fish will recover without any

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430 administration when over 22 oC. Therefore, temperature control is an efficient method
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431 to fight against CyHV-1. Carp pox is an endemic chronic skin disease. In the early

432 days after infection, the fish become thin and transparent. Smooth gray and white
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433 patchy growth organisms appear and is covered with a thin layer of white mucus.
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434 With the development of the disease, white spots gradually expand and thicken, the
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435 number gradually increases and interconnects into pieces. Smooth, creamy to brown

436 appears as pink "paraffin-like or glass-like" pox and sores on blood vessels. These
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437 growth organisms are very tightly bound to the surface of infected fish body. The
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438 main component of the growth organisms is collagen fibers, which do not transfer but
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439 can naturally fall off and reappear in the original affected area. The back, head, fins

440 and caudal peduncle are dense areas of pox and sores. The severely diseased fish is

441 full of pox and sores, and the lesions often have bleeding. When the proliferation of

442 organisms spreads to the bulk of the fish body, it will affect the growth and

443 development of fish. The spine is damaged, the bones are softened, and the appetite
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444 diminishes, but generally no death.

445 The occurrence and disappearance of pox can be controlled by changing certain

446 environmental factors, such as improvement of water quality or rise of temperature

447 leading to the disappearance of symptoms, but affecting the growth and value of the

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448 fish. Inoculation of the pox with scratches on healthy common carp can produce the

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449 same symptoms as natural conditions [142]. In natural conditions, when water quality

450 deteriorates, toxic substances stimulate the surface of fish and tissues, secreting large

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451 amounts of mucus, and finally the mucus falls off.

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452 2.6. Herpesviral haematopoietic necrosis
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453 Herpesviral harmatopoietic necrosis was firstly detected in farming goldfish in

454 Japan [143]. It is also found in Australia [144] and New Zealand [145], spreading to
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455 global. The disease caused a large-scale death of goldfish fry in Taiwan in 1995, and
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456 CyHV-2 is responsible [146]. CyHV-2 infects crucian carp in many provinces in
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457 China [147, 148], commonly known as crucian carp gill hemorrhagic disease. The

458 influenced species only include goldfish [149], crucian carp and its common variety
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459 [150], and hybrids of goldfish [14].

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460 CyHV-2 is harmful to fish eggs, fry, fingerlings, broodstock, etc., while juveniles
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461 are more susceptible to infection than adult fish, and are more likely to cause

462 fulminant deaths of juveniles less than 1 year old [151]. In addition, like other

463 herpesviruses, CyHV-2 can form latent infection and becomes a potential source of

464 transmission [152]，while temperature is a key factor for virus replication. At the

465 beginning of the disease, the fish appear dying, poor appetite and slow swimming,
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466 staying at the bottom of the pond or tank. After the skin is pale with mucus, pustules

467 appear on fins. It appears part of the abdomen swells, the eyeballs protrude from both

468 sides and there is freckle bleeding on the sputum [153], blisters on the fins [149],

469 ascites, etc. [3]. The hepatopancreas, spleen and kidney are pale and the spleen and

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470 kidney are swollen [154]. Hematopoietic cells in the head kidney and body kidney

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471 show obvious nuclear pyknosis and nuclear lysis necrosis, with spleen large areas of

472 necrosis, sometimes accompanied by hemorrhage as well as pancreatic, thymus,

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473 intestinal development from multiple lesions to diffuse necrosis, epithelial cell

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474 proliferation, degeneration and necrosis of the oropharynx and epidermal cells, focal
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475 necrosis of the heart. Other tissues and organs including muscle tissue, brain tissue are

476 found no pathological changes [143, 144, 146, 151].

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477 The partial or complete nucleotide sequences of CyHV-2 such as helicase gene,
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478 DNA polymerase gene, terminal enzyme gene, and capsid protein genes have been
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479 reported, but the complete genome sequence and gene map have not been determined

480 yet, requiring further researches and improvement. Crucian carp Carassius auratus
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481 herpesvirus (CaHV) was isolated and its genome was sequenced and analyzed,
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482 showing that CaHV is most closely related to CyHV2 and clusters closely with
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483 CyHVs of the family Alloherpesviridae [155].

484 At present, PCR is a relatively accurate method for detecting CyHV-2 infection,

485 even a very small amount of viral DNA in tissue [145]. Conventional PCR method

486 based on CyHV-2 helicase gene have high specificity, with no amplification of the

487 genomic DNA of CyHV-1, CyHV-3 and IcHV-1. It can effectively detect CyHV-2
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488 isolates in different regions. A real-time quantitative PCR is established [153], which

489 has high specificity and high sensitivity. It can not only detect CyHV-2 from fish

490 exhibiting clinical symptoms, but also detect carriers. The diagnosis can detect the

491 sick fish in the incubation period in advance so that it can prevent the breakout in time

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492 and take appropriate measures to minimize the loss of the disease.

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493 3. Vaccine Development

494 The first successful use of inactivated Aeromonas salmonicida for oral

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495 immunization in rainbow trout (Oncorhynchus mykiss) in 1942 opens up a precedent

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496 for the utilization of vaccines in fish applications [156]. In the following decades,
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497 many researchers make extensive explorations of the preparation of fish vaccines to

498 prevent the sudden emergence of fish diseases that are difficult to control with drugs.
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499 According to incomplete statistics, the number of commercial vaccines for global
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500 commercial production has been more than 140 by the end of 2012 [157]. Currently,
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501 there are more than 50 aquaculture vaccines studied in China and nearly 30 species of

502 pathogens are involved, according to incomplete statistics [7]. However, only 5
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503 aquatic vaccines have obtained the national new veterinary drug certificate up to now.
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504 3.1. Vaccine types

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505 According to the preparation method, aquaculture vaccines are classified into

506 live vaccine, inactivated vaccine and gene engineering vaccine (Table 2).

507 3.1.1. Live vaccine

508 At present, most of the aquaculture live vaccines are prepared with attenuated or

509 mutated attenuated virus strains whose pathogenicity has been greatly weakened, such
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510 as VHSV [158], Canine coronavirus (CCV) [159], IHNV [160], and GCRV [161]

511 vaccines.

512 3.1.2. Inactivated vaccine

513 The inactivated vaccine is a managerial method that inactivates pathogenic

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514 microorganisms, but it remains immunogenic, providing the ability to induce specific

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515 resistance of aquatic animals after inoculation. These vaccines include a variety of

516 tissue fluid inactivated vaccines, vibrio inactivated vaccines, Aeromonas hydrophila

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517 vaccines, and streptococcal vaccines, for example, VHSV vaccine [162] which is

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518 commonly used in trout farming in Europe and the United States.
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519 3.1.3. Gene engineering vaccine

520 Gene engineering vaccine is further divided into recombinant subunit vaccine,
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521 DNA vaccine, gene deletion/mutant vaccine and living-vector vaccine.

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522 3.1.3.1. Recombinant subunit vaccine

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523 The protective antigen genes of pathogens is expressed in different expression

524 systems in vitro using recombinant DNA technology to produce pathogenic proteins
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525 that can induce protective immune responses in hosts. After separation and
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526 purification, recombinant subunit vaccine is completely prepared. Recombinant

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527 subunit vaccines do not contain pathogenic virulence factors and are expressed by

528 genetically engineered bacteria. At present, there are many studies on the preparation

529 of subunit aquatic vaccines based on immunogenic components such as bacterial

530 outer membrane proteins, lipopolysaccharides and other protective antigens, but most

531 of them are still in the experimental stage and have not been commercialized. Gene
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532 engineering vaccines in aquaculture include IHNV [163], infectious pancreatic

533 necrosis virus (IPNV) [164, 165], among which the IPNV VP2 recombinant subunit

534 vaccine is currently the only commercially available fish recombinant vaccine [165].

535 3.1.3.2. DNA vaccine

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536 A DNA vaccine is a recombinant eukaryotic expression vector that encodes a

537 certain protein antigen and is directly injected into animals. After being taken in by

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538 host cells, it expresses an antigen protein, thereby inducing non-specific and specific

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539 immune responses, playing an important role in immune protection effect. The

540 research on aquatic DNA vaccine was firstly reported in 1996 [166, 167]. Although

U
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541 the DNA vaccine researches start late, encouraging achievements have been made

542 [168]. Till nowadays, DNA vaccines mainly focus on the prevention and control of
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543 infectious virus diseases such as IHNV [169], VHSV [170], SVCV [171], and
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544 snakehead rhabdovirus (SHRV) [172].

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545 3.1.3.3. Gene deletion/mutant vaccine

546 Gene deletetion or mutant vaccine is a type of vaccine made by genetic

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547 engineering removing a fragment of a virulence gene in a viral or bacterial genome,

548 making it a defective strain. Vaughan reported the first gene mutation vaccine, the
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549 attenuated strain of Aeromonas salmonicida to prevent salmon furunculosis in 1993

550 [173]. Gene deleted/mutant vaccine could induce T cell immune responses in rainbow

551 trout [174, 175]. This type of vaccine is also researched in channel catfish against

552 channel catfish virus (CCV) [176] and in zebrafish against Erwinia [177].

553 3.1.3.4. Live-vehicle vaccine

554 Live-vehicle vaccines are genetically engineered vaccines that use a

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555 non-pathogenic virus or bacteria to carry and express other highly pathogenic or

556 protective immune-related antigen genes, creating a multivalent vaccine. Under this

557 conditions, attenuated pathogens have become excellent carriers of heterologous

558 antigens in prevention and treatment [178]. The effect of a bacterial live vector

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559 vaccine is mainly concentrated on whether it can express a sufficient amount of

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560 protein to cause the host immune responses and produce a protective immune

561 response [179]. The initial report of living-vehicle vaccine in aquatic organism was in

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562 1995, preventing Salmonella in salmon [180]. Subsequently, the application of

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563 living-vehicle vaccine was reported to prevent Streptococcus [181], Edwardsiella
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564 tarda, A. hydrophila [182] and CCV [183].

565 3.2. Delivery methods

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566 Common immunization delivery methods of aquaculture vaccines include

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567 injection, immersion and oral administration (Table 3).

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568 3.2.1. Injection

569 Global aquaculture vaccines are mainly immunized with injection. Aquaculture
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570 vaccine injection mainly includes intramuscular injection and intraperitoneal injection.
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571 Intramuscular injection is mainly used for vaccination of DNA vaccines, while
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572 intraperitoneal injection is mainly used for inoculation of traditional inactivated

573 vaccines and attenuated vaccines [184].

574 3.2.2. Immersion

575 There are four types of immersion immunization, direct immersion, hypertonic

576 soak, spraying immunization and immersion bath. After the first successfully
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577 hypertonic immunized fish in 1976 [185], vibrio vaccine achieves success in soaking

578 immunization in salmon [186] and rainbow trout [187]. However, the mechanism on

579 vaccination into fish during immersion immunization is still unclear, such as whether

580 the vaccine enters the body through the skin, tendon, lateral line, or other sites,

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581 whether the vaccine-induced immunity works through the blood circulation system or

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582 the mucosal system, etc. In addition, a variety of factors affect the host's uptake of

583 soaked immune antigens, including vaccine concentration, immersion time, aquatic

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584 animal size, adjuvants, antigenic forms, water temperature and so on [188].

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585 3.2.3. Oral administration
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586 Traditionally, adjuvant vaccine, biofilm vaccine and antigenic component

587 vaccine can be implemented by oral immunization. Along with the development of
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588 new technology, new oral immunization more prefer to slow-releasing micro capsule
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589 vaccine, yeast carrier vaccine and transgenic plant vaccine [189]. Oral vaccines are
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590 considered to be the most operative immune modes for aquatic vaccines. The main

591 consideration is that the living environment of aquatic animals is inseparable from the
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592 water body and the cultured animal scale is large.

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593 3.3. Cyprinid viral disease vaccine

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594 There are only four validation productions for cyprinid viral vaccines in global,

595 which are KHV inactivated vaccine approved in Israel in 2003, carp herpesvirus live

596 vaccine approved in the United States in 2012, SVCV inactivated vaccine approved in

597 Czech Republic in 2012 and GCRV live vaccine in China in 2011. GCRV gene

598 engineering injection vaccine is still under clinical trials and other cyprinid viral
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599 disease vaccines are under laboratory researches (Table 4).

600 A KHV vaccine patent is granted in the United States in 2018. A attenuated

601 recombinant KHV comprising a genome in which ORF56 and ORF 57 are mutated,

602 wherein the herpesvirus is capable of replication, got the patent granted to protect

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603 common carp and koi against KHV indicating excellent immunoprotection capability

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604 [190]. KHV liposome oral vaccine can protect common carp from KHV infection

605 [191]. Abundant immune genes are found to participate in the antiviral responses

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606 against KHV, indicating that the immune response to KHV is largely controlled by the

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607 genetic factors of the host [54].
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608 There are various GCRV vaccines reported in the past decades. GCRV

609 histoplasmic inactivated vaccine was attempted in China since 1960s. Until 1986,
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610 Chinese researchers exploited high-efficiency attenuated vaccine with good immune
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611 effect and long protection period. GCRV is detoxified by eucalyptus liquid and
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612 injected to immunize grass carp demonstrating long-term immunoprotection and up to

613 90% protection ratio [161]. The immunogenicity of VP5 and VP7, which are the
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614 major outer capsid protein of GCRV subtype I, are researched a lot to evaluate the
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615 potential of function as vaccine [192]. Recombinant of VP5 and VP7 vaccine is
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616 synergistically orally administrated to grass carp inducing good immunoprotection

617 and less than 10% cumulative mortality [193]. Outer capsid protein VP4 in GCRV

618 subtype II is also used to prepare GCRV vaccine. The immune protection assay shows

619 that IgM and other antiviral immune responses are significantly triggered and 82%

620 protection ratio is achieved [107]. Other researches on GCRV gene engineering
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621 vaccines mainly concentrate on the cell proliferation enhancing activity and

622 immunoprotection of GCRV VP6 and VP7 recombinant plasmid [194-197]. A GCRV

623 VP6 DNA vaccine obtained by baculovirus expression system can achieve a

624 protection ratio of 95% [198]. Using Bacillus subtilis as the carrier of GCRV VP4, the

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625 recombinant vaccine is orally immunized grass carp. Challenge experiment reveals

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626 that the oral recombinant subunit vaccine can protect 50%-60% grass carp from

627 infection and generate immunity against GCRV [199]. GCRV VP5 expressed by

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628 Escherichia coli is proved to be relative to virus infection and have the potential to

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629 produce subunit vaccine [200]. The GCRV inactivated vaccine induces the IFN
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630 system of grass carp and strengthens the IgM expression continuously [201]. There

631 are still hundreds of researches unlisted, working on the preparation of GCRV vaccine
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632 to improve the utility of vaccines to protect grass carp from GCRV infection. Immune
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633 adjuvant is pivotal to enhance the potency of vaccines against GCRV. New type
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634 immune adjuvant like CpG [202, 203] develops rapidly in the past decades.

635 Inactivated viral vaccine against SVCV provides limited protection, whereas
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636 attenuated vaccine has not been pursued because of a number of limitations, including
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637 improper attenuation of the virus, lack of quantitative evaluation for the protection
AC

638 activity provided by the vaccine, and restrictive market and legal regulations [62].

639 Regardless of the obstacles of technologies and regulatory approvals [204], researches

640 on DNA vaccines against SVCV has been steadily increasing in the last decade

641 [205-207]. The initial SVCV DNA vaccine did not exhibit an expected effectiveness

642 like other fish DNA vaccines against Novirhabdoviruses, including IHNV and VHSV
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643 [208]. Therefore, multiple trials have been conducted to develop an efficacious SVCV

644 DNA vaccine. The G gene of SVCV encodes a surface glycoprotein which is

645 considered to be a major antigen for inducing the primary host immune responses and

646 is frequently used in DNA vaccine preparation [171, 209]. Ten SVCV DNA vaccines

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647 carrying SVCV G gene are tested in carp [171]. The majority of treated groups show

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648 little protection, with relative survival ranging from 21% to 33%. The strongest

649 protection (48% relative survival) is observed in a group injected with a combination

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650 of two constructs expressing the full-length G gene. However, the relative

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651 contribution of the two constructs to this protection rate is not determined [171]. The
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652 efficacy of another DNA vaccine expressing the G gene of a North American SVCV

653 isolate is investigated in ornamental koi and goldfish [209]. In all trials, immunized
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654 fish demonstrate a strong protective response against SVCV, with relative survival
D

655 ranging from 50% to 88% [209]. Vaccination with an oral or injection subunit vaccine
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656 based on the baculovirus recombinant expression of transmembrane SVCV-G protein

657 in insect cells shows positive immune protection [210]. Taken together, these studies
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658 put forward the validation for the potential utilization of G gene for DNA vaccine as a
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659 prophylactic therapy against SVCV infection.

AC

660 Although DNA vaccination via the parenteral route has been used as an efficient

661 strategy to elicit antiviral immunity, it is not convenient for large-scale immunization

662 due to the handling stress on fish, high labour and production cost [211]. Furthermore,

663 there is limited scientific literatures about the condition of DNA vaccines after

664 injection into fish [212]. Intestine, gills, skin and other mucosal tissues of fish, are
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665 deemed important for immune protection against pathogen invasion [213, 214].

666 Activation of mucosal immunity prevents pathogen infections and replications in

667 mucosa, whereas intramuscular vaccines fail to do this [214]. Oral vaccination is an

668 effective way to induce mucosal immunity [215] and this strategy has shown a

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669 successful induction of the antiviral responses against viral diseases in different fish

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670 species [165]. Lactobacilli possess multiple properties to be suitable candidates for

671 vaccine antigen delivery vectors [216]. Lactobacilli also play a non-specific immune

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672 adjuvant effect [217]. A genetically engineered Lactobacillus plantarum

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673 co-expressing SVCV G protein and KHV ORF81 protein is investigated for protective
AN
674 immunity in carp and koi through oral vaccination [218]. Immunized carp and koi

675 show effective protection rates of 71% and 53%, respectively [218]. Moreover,
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676 immunized carp shows significantly elevated expression of IgM [218]. These results
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677 demonstrate that the recombinant L. plantarum is able to induce a protective immune
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678 response in fish against SVCV and KHV, suggesting a practical approach for

679 large-scale control of cyprinid viral disease [218].

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680 4. Conclusion and prospects

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681 Along with the rapid development of freshwater fish farming industry, increasing
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682 number of diseases break out, especially viral diseases. The huge economic losses,

683 food and environment safety risks caused by the diseases lead to the urgent need of

684 vaccine. The variation of pathogens and the diversity of antigens are the main

685 obstacles of efficient vaccine development. At the same time, the immune response

686 mechanisms of vaccines are still uncertain, which not only increases the difficulty of
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687 suitable programming, but also leads to the blindness of vaccine researches and

688 applications. The use of fish vaccines lacks effective and convenient ways of

689 administration as well as measures for enhancing vaccine immunity and effective

690 vaccine efficacy assessment systems and methods. More factors restrict the utilization

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691 of vaccines, for example, water temperature, water quality, illumination and season. In

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692 addition, the organism conditions (age, weight, nutriture, physiological status, group

693 effect) and vaccine quality (immunogenicity, adjuvant, preparation, storage,

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694 transportation, administration method, dosage and time) also have a great influence on

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695 the potency of vaccine.
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696 In the future, the studies on fish vaccine are needed to improve the theoretical

697 basis. The tendency is to concentrate efforts to solve one or two severe aquatic animal
M

698 diseases as a breakthrough and promote the development of fish vaccines. Meanwhile,
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699 the development of classic and new fish vaccines need to cooperate with each other,
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700 with the help of human and veterinary vaccine researches. The research on the

701 administration in relation to the effectiveness of fish vaccines is an important part of

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702 vaccine development. It is urgent to establish a simple and effective fish vaccine
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703 evaluation system and accelerate the construction of pilot test bases for fish vaccines.
AC

704 The developments of appropriate immunopotentiators and immune adjuvants also

705 promote the potency of fish vaccines.

706 As the worldwide rapid development of science and technology as well as the

707 positive attempts in fish and other aquatic animals, aquaculture industry is showing

708 the tendency to flourish. Taking full advantage of the immune responses to diseases,
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709 vaccination is a vital strategy to prevent and control the potential diseases, solving the

710 problems of food quality and safety as well as environmental pollutions.

711 Acknowledgements

712 This work was supported by National Natural Science Foundation of China

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713 (31873044) and Huazhong Agricultural University Scientific & Technological

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714 Self-Innovation Foundation (2014RC019).

715

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716 References

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718 2. Parslow C, Higginbotham J. Piscinae: Artificial Fishponds in Roman Italy; 1997.

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722 4. Ariel E. Ornamental fish as trans-boundary vectors of viral diseases. Diseases in Asian

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740 12. Gilad O, Yun S, Adkison M, Way K, Willits N, Bercovier H, et al. Molecular comparison of

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746 14. Hedrick R, Waltzek T, Mcdowell T. Susceptibility of koi carp, common carp, goldfish, and
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752 16. Fabian M, Baumer A, Steinhagen D. Do wild fish species contribute to the transmission of koi
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753 herpesvirus to carp in hatchery ponds? Journal of Fish Diseases. 2012 36:505-14.

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755 naiive common carp by cohabitation. Research in Veterinary Science. 2011 90:536-9.

756 18. Bergmann S, Lutze P, Schütze H, Fischer U, Dauber M, Fichtner D, et al. Goldfish (Carassius

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1205 enhance the immunoprotective effect of a DNA vaccine against spring viremia of carp virus in
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1273
C EP
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60

Table 1
Cyprinid viral diseases.
Disease Pathogen Genome Taxonomic classification1 Year emerged Major influenced Known geographic OIE listed2

PT
species distribution
Koi herpesvirus disease Koi herpesvirus (KHV); dsDNA Herpesvirales 1997 [8] Koi; Asia; Yes

RI
Cyprinid herpesvirus-3 (CyHV-3) Alloherpesviridae, Common carp Europe;
Cyprinivirus North America;

SC
Africa

Spring virenis of carp Spring virenis of carp virus (SVCV) (-) ssRNA Mononegavirales, 1971 [64] Common carp; Asia; Yes

U
Rhabdoviridae, Grass carp; Europe;

AN
Vesiculovirus Bighead carp; North America;
Silver carp; South America
Goldfish

M
Grass carp hemorrhagic disease Grass carp reovirus (GCRV) dsRNA Reoviridae 1954 [98] Grass carp; Asia; No

D
Spinareovirinae Black carp; Europe;
Aquareovirus Africa

Koi sleepy disease Carp edema virus (CEV)

TE
dsDNA Poxviridae 1974 [28] Common carp;
Koi
Asia;
Europe;
No
EP
North America;
South America
C

Carp pox disease Carp pox herpesvirus (CPHV); dsDNA Herpesvirales 1563 [1] Common carp; Asia; No
AC

Cyprinid herpesvirus-1 (CyHV-1) Alloherpesviridae, Crucian carp; Europe

Cyprinivirus Orfe

Herpesviral haematopoietic necrosis Herpesviral haematopoietic necrosis dsDNA Herpesvirales 1995 [146] Goldfish; Asia; No
virus (HVHNV); Alloherpesviridae, Crucian carp and Australia
Cyprinid herpesvirus-2 (CyHV-2) Cyprinivirus its common varient
1 2
Note: ICTV, 2017. OIE, 2018.
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61

Table 2
Frequently-used fish vaccine types.
Classification Merit Defect

PT
Live vaccine Generally attenuated vaccine; Close to natural infection; Poor safety under natural conditions; Short shelf life;
Effectively stimulate immune system; Not convenient storage and transportation

RI
Pathogen can reproduce in vivo;
Low dosage; Long protection duration; No adjuvant needed

SC
Inactivated vaccine Short development cycle; Safe to use; Easy to preserve Cannot reproduce in vivo after immunization；

U
Large immunize dosage; Short duration;
Appropriate adjuvant is needed

AN
Gene engineering vaccine Recombinant subunit vaccine Excellent safety; Simple production; Easy control; The expression is affected by the expression system;

M
High stability; High purity Weak immunogenicity; Short protection duration;
Difficult to effectively remove intracellular pathogens

D
DNA vaccine High stability; Induce comprehensive immune response; Uncertain immune response caused by the vaccine;

TE
Easy production in large scale; No risk of return of the virus; Tissue distribution and expression still unknown;
Low cost; The most promising vaccine More researches are needed to confirm the stability;
EP
Immune tolerance; Risk of integrating into the genome
C

Gene deletion/mutant vaccine High stability; Not easy to reverse under natural conditions; Not many successful cases;
Good immunogenicity; Guaranteed safety;
AC

Important direction of vaccine development

Living-vector vaccine Can be administered through mucous membrane system; Restricted by its expression level and its persistence;
Easy control; Risk of plasmid loss reducing vaccine function;
Environmental safety needs to consider
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62

Table 3
Comparison of delivery methods of fish vaccines.
Infection Immersion Oral

PT
Target Systemic Mucosal system Digestive system
Administration ease Laborious Less convenient Convenient

RI
Stress Severe (requires anaesthetization and Moderate for spraying; Negligible
handling); Severe for bath

SC
Moderate for automated vaccination
Potency Best with adjuvant Less efficient Lowest

U
Immunity duration 6-12 months Shorter Shortest
Cost Cost-effective for high value species Cost-effective for fish < 10g Moderate

AN
Merit Best immune effect; High serum antibody titer; Suitable for multiple times immunization; Suitable for small size fish
Low vaccine dosage Not restricted by fish body size species and large-scale

M
immunization;
Save time and effort

D
Defect Large fish stress response; Damage vaccine to a certain extent; Synergistic measures are needed;
Suitable for large size fish body; Large dosage of vaccine; Large dosage

TE
Time-consuming and laborious Short immune effect duration
C EP
AC
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Table 4
Cyprinid viral disease vaccine production licensed in global.
Vaccine Immune object Preparation Country Approved time

PT
method
KHV vaccine Koi Inactivated Israel 2003

RI
Carp herpesvirus Cyprinid Live Unite States 2012
vaccine

SC
SVCV vaccine Common carp Inactivated Czech Republic 2012
GCRV (strain-892) Grass carp Live China 2011

U
AN
M
D
TE
C EP
AC
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Highlights

1. Reviewed the six most popular cyprinid viral diseases.

2. Summarized the development of aquaculture vaccine.

3. Characterized the vaccines specifically against KHV, GCRV and SVCV in detail.

PT
RI
U SC
AN
M
D
TE
C EP
AC