Shengyu Lio, Chn Zhu, Dingji Shi,b, Peimin He, Rui Ji,＊

aCollege of Marine Ecology and Environment, Shanghai Ocean University, Shanghai, 201306, China

bInstitute of Botany, Chinese Academy of Sciences, Beijing, 100093, China

Keywords:

ABSTRACT

1.Introduction

Shrimp is a nutritious and delicious seafood that has become a major source of animal protein produced by marine aquaculture (Stokstad,2010). Since 1970, shrimp farming has grown at an average annual rate of at least 18% (Pradeep, Rai, Mohan, Shekhar, & Karunasagar, 2012).Approximately 75% of the shrimp consumed by humans is produced by a global industry that is dominated by two species: the black tiger shrimp (

Penaeus monodon

) and the white pacific shrimp (

Penaeus vannamei

) (Walker and Winton, 2010). The reliance on such a small number of species together with the large growth in commercial shrimp aquaculture has led to a substantial increase in diseases that can diminish productivity and yield. White spot disease, which results from infection by the white spot syndrome virus (WSSV), causes the most severe damage in shrimp farming and has the potential to restrict the long-term and sustainable development of this industry (Walker et al., 2011).

White spot syndrome virus (WSSV) was first discovered in Taiwan Province, China, in 1992, and has subsequently spread rapidly across the entire Asian region (Chinese mainland, South Korea, Indonesia, Japan,Thailand, and India) and to Latin america (Mexico and Ecuador) due to poorly controlled farming methods and inadequate sanitary conditions(Walker and Winton, 2010; He et al., 2006, 2016; Kim, Kosuke, Nam,Kim, & Kim, 2007; Valdez, Yepiz-Plascencia, Ricca, & Olmos, 2014). The virus has a spread to the Middle East, Europe, Central and South America(He et al., 2016; Moser et al., 2012).

White spot syndrome virus is the sole species of the genus

Whispovirus

, which itself is the only genus of the family Nimaviridae. WSSV is a large, enveloped, ovaloid DNA virus with a flagellum-like tail and helical nucleocapsid (Huang, Li, Wu, & Yang, 2015; van Hulten et al.,2001). The 300 kb viral genome contains at least 181 predicted open reading frames (ORF), most of which encode polypeptides with no discernible homology to other known proteins (Durand, Lightner, Redman, & Bonami, 1997; Lo et al., 1997).WSSV infects a wide range of species, including prawns, crabs, lobsters, crayfish, marine copepods and freshwater animals including the giant river prawn

Macrobrachium rosenbergii

(Chakraborty et al., 2002;Corbel, Zuprizal, Huang, Sumartono, & Bonami, 2001; Pradeep et al.,2012; Wang, Lo, Chang, & Kou, 1998). The infected animals show signs of lethargy, decreased feeding, anorexia, a loose cuticle, and their body color changes from red to pink. White spots due to calcium deposits appear in the exoskeleton, especially in the carapaces and last abdominal segments (Chou, Huang, Wang, Chiang, & Lo, 1995).

In situ hybridization and transmission electron microscope (TEM)have shown that WSSV particles are located mainly in the connective tissue around the vas deferens of the male reproductive system and the oocytes in shrimp ovaries. No WSSV has been detected in mature eggs suggesting that the virus kills the infected egg cells before they mature(Lo et al., 1997). No penaeid shrimp species is entirely resistance to WSSV, and even no sign of WSSV shrimp have been observed as asymptomatic WSSV carriers (Khadijah et al., 2003; Pradeep et al.,2012).

Invertebrates lack an adaptive immune system and thus rely completely on their innate immune system (Hoffmann & Reichhart,2002). Lin et al. (2013) exposed shrimp to heat- or formalin-killed

Vibrio alginolyticus

and then to living

V. calginolyticus

showing that several immune parameters (PO, SOD and lysozyme activities) were greatly improved, and their subsequently verified that shrimp have a specific immuno-memory. Sudheer et al. (2015) have shown that binary ethyleneimine inactivated WSSV protects

Penaeus clarkii

from WSSV infection. Recombinant VP26 can, to some extent, immunize shrimp to WSSV(Satoh, Nishizawa, & Yoshimizu, 2008). Feeding

P. vannamei

with

Bacillus subtilis

expressing a VP26 fusion protein protected the shrimp from subsequent WSSV infection (Valdez et al., 2014). Together these experiments show that shrimp immunized with WSSV VP26 exhibit resistance to WSSV. Here we review the

vp26

gene, VP26 protein structure, VP26 binding proteins and VP26 immune adjuvant to provide a reference for research aimed at the prevention and treatment of WSSV.Moreover, our next experiments focused on the prevention and treatment of WSSV by the tegument protein VP26.

2.The WSSV genome and vp26 gene

2.1.WSSV genome

To date the genomic sequences of ten different WSSV isolates have been deposited in GenBank (Oakey & Smith, 2018). The most studied isolates are from mainland China (WSSV–CN, AF-332093 (Yang et al.,2001)), Taiwan (WSSV-TW, AF440570 (Tsai et al., 2000)), Thailand(WSSV-TH, AF-369029 (van Hulten et al., 2001)), Korea (WSSV-KR,JX-515788 (Chai, Yoon, Lee, Kim, & Choi, 2013)), and Australia(WSSV-AU, MF768985 (Oaky et al., 2018)). Table 1 is some information of the five isolates.

Sequence alignment showed that the five virus isolates have genomes that are highly homologous (Cai, Sun, & Liu, 2018). The Thailand isolates is 99% homologous with the Chinese mainland isolate with only a 12 kb fragment deleted (van Hulten et al., 2001). Compared to the Taiwan isolates, the Thailand isolates has a 14 kb fragment deleted. Both Taiwan and mainland China isolates have one more sequence at 31135 site than the Thailand isolates, the remaining sequences are virtually indistinguishable(He et al., 2016).

2.2.The vp26 gene

The

vp

26 gene has a total length of 612 bp (van Hulten et al., 2000a).

vp

26 is located in ORF311 in mainland China isolates (WSSV–CN,AF-332093), in ORF367 in the Taiwan isolate (WSSV-TW, AF440570),and in ORF153 in the Thailand isolate (WSSV-TH, AF-369029) (Li, Xu,Li, Zhou, & Yang, 2011). Nucleotide sequence alignments show that

vp

26 and

vp

24 have 40% homology, and that

vp

26 and

vp

28 have 48%homology. Such data indicate that these three WSSV structural protein genes have a common ancestor, and that the genes evolved through gene duplication and differentiation (van Hulten et al., 2000b). The eighteen

vp

26 sequences published in GenBank (Table .2) show that

vp

26 is highly conserved in each isolate. 14 of the published

vp26

sequences have 100% homology. In the Chinese isolate (sequence: AY220746) a Gto A change at 575 results in conversion of Arginine to Lysine. In the Vietnamese isolate, (sequence: AJ551446) the change from T to C at 345 does not alter the transcribed amino acid (Joseph, Anbu, James, Lalitha,& Surendran, 2015).

Table 2 Comparison of vp26 gene sequences in GenBank.

3.The VP26 protein

3.1.The location of VP26

Marks et al. (2003) used transcriptional analysis to show that

vp2

6 has no conserved transcriptional initiation sequence and that its 5terminal is located 25 nt downstream of an A/T-rich region. The

vp26

gene is expressed in the late stage of infection. van Hulten et al. (2000) used the results of Western blot analyses to propose that VP26 is a nucleocapsid protein. By contrast, the immunogold labeling and electron microscopy data of Zhang et al. (2002) suggested that VP26 exists in the WSSV envelope. Xie and Yang (2005) reported that VP26 interacts with host actin and with VP28 and have proposed that VP26 is the connecting protein between the envelope and nucleocapsid. The location of colloidal gold particles observed by electron microscopy led Tsai et al.(2006) to propose that VP26 is a tegument protein located between the viral envelope and the nucleocapsid.

3.2.The structure of VP26

VP26 has no discernible sequence homology with any of the proteins currently deposited in GenBank. VP26 contains 204 amino acids and has a calculated molecular mass of 22 kDa. This value differs from the experimentally obtained value by 4 kDa, which may reflect posttranslation modifications, such as glycosylation, phosphorylation, or splicing. The protein has an isoelectric point of 9.3 (van Hulten et al.,2000a). The N-terminal of VP26 (MEFGNLTNLDVAIIAILSIAIIIVIMVIMVFNTRVGRSVVAN) is somewhat hydrophobic and may be a transmembrane domain or participate in the interaction of VP26 with itself or with other structural proteins to form homo- or heteromers (van Hulten et al., 2000a; Marks et al., 2003). The N-terminal region also contains a putative transmembrane anchor composed of a helix formed by amino acids 12–34. There is a positively charged region with two Arginines behind the anchor, indicating that the C-terminal end of the protein is on the cytoplasmic side (van Hulten et al., 2000a). There are three potential N-glycosylation sites in the VP26 amino acid sequence.Hansen et al. (1998) predicted three O-glycosylation sites and 14 possible phosphorylation sites. The presence of only one Cysteine, which is located in the C-terminal domain, indicates that disulfide bonds are not formed (van Hulten et al., 2000a). Naturally occurring VP26 exists as a monomer, a dimer or a trimer, and can also interact with other low-abundance envelope proteins to form various complexes (Li et al.,2011). Using biotin marker transfer technology, VP26 was shown to have a role as a connecting protein between the viral envelope and the nucleocapsid by binding to the viral tegument protein VP51, indicating that VP26 is involved in the viral envelope process (Wan, Xu, & Yang,2008). VP26 is a tegument protein (or stromal-like junction protein between the viral envelope and the nucleocapsid) that may help transport the WSSV nucleocapsid to the host nucleus via the cytoskeleton (Xie and Yang, 2005). In addition, VP26 also interacts with a variety of protein molecules in WSSV which plays a major role in the later stages of the virus invasion (Table .3).

Table 1 Information of the five isolates.

3.3.The role of VP26 in the process of invading the host

WSSV transmission involves three main routes: horizontal transmission between individuals through direct contact (Chou, Huang, Lo, &Kou, 1998), vertical transmission from parent to offspring (Jiang et al.,2000) and interspecific transmission between different kinds of animals through direct contact (van Hulten et al., 2001). Shrimp cannibalism is one of the most important routes for WSSV transmission. Healthy shrimp die after oral consumption of WSSV- infected shrimp. The shrimp digestive tract is the main site of WSSV infection. Virus particles that reach the digestive tract of healthy shrimp must bind to the surface of host cells to initiate viral infection (Verma, Gupta, Singh, & Nagpure,2017). During the interaction between WSSV and its host, VP26 interacts with several host molecules to facilitate WSSV invasion(Table .4). For example, VP26 may bind to actin (Xie and Yang, 2005) or to beta-integrin to facilitate the virus entry (Zhang, Liu, & Huang, 2014).

Table 3 Interaction between VP26 and other proteins in WSSV.

Table 4 Molecules binding to VP26.

4.Studies of immune control of WSSV by VP26 immune adjuvant

No effective methods to prevent, treat or control WSSV infection have been developed. Several approaches are used to minimize infection including utilizing innate immune responses (Vidya et al., 2016), environmental control (Raj, Vijayan, Alavandi, Balasubramanian, & Santiago, 2012), and pre-exposure to pathogens (Melena et al., 2006).Nevertheless, WSSV-infected shrimp still have a high mortality rate.

Kurtz and Franz (2003) provided evidence that the invertebrate defense system has a specific memory capacity. These authors used a copepod marine animal experiment to show that the success of re-infection depends on the similarity of antigens between continuously encountered parasites. Witteveldt, Cifuentes, Vlak, and van Hulten(2004) then reported that immune adjuvant may prevent viral infection of shrimp. Subsequently, efforts to control WSSV using an inactivated virus (Lu et al., 2008), antisense RNA (Akhila et al., 2015), a protein subunit (Satoh et al., 2008), or a DNA fragment (Li, Liu, Hou, & Huang,2010) have been explored. Together these studies have established that the shrimp immune response can be enhanced to improve survival rates against WSSV.

4.1.The development of a VP26 protein subunit

Namikoshi et al. (2004) were the first to report that the VP26 protein subunit could be used to immunize shrimp against WSSV. The survival rate of shrimp was 60% after a direct challenge on second stage of 10th days post-administration (dpa) after first stage of 20th days post-administration, and direct challenge after 30th days post-administration resulted in a survival rate of 57%. The LDof WSSV introduced orally, by immersion or by intramuscular injection is 10,10and 10g shrimp, respectively, indicating that oral infection efficiency is substantially lower than that of the other two treatments. In shrimp farms, however, WSSV is easily transmitted by the oral route because the infected shrimp are eaten (Satoh et al., 2008). It is notable that feeding

P. vannamei

with spores of

Bacillus subtilis

expressing the VP26 fusion protein protected

P. vannamei

100% from WSSV infection(Valdez et al., 2014). A summary of the methods using the VP26 protein subunits to immunize shrimp against WSSV is given in Table .5.

Table 5 The VP26 protein subunit.

4.2.VP26-dsRNA

RNA interference (RNAi) is a gene silencing mechanism involving double-stranded RNA (dsRNA) (Hannon, 2002). Gene expression is suppressed by blocking the translation of the target gene or by inducing the degradation of the transcribed mRNA. RNAi is an efficient and specific gene silencing technique that has become a powerful tool for studying the functions of specific genes and for inhibiting pathogen infections or replication (Lima, Harris, & Cook, 2013).

Fu, Shuai, and Xu (2005) verified that the dsRNA of viral outer membrane protein is taken up by shrimp cells where it binds to the mRNA encoding the viral outer membrane protein and prevents the mRNA from being translated. This inhibits WSSV replication and proliferation and improved the survival rates of the infected shrimp. The immune system of invertebrates is similar to that of vertebrates since it can use dsRNA as a virus-associated molecule to trigger an innate antiviral response (Robalino et al., 2004). Subsequently, Mejia-Ruiz,Vega-Pena, Alvarez-Ruiz, and Escobedo-Bonilla (2011) verified the antiviral effect of VP26-dsRNA on single or continuous intramuscular injection of WSSV at a high infection dose. Their data showed that shrimp exposed to WSSV continuously had stronger and longer antiviral responses than shrimp exposed to WSSV once 10 or 20 days post-administration. Cesar, Escobedo, and Sergio (2015) immunized

P. vannamei

with VP26-dsRNA to explore its anti-WSSV effect. Their results show that mortality rate was 21% after a viral challenge on the 10th day post-administration. The use of VP26-dsRNA as an adjuvant summarized in Table .6.

5.Discussion and outlook

Feng et al. (2018) have compared various types of adjuvants and found that dsRNA gives the highest protection rates. Among the subunit protein, the VP26 subunit protein has a greater protective effect than the other subunit protein. The protection rate of the protein adjuvant expressed in eukaryotes are higher than protein expressed in prokaryotes (Feng et al., 2014; Jha et al., 2007). Of the three immunization methods used for subunit adjuvants, oral administration was somewhat less effective than immersion or injection. Oral adjuvants are however more suitable for use in shrimp farming (Valdez et al., 2014). In terms of virus attack patterns, the results showed that the mortality rate caused by oral administration was comparable to injection, but lower than virus immersion (Feng et al., 2018; Satoh et al., 2008). In addition, the dose of the adjuvant and the dose of the WSSV have a substantial influence on the immune effect (Zhu, Du, Miao, Quan, & Xu, 2009; Amar and Faisan,2011).

WSSV adjuvants can substantially enhance the immunity of shrimp.However, the practical application of this approach is hindered by our limited knowledge of immunization time, viral attack time, and viral attack mode, all of which have a decisive impact on the immunization effect (Valdez et al., 2014; Kurtz & Franz, 2003). Adjuvants for use in shrimp farming must be easy to produce, convenient to administer,environmentally stable, and safe for humans. Moreover, the whole process must be economically viable (He et al., 2016). Degradation of soluble antigens in the stomach, which lead to adverse immune responses and the development of carrier systems that display exogenous antigens on their surfaces has long been a focus of research due to their found the adjuvant is more stable using Bacillus subtilis spores carrying VP26 (Namikoshi et al., 2004; Valdez et al., 2014).

The

vp26

gene, the structure of VP26 protein and its role in the process of virus invasion into the host have been studied in some detail.However, there are only a few studies on the use of VP26 as an adjuvant and the results of previous studies on VP26 anti-WSSV infection are inconsistent (Namikoshi et al., 2004; Satoh et al., 2008; Valdez et al.,2014). Valdez et al. (2014) used

B. subtilis s

pores to carry the VP26 protein and achieved up to 100% protection rates against WSSV.Although envelope proteins such as VP28 and VP19 have a primary role in immediate early infection of the host, VP26 is tegument protein and has also an important role in early infection (Tsai et al., 2006). In the following, we will briefly describe research directed toward the development of the VP26.

5.1.Search for a novel immune adjuvant for VP26

Despite studies of several VP26 subunit, there have been no systematic investigations into the most effective combinations of VP26 subunit and immune adjuvant (Namikoshi et al., 2004; Satoh et al.,2008; Valdez et al., 2014). In the study of inactivated WSSV, only the traditional immune adjuvants including saccharides and polypeptide were used, while conventional adjuvants (Beta-1,3-glucan and killed Vibrio penaeicida) are toxic (Namikoshi et al., 2004; Tafalla, Bogwald,& Dalmo, 2013).

The responses of the immune system and the immune signaling pathways of cultured shrimp induced by WSSV infection have been the subject of mumerous studies. Therefore, changes in gene expression of cultured shrimp following WSSV infection may provide a basis for the development of new immune adjuvants (Li and Xiang, 2013a; 2013b).

Table 6 The VP26-dsRNA.

5.2.A new safe and reliable expression system of VP26 protein

Escherichia coli

and

B. subtilis

are currently used as expression systems for the production of VP26 protein subunits. Both have disadvantages in terms of effectiveness, biological safety, cost, time and yield (Namikoshi et al., 2004; Satoh et al., 2008; Valdez et al., 2014). The eukaryotic green algae

Dunaliella salina

has been developed to produce exogenous proteins and has many advantages over the bacterial expression systems(Feng et al., 2014). The VP28 protein subunit produced in

D. salina

is an effective anti-WSSV for cultured shrimp (Feng et al., 2014).The use of food-grade organisms including bacteria (Lactobacillus and Bacillus), yeast, algae, plants and insects to produce recombinant adjuvants and as delivery vectors is a relatively mature technology. Such organisms are beneficial for adjuvant productions in terms of cost, ease of management and safety (Rosales-Mendoza, Angulo, & Meza, 2016).For example, Jia et al. (2016) used the cyanobacterium

Anabaena

sp

.pcc

7120 as an expression system to produce the envelope protein VP28, and achieved effective anti-WSSV response.

5.3.Shrimp can be immunized with VP26-DNA

The stability of DNA fragment and its relatively low cost and easy production have been extensively studied for its use against WSSV infection. DNA can be transmitted vertically from parent to offspring,which is a good strategy for producing pathogen-free shrimp and for protecting shrimp from pathogens (Chowdhury, Gireesh-Babu, Pavan--Kumar, Babu, & Chaudhari, 2014). To date, no researchers have explored the ability of VP26-DNA to induce immune responses. By contrast, high protection rates have been obtained using plasmids containing VP28-DNA to induce immune protection of cultured shrimp against WSSV (Kumar, Ahamed, Sarathi, Basha, & Hameed, 2008). Developments on chitosan coating technology for plasmid DNA makes oral gene transfer feasible and facilitates the practical application of DNA fragment (Rajeshkumar et al., 2009).

At present, white spot syndrome virus is still a disaster in shrimp culture. Although researchers have done some research on the structure of the virus and adjuvant control, there are no ways to completely control its spread, and we still need to do more to prevent and treatment of the WSSV. We also know very little about mechanism virus invades.This paper reviews the related research of VP26 to provide reference for the prevention and treatment of WSSV.

Acknowledgments

This research work was financially supported by the Shanghai agriculture Science and Technology Innovation Project (2017, No 1-13), the Shanghai Science and Technology Commission Innovation Project (No.17391902200), and the National Marine 863 Project (No.2014AA093506). We thank Malcolm O’Neill of the Complex Carbohydrate Research Center at the University of Georgia for advice during the writing of the manuscript.