A model for apoptotic interaction between white spot syndrome virus and shrimp

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.     

Enzyme E2 from Chinese white shrimp inhibits replication of white spot syndrome virus and ubiquitinates its RING domain proteins

Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.     

Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei challenged with a low dose of white-spot syndrome virus

Apoptosis has long been observed in viral target organs of white-spot syndrome virus (WSSV)-infected shrimp and whether the phenomenon helps the shrimp to survive the infection or is a factor leading to mortality is still controversial. If the shrimp mortality is a result of triggered apoptosis, then inactivation of caspase-3, a key protein in the induction of apoptosis, should improve shrimp survival upon challenge with WSSV. To test this prediction, we identified and characterized a caspase-3 homologue (cap-3) from the Pacific white shrimp Penaeus (Litopenaeus) vannamei and used this information to silence cap-3 expression by RNA interference prior to WSSV challenge. After confirming the efficacy of cap-3 silencing, its effects on mortality at high and low doses of WSSV were evaluated. In a high-dose WSSV challenge, cap-3 silencing had no significant effect on WSSV-induced mortality, except for a delay in mean time to death. However, at a low-dose WSSV challenge, cap-3 silencing correlated with a lower level of cumulative mortality, relative to silencing of a control gene, suggesting that apoptosis may exacerbate rather than decrease mortality in WSSV-challenged shrimp.     

RING-H2 protein WSSV249 from white spot syndrome virus sequesters a shrimp ubiquitin-conjugating enzyme, PvUbc, for viral pathogenesis

Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.     

Blocking the large extracellular loop (LEL) domain of FcTetraspanin-3 could inhibit the infection of white spot syndrome virus (WSSV) in Chinese shrimp, Fenneropenaeus chinensis

Tetraspanins belong to the transmembrane 4 superfamily (TM(4)SF), which span the cell membrane 4 times and act as bridges or connectors. Increasing evidences have shown that tetraspanins play important role in virus infection. The large extracellular loop (LEL) of a tetraspanin is considered as a possible target of some virus. Tetraspanins are widely found in invertebrates, but the functional roles of most invertebrate tetraspanins have remained unknown. Recently, a tetraspanin, called FcTetraspanin-3, was cloned from the cDNA library of Chinese shrimp, Fenneropenaeus chinensis. The FcTetraspanin-3 constitutive expression in all examined tissues and the expression of the gene were highly induced in hepatopancreas, lymphoid organ and intestine by white spot syndrome virus (WSSV) challenge. In this study, we expressed and purified the recombinant peptide containing the LEL domain of FcTetraspanin-3, and produced the anti-LEL polyclone antibody. The expression of FcTetraspanin-3 was observed by real-time PCR and Western blot. Also, the localization of FcTetraspanin-3-positive cells in intestine and hepatopancreas were revealed by immunofluorescence. The results of anti-LEL antibody blocking experiments shown that the antibody can significantly reduce the mortality of shrimp challenged by WSSV. Additionally, dsRNA interference was utilized to examine the functional role of FcTetraspanin-3 in response to WSSV infection, and a sensible decrease of the viral copy number in the tetraspanin knockdown shrimp. These results suggested the blocking of LEL domain of FcTetraspanin-3 could inhibit the infection of WSSV. FcTetraspanin-3 might play an important role in response to WSSV infection, and the LEL domain of FcTetraspanin-3 might mediate the entry of WSSV.     

Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine

Although invertebrates lack a true adaptive immune response, the potential to vaccinate Penaeus monodon shrimp against white spot syndrome virus (WSSV) using the WSSV envelope proteins VP19 and VP28 was evaluated. Both structural WSSV proteins were N-terminally fused to the maltose binding protein (MBP) and purified after expression in bacteria. Shrimp were vaccinated by intramuscular injection of the purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection. As controls, purified MBP- and mock-vaccinated shrimp were included. VP19-vaccinated shrimp showed a significantly better survival (p<0.05) as compared to the MBP-vaccinated control shrimp with a relative percent survival (RPS) of 33% and 57% at 2 and 25 days after vaccination, respectively. Also, the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival when challenged two days after vaccination (RPS of 44% and 33%, respectively), but not after 25 days. These results show that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine. This suggests that the shrimp immune system is able to specifically recognize and react to proteins. This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, opening the way to new strategies to control viral diseases in shrimp and other crustaceans.     

An immersion of Gracilaria tenuistipitata extract improves the immunity and survival of white shrimp Litopenaeus vannamei challenged with white spot syndrome virus

The innate immunity and resistance against white spot syndrome virus (WSSV) in white shrimp Litopenaeus vannamei which received the Gracilaria tenuistipitata extract were examined. Shrimp immersed in seawater containing the extract at 0 (control), 400 and 600 mg L(-1) for 3 h were challenged with WSSV at 2 × 10(4) copies shrimp(-1). Shrimp not exposed to the extract and not received WSSV challenge served as unchallenged control. The survival rate of shrimp immersed in 400 mg L(-1) or 600 mg L(-1) extract was significantly higher than that of challenged control shrimp over 24-120 h. The haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity, and lysozyme activity of shrimp immersed in 600 mg L(-1) extract were significantly higher than those of unchallenged control shrimp at 6, 6, 6, 6, and 6-24 h post-challenge. In another experiment, shrimp which had received 3 h immersion of 0, 400, 600 mg L(-1) extract were challenged with WSSV. The shrimp were then received a booster (3 h immersion in the same dose of the extract), and the immune parameters were examined at 12-120 h post-challenge. The immune parameters of shrimp immersed in 600 mg L(-1) extract, and then received a booster at 9, 21, and 45 h were significantly higher than those of unchallenged control shrimp at 12-48 h post-challenge. In conclusion, shrimp which had received the extract exhibited protection against WSSV as evidenced by the higher survival rate and higher values of immune parameters. Shrimp which had received the extract and infected by WSSV showed improved immunity when they received a booster at 9, 21, and 45 h post-WSSV challenge. The extract treatment caused less decrease in PO activity, and showed better performance of lysozyme activity and antioxidant response in WSSV-infected shrimp.     

Passive protection of shrimp against white spot syndrome virus (WSSV) using specific antibody from egg yolk of chickens immunized with inactivated virus or a WSSV-DNA vaccine

White spot syndrome virus (WSSV) causes high mortality and large economic losses in cultured shrimp. The VP28, VP19 and VP15 genes encode viral structural proteins of WSSV. In this study, hens were immunized with recombinant plasmid (pCI-VP28/VP19/VP15) with linkers or with inactivated WSSV, which used CpG oligodeoxynucleotides (CpG ODNs) and Freund's adjuvant as adjuvant, respectively. Egg yolk immunoglobulin (IgY) from hens immunized with inactivated vaccine and DNA vaccine was obtained, purified and used for protection of Metapenaeus ensis shrimp against WSSV. The data showed that the antibody response of the hens immunized with the DNA vaccine was improved by CpG ODNs as adjuvant, but was still inferior to inactivated WSSV in both sera and egg yolks. Using specific IgY from hens immunized with inactivated WSSV and DNA vaccine to neutralize WSSV, the challenged shrimp showed 73.3% and 33.3% survival, respectively. Thus, the results suggest that passive immunization strategy with IgY will be a valuable method against WSSV infection in shrimp.     

Protection of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV) challenge by double-stranded RNA

To determine whether Penaeus chinensis can be protected against white spot syndrome virus (WSSV) infection by intramuscular injection with long double-stranded RNAs (dsRNAs) as in other shrimp species and whether the protection degree by WSSV-specific dsRNAs is correlated with the roles of viral genes, P. chinensis juveniles were intramuscularly injected with long dsRNAs corresponding to VP28, VP281, protein kinase genes of WSSV, and an unrelated long dsRNA corresponding to a green fluorescence protein (GFP) gene. All shrimp injected with long dsRNAs including GFP dsRNA showed higher survival rates against WSSV infection than shrimp injected with PBS alone. Furthermore, shrimp injected with dsRNAs corresponding to VP28 and protein kinase showed higher survival rates than those injected with dsRNAs corresponding to VP281 and GFP. These results indicate that the introduction of long dsRNAs corresponding to viral proteins, which are essential for WSSV infection, is quite effective in blocking WSSV infection in P. chinensis, and suggest that dsRNA-mediated protection is a common feature across shrimp species.     

Involvement of Viral MicroRNA in the Regulation of Antiviral Apoptosis in Shrimp

Viruses, in particular DNA viruses, generate microRNAs (miRNAs) to control the expression of host and viral genes. Due to their essential roles in virus-host interactions, viral miRNAs have attracted extensive investigations in recent years. To date, however, most studies on viral miRNAs have been conducted in cell lines. In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp in vivo. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis and Northern blotting, the expression of viral miRNAs was tissue specific in vivo. The results indicated that the viral miRNA WSSV-miR-N24 could target the shrimp caspase 8 gene, and this miRNA further repressed the apoptosis of shrimp hemocytes in vivo. As a result, the number of WSSV copies in shrimp in vivo was significantly increased compared with the control level (WSSV only). Therefore, our study presents the first report on the in vivo molecular events of viral miRNA in antiviral apoptosis.