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Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii
Virus Research 214 (2016) 65–70
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Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii Rod Russel R. Alenton a , Hidehiro Kondo d , Ikuo Hirono d , Mary Beth B. Maningas a,b,c,∗ a
The Graduate School, University of Santo Tomas, Espa˜ na, 1015 Manila, Philippines Department of Biological Sciences, College of Science, University of Santo Tomas, Espa˜ na, 1015 Manila, Philippines Molecular Biology and Biotechnology Laboratory, Research Cluster for Natural and Applied Sciences, University of Santo Tomas, Espa˜ na, 1015 Manila, Philippines d Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, Japan b c
a r t i c l e
i n f o
Article history: Received 6 October 2015 Received in revised form 19 January 2016 Accepted 20 January 2016 Available online 23 January 2016 Keywords: VP9 RNA interference Macrobrachium rosenbergii Gene silencing WSSV
a b s t r a c t White Spot Syndrome Virus (WSSV) remains the most widespread and devastating infectious agent that hit the shrimp aquaculture industry worldwide. To date, there are no known effective strategies yet to combat WSSV infection. Hence, functional studies on genes critical for viral infection is essential in elucidating shrimp–virus interaction. Here we report the function of a gene from WSSV coding for a non-structural protein, VP9, utilizing RNA interference. Silencing of VP9 gene also effectively suppressed other gene region in the WSSV genome (wsv168 gene) as early as day 1 post infection (dpi). Three setups using Macrobrachium rosenbergii shrimp were prepared for treatment using VP9-dsRNA, GFP-dsRNA, and PBS. Each shrimp was challenge with WSSV, and survival rate was recorded. VP9- and GFP-dsRNA injected shrimps showed a significant survival rate of 80% and 70%, respectively, in contrast to 0% of the PBS injected shrimps at 25 dpi. Re-infection of shrimp survivors using a higher viral titer concentration, concurrent with the infection of new shrimp samples for the PBS control group, resulted in a significant 67% survival rate for VP9-dsRNA compared to 0% with that of GFP-dsRNA and PBS group. Challenge test on two more species, Penaeus monodon and Marsupenaeus japonicus, also significantly increased survival after VP9-dsRNA treatment. Our results provided evidence that VP9 gene plays an essential role in WSSV replication and it can be a potent target gene in the development of RNAi therapeutics for shrimps. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Shrimp is an indispensable source of revenue, as it accounts for almost 15% of the total aquaculture commodity in trade worldwide (FAO, 2014). However, the inflicting diseases on today’s aquaculture hampered the sustainable growth of the shrimp culture industry worldwide. In Asia-Pacific countries, the industry reported annual losses of about 4 billion US dollars (FAO, 2008). A large fraction of the damage to the industry is undoubtedly caused by viruses, which were accounted to have over 20 strains that of which affected both penaeid shrimp wild stocks and commercial production (Flegel, 2006). Among the known viruses to affect shrimp culture, the White Spot Syndrome Virus (WSSV) is one of the most potent and
∗ Corresponding author at: Molecular Biology and Biotechnology Laboratory, Research Cluster for Natural and Applied Sciences, University of Santo Tomas, ˜ 1015 Manila, Philippines. Fax: +63 2 7409730/7314031. Espana, E-mail addresses: [email protected], [email protected] (M.B.B. Maningas). http://dx.doi.org/10.1016/j.virusres.2016.01.013 0168-1702/© 2016 Elsevier B.V. All rights reserved.
widespread pathogen, as this virus can spread the disease rapidly in a span of 2–10 days post-infection and can bring 100% mortality (Flegel, 2006; Peinado-Guevara and Lopez-Meyer, 2006). Although there are some methods that displayed efficacy against the virus under experimental conditions, no effective treatments have been available to address WSSV problem in the field (Gitterle et al., 2006a,b; Dang et al., 2010; Cock and Gitterle, 2009). Understanding the underlying molecular interaction between the host and pathogen is critical in creating strategies to prevent diseases. A novel approach to understand host–pathogen interaction is the utilization of RNA interference (RNAi) technology. RNAi is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) triggers the silencing of a cognate gene. Unequivocal evidences pointed out the efficiency of RNAi in studying gene function and its implication in mounting antiviral responses in eukaryotes. In the shrimp system, a number of studies have demonstrated the effectiveness of RNAi in studying shrimp–pathogen interaction. Injection of WSSV genespecific dsRNA efficiently suppressed viral replication in penaeid shrimp (Kim et al., 2007; Robalino et al., 2007) and suppression of yellow head virus (YHV) replication by cognate-dsRNA, signifi-
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cantly reduced mortality in the black tiger shrimp (Tirasophon et al., 2007; Yodmuang et al., 2006). VP9, also known as ICP11, is a WSSV nonstructural protein encoded by ORF wssv230, which is suspected to be involved in the replication of viral genome, production of viral particles, and inhibition of host cell functions (Liu et al., 2006b; Wang et al., 2008; Tonganunt et al., 2009; Carpp et al., 2011). VP9 was identified and characterized to have DNA recognition folding and specific metal binding sites (Liu et al., 2006a,b; Chen et al., 2009). This DNA binding sites suggests that VP9 acts as DNA mimic that specifically binds to histone proteins H2A, H2B, and H3 (Wang et al., 2008). These studies showed that VP9 of WSSV plays an important role in the early infection of the virus. However the function of VP9 in vivo is yet to be elucidated (Liu et al., 2006a,b). Understanding the function of VP9 in WSSV infectivity will contribute to the existing knowledge on the virus and for drug development and prevention strategies. Here, we report the function of VP9 gene in vivo by utilizing RNA-interference technology. We further demonstrate that silencing VP9 gene impairs WSSV infectivity and significantly increase survival rate upon challenge with WSSV, at least in three shrimp species.
2. Materials and methods 2.1. Laboratory set-up and shrimp acclimatization Macrobrachium rosenbergii shrimp were purchased from Southeast Asian Fisheries Development Center (SEAFDEC) Binangonan, Rizal, Philippines, where the shrimps were hatched and reared in a closed system free from any history of infection. The samples were then brought to the laboratory placed in sealed polyethylene bags inflated with oxygen, followed by a week-long acclimatization period before the actual experiment. One hundred (100) juvenile shrimp, weighing 3–5 g were used in this study. The shrimps were maintained in filtered re-circulating de-chlorinated tap water tanks system maintained at 22–25 ◦ C and 0 ppt salinity. Water was replaced twice a week, however only during acclimatization period, and same water was used throughout the experiment starting from viral infection. The amount of feeds given was 1% of the total body weight (g) of the shrimp samples per tank on a daily basis.
2.2. PCR and RT-PCR primers synthesis For the optimization of PCR conditions, wsv168 primers (Flegel, 2006) were used for the confirmation of WSSV infection on shrimp samples. The elongation factor-1␣ (EF-1␣) used as an internal control (Table 1).
The primers used in the production of double stranded RNA using T7 RiboMAXTM Express Large Scale RNA Production System require a T7 promoter. Hence, T7 promoter was incorporated to gene specific primers (WSSV, GFP, & VP9) (Table 1). All primer sets were synthesized by 1st Base company (Singapore). RT-PCR analysis made use of primers specific for VP9 gene. Likewise, wsv168, EF-1␣ and primers were used as mentioned above (Table 1). 2.3. In vitro production of double-stranded (ds)RNA using T7 polymerase Double stranded RNA (dsRNA) was generated in vitro using T7 RiboMAXTM Express (Promega, USA) following the manufacturer’s instructions. The cDNA template used was synthesized from extracted total RNA using TRIzol Reagent (Invitrogen Life Technologies, USA) from WSSV-infected shrimp. The target genes, VP9 and GFP, were amplified using T7GFP and T7VP9 primers sets (Table 1), producing sense and anti-sense strand separately. Thermal cycling conditions were: Initial denaturation at 95 ◦ C for 5 min, followed by 30 cycles of denaturation at 95 ◦ C for 30 s, annealing at 50 ◦ C for 30 s, and extension at 72 ◦ C for 30 s, then a final extension of 72 ◦ C for 10 min. Products were then viewed through 1% Agarose gel electrophoresis prior to purification using ethanol precipitation, and then were quantified through UV–vis spectrophotometer. Purified PCR products with 1 g/L concentration were transcribed to yield single stranded RNAs. Equal volume of single stranded RNAs were allowed to anneal by incubation at 70 ◦ C for 10 min to produce double stranded RNA. dsRNA were further purified following the protocol provided by RiboMAX Express (Promega, USA) and then quantified through spectrophotomer. dsRNA stocks were prepared to a final concentration of 1 g/L. 2.4. Preparation of viral stock inocula and median lethal dosage (LD50 ) WSSV stock was isolated from WSSV-infected shrimp obtained from SEAFDEC, Iloilo, Philippines. WSSV infection was confirmed through Polymerase Chain Reaction (PCR) using wsv168 (Table 1). WSSV isolation was done as previously described by Rout et al. (2007). Viral isolates were obtained from gills of WSSV infected shrimp. 10–20 mg of tissues were homogenized in a 1.5 microcentrifuge tube with 600 L of PBS previously stored in 4 ◦ C freezer, and then the homogenate was centrifuged at 8000 rpm. The supernatant was then transferred to a new tube and filtered through 0.2 m Nylon Filter Media (Whatman® ) to a new tube. 100 L of the viral isolate was injected to 5 healthy M. rosenbergii shrimps, from which a new viral stockwas isolated for the challenge test,
Table 1 Primer sequences for PCR and RT-PCR. Primer name
Nucleotide sequence (5 -3 )
VP9
F-CCAGACTGACGCCGATTTCTT R-CGATGCCTCCATTGAGGACAAA
T7VP9
Liu et al. (2006a,b) Liu et al. (2006a,b)
F-TAATACGACTCACTATAGGCCAGACTGACGCCGATTTCTTRTAATACGACTCACTATAGGCGATGCCTCCATTGAGGACAAA
GFP Maningas et al. (2008)
F-ATGGTGAGCAAGGGCGAGGA R-TTACTTGTACAGCTCGTCCA
T7GFP Maningas et al. (2008)
F-TAATACGACTCACTATAGGATGGTGAGCAAGGGCGAGGA R-TAATACGACTCACTATAGGTTACTTGTACA GCTCGTCCA
WSSV/wsv168 Flegel (2006)
F-GTACGGCAATACTGGAGGAGGT R-GGAGATGTGTAAGATGGACAAG
EF-1␣ Maningas et al. (2008)
F-ATGGTTGTCAACTTTGCCCC R-TTGACCTCCTTGATCACACC
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Fig. 1. (A–C) RT-PCR analysis of shrimp samples treated with VP9-dsRNA, GFP-dsRNA, and PBS from representative tissue gills (g) and hemocyte (hm) sampled on 0, 1, 3, 7 dpi; using primers for VP9 and control gene EF-1␣, together with DNA marker (M) and blank control reaction (B).
following the same procedure mentioned above. The WSSV viral stockwas stored at −80 ◦ C freezer until the commencement of the challenge test. Presence of the virus was confirmed through PCR using DNA of the infected shrimp samples as template. The virus concentration used in the challenge test was determined based on the median lethal dosage (LD50 ). To determine the concentration, a serial dilution (10−1 up to 10−7 ) of the viral stock was performed by diluting 100 L of the viral isolate to 900 L PBS. 100 L of virus solution with the said dosages were intramuscularly injected to four shrimps per concentration, between the 2nd and 3rd tail segment. To confirm WSSV infection on dead shrimps, PCR was done using wsv168 primers (Table 1). Results of LD50 indicated 10−2 was the optimum concentration for the challenge test. Aliquots of 10−2 viral isolates were prepared and 100 L of the viral inoculum was injected to each shrimp. 2.5. RNAi & challenge test with WSSV 5 g of both VP9- and GFP-dsRNA were separately suspended in 100 L PBS, and were intra-muscularly injected between the 2nd and 3rd tail segment of each shrimp. PBS was also injected to serve as control. Twenty-two (22) shrimps were injected per treatment (VP9-, GFP-dsRNA, and PBS) were divided and placed in three separate tanks, 12 shrimps (for gene expression samples) on one tank and 10 shrimps (for mortality data) on the other. Twenty-four (24) hours after treatment, the viral challenge test followed by injecting in the same manner mentioned above 100 L of 10−2 concentration of WSSV viral stock. Shrimp mortality was recorded daily up to the 10th day-post-infection (dpi). For the determination of gene expression, 3 shrimp samples were randomly taken on 0, 1, 3, and 7 dpi. After the first challenge test, survivors from VP9- and GFPdsRNA treatment remained healthy until 30 dpi and thus subjected to re-infection using a higher (10−1 ) concentration of the viral stock. The control set-up (for PBS) was replenished with naïve M. rosenbergii samples and were injected with 100 L of PBS. Survival data was recorded until 19 dpi. To confirm the anti-viral property of VP9-dsRNA treatment, challenge test was likewise done on two other shrimp species Penaeus monodon and Marsupenaeus japonicus. 2.6. cDNA synthesis and RT-PCR analysis Relevant tissues (gills, heart, hepatopancreas, muscles, intestine lymphoid, and hemocyte) were dissected out from three individual shrimp samples of each treatment groups. Total RNA was extracted using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s protocol. Extracted RNA was then viewed and quantified
using UV–vis spectrophotometer. Extracted RNA was then converted to cDNA by adding 1 L of oligo(dT), 10 mMdNTP Mix, then the solution was incubated at 65 ◦ C for 5 min and chilled on ice for 1 min and centrifuged briefly using table-top centrifuge. To each mixture, 4 L of 5× First-Strand Buffer, and 1 L of 200 Units/L SuperScript III reverse transcriptase and 1 L RNase-Out (Invitrogen, USA). The solutions were mixed and allowed to stand for 5 min at room temperature, then incubated at 50 ◦ C for 60 min to stop the reaction. Viral clearance was tested using RT-PCR analysis from cDNA templates of the infected shrimp samples from VP9-, GFP-dsRNA, and PBS on 0, 1, 3, and 7 dpi. Primers specific for WSSV (wsv168) detection were used for RT-PCR analysis (Table 1). Likewise, expression of gene transcripts of EF-1␣ and VP9 were observed through RT-PCR with 10 L reactions containing 1X Buffer (with MgCl2 ), 2 mM dNTP’s and 1 g cDNA template, 2 M of sense and ant-sense primers, 1 unit of Taq polymerase, 5.2 L RNase-free water. Thermal cycling conditions were: initial denaturation at 95 ◦ C for 5 min, followed by 28 cycles of denaturation at 95 ◦ C for 30 s, annealing at 50 ◦ C for 30 s, and extension at 72 ◦ C then a final extension of 72 ◦ C for 10 min. Products were then viewed through Agarose gel electrophoresis, with 100 bp DNA marker. 2.7. Statistical analysis Kaplan–Meier survival curve with a chi-square test with 99% confidence interval using GraphPad Prism software 5 was used to analyze the survival data. 3. Results 3.1. In vivo gene silencing of VP9 RT-PCR analysis showed that the VP9 gene is expressed more in the gills and hemocyte using WSSV infected shrimps. Hence, hemocyte and gill tissues were utilized for the RT-PCR experiments. On both control groups, GFP-dsRNA and PBS treated shrimp VP9 gene transcript was consistently expressed in gills and hemocyte on 0–7 dpi (Fig. 1A and B). Expression of housekeeping gene EF-1␣ transcript was also consistent as shown in Fig. 1A–C. However for VP9-dsRNA treated shrimps, VP9 gene was silenced on both tissues at 0 (3 hpi) up to 7 dpi. To further investigate the effect of silencing the VP9 gene on the virus’ infection on shrimp, wsv168 primers (Table 1) were used to detect WSSV on the tissue samples. This set of primer is routinely used for WSSV detection (Flegel, 2006). For VP9dsRNA (bottom row), wsv-168 transcript was detected on 0 dpi on gills and hemocyte (Fig. 2). However, on 1 dpi wsv168 transcript was only detected on gills (g) samples and no longer on hemocytes
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Fig. 2. Gene expression using WSSV primers from tissues gills (g) and hemocyte (hm) of VP9-dsRNA, GFP-dsRNA, and PBS treatment groups sampled on 0, 1, 3, and 7 dpi.
(hm). wsv-168 transcript was no longer detected on 3 and 7 dpi. For the control groups, PBS and GFP-dsRNA treated shrimp wsv168 transcript was consistently expressed from 0 to 7 dpi. 3.2. Effects of silencing VP9 gene on shrimp survival upon WSSV infection To determine the involvement of the viral protein (VP9) gene in the interaction of WSSV and its shrimp hosts, a viral challenge test was conducted using WSSV. Challenge test data showed that VP9and GFP-dsRNA treated shrimps had a higher survival rate of 80%, and 70% respectively compared to 0% in the PBS treated shrimp at 25 dpi. Survival rates were maintained at 15 dpi for both VP9- and GPF-dsRNA treatment, while the PBS reached 0% (Fig. 3A). After 31 days, re-infection of shrimp survivors from challenge test with WSSV using ten times higher viral concentration (10−1 ), shrimps injected with VP9-dsRNA sustained 67% survival, while GFP-dsRNA and PBS treated reached 0% survival rate at 18 dpi (Fig. 3B). Challenge test using P. monodon and M. japonicus displayed increase in survival of 70% and 65%, respectively at 7 dpi, compared to that of the control groups with 10% and 20% at 7 dpi for PBS and GFP, respectively (Fig. 4A and B). Statistical analysis, Kaplan–Meier survival curve using chisquare test, revealed a significant difference on survival curves. A significant protective effect against the virus was observed for VP9-dsRNA and GFP-dsRNA treated shrimps compared with the PBS control group. 4. Discussion VP9 was identified as a non-structural protein having DNA recognition folding and specific metal binding sites which allows it to bind to nucleosomes acting as a DNA mimic which may enable the virus to unwind host DNA making it vulnerable that may lead to cell death (Liu et al., 2006a,b; Chen et al., 2009; Wang et al., 2008). Another implication of the previous results would be that host DNA is left unwounded by VP9 for WSSV to incorporate its
genome to the infected host. Taken together, it can be inferred that VP9 is involved in the early stage of WSSV infection and to confirm this and the previous results, we hypothesize that an in vivo knockdown of the gene would impair or inhibit WSSV infection. In this study, gene silencing through RNA interference was successfully utilized to elucidate the function of VP9 gene in vivo, confirming its involvement in WSSV infectivity as demonstrated by RT-PCR and its effect on shrimp survival after challenge test with WSSV. Results of RT-PCR (Fig. 1A) demonstrated that VP9 transcripts could be suppressed as early day 0 post-infection in WSSV infected shrimp samples (taken 3 hpi). This confirms the transcriptional analysis by real-time RT-PCR which showed that VP9 transcript is expressed during the early (2 hpi) up to the late stage of the virus’ infection which may account for it is early silencing at 3 hpi (2006b). Silencing of VP9 gene caused inhibition of wsv168 gene expression at 1 dpi (Fig. 2). In this study, we used wsv168 primers for the detection of viral clearance as this gene has been widely used as a target for detection methods for shrimp farms in countries like Thailand and the Philippines to screen broodstock and postlarva for WSSV infection (Flegel, 2006; Caipang et al., 2012; Muegue et al., 2013). Interestingly, blast results of wsv168 (AF536176) primer target sequence (Flegel, 2006), using NCBI BLAST program (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi), showed 99% identity to WSSV ribonucleotide reductase (RR1) gene (AF099142), which is an enzyme involved in nucleic acid metabolism, and was found to be involved in WSSV infection. Also, similar to VP9, wsv168/RR1 is one of WSSV “early genes” (van Hulten et al., 2000; Tsai et al., 2000; Lin et al., 2002). This result implies that silencing of VP9 also inhibit the expression of another gene (wsv168/RR1) needed by the virus for its DNA metabolism and synthesis. Using wsv168 as indicator of viral clearance, results show that whenVP9 was silenced at 3 hpi, it was not enough for the dsRNA to completely eliminate viral load, as viral clearance started at 1 dpi for hemocyte samples (Fig. 2). In another study involving silencing of viral genes, the earliest reported viral clearance started at 1 dpi (Dang et al., 2010). This suggests that VP9-dsRNA caused an early interruption in the viral infection mechanism. These findings confirms previous reports that early-transcribed viral genes are most likely to be involved in the viral replication and modification of the host’s cellular mechanism at the beginning of the infection, leading to viral inhibition (Wang et al., 2008; Wu et al., 2007). The molecular events corroborate the results of the challenge tests. Silencing of VP9 and WSSV clearance is confirmed by the challenge test data where survival of M. rosenbergii increased for VP9and GFP-dsRNA treated shrimps while the PBS treated shrimps decreased to 0% at 15 dpi. The VP9-dsRNA treated shrimp maintained 60% survival until 31 dpi (Fig. 3A). Treatment with GFP-dsRNA, although no viral genes were suppressed, displayed a protective effect. Antiviral protection by
Fig. 3. Challenge test with WSSV on M. rosenbergii shrimps treated with VP9-dsRNA, GFP-dsRNA, and PBS (A). Re-infection of shrimp survivors from the 1st challenge test from VP9-& GFP-dsRNA treated. Naïve shrimps were added for the PBS treatment (B).
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Fig. 4. Challenge test on Penaeus monodon (A) Marsupenaeus japonicus (B) shrimps treated with VP9-dsRNA, GFP-dsRNA, and PBS.
GFP-dsRNA demonstrates the non-specific anti-viral protection by arbitrary-sequence dsRNA against viruses (White spot syndrome and Taura syndrome virus) observed in shrimp (Maningas et al., 2008; Robalino et al., 2004). This phenomenon therefore illustrates that the shrimp’s anti-viral system recognizes dsRNA as a virusassociated molecular pattern present among shrimp (Robalino et al., 2004, 2007; Labreuche et al., 2010). However, during the first infection (Fig. 3A) the non-specific protection displayed by GFP-dsRNA in M. rosenbergii appeared to be as effective as VP9-dsRNA, making it difficult to discriminate the effectiveness of protection obtained from silencing VP9 from the non-specific antiviral protection. Thus, a re-infection experiment was done to clarify which treatment could withstand another challenge test (with 10 times higher viral concentration). Reinfection data showed that only VP9-dsRNA sustained its antiviral activity confirming the long-term protection sequence-specific antiviral immunity (Fig. 3B). These results support the other sequencespecific dsRNA treatment targeting viral proteins where efficiency of antiviral protection brought by RNAi lies in the genes targeted for silencing (Mejía-Ruíz et al., 2011). A recent review by Lima et al. (2013) mentioned the possibility of a dual-stimulation of innate immunity and antiviral silencing by dsRNA among invertebrates, which may therefore make specific RNAi-mediated anti-viral protection more effective and have a long-term effect. In contrast, GFP-dsRNA only triggered innate immune response, which did not persist upon re-infection. This is in accord with the study of Bartholomay et al. (2012) and Robalino et al. (2005) where nonspecific dsRNA triggers solely the innate immunity against viruses, which can only delay the onset of infection or provide protection in a short-term manner, which was also observed in the challenge test with P. monodon and M. japonicus (Fig. 4A and B). The protection rendered by silencing VP9 gene was evident in at least three other shrimp species thriving in different environment conditions namely; the freshwater—M. rosenbergii, the brackish water—P. monodon (Fig. 4A) and the marine species—M. japonicus (Fig. 4B). However, the results of GFP-dsRNA treatment were seemingly inconsistent among M. rosenbergii and the two other species. Survival was sustained by GFP-dsRNA at 50% until 31 dpi for M. rosenbergii, as compared to brackish water—P. monodon and the marine species—M. japonicus where only 20% survived at 7 dpi for both species. This difference in the protective effect of GFP-dsRNA treatment may be associated with the variability in susceptibility to WSSV infection among shrimp species (Bateman et al., 2012). Freshwater shrimp, in general, are known to be less susceptible compared to species thriving in waters with higher salinity as this make the optimal condition for WSSV infection (SanchezPaz, 2010; Jiravanichpaisal et al., 2004; Gao et al., 2011). Moreover, M. rosenbergii was specifically reported to possess tolerance to WSSV infection confirming the species-dependent susceptibility to WSSV (Hameed et al., 2000; Yoganandhan and Hameed, 2007;
Sarathi et al., 2008). These accounts for the increased protection rendered by GFP-dsRNA during the first challenge test (Fig. 3A). Taken together, all challenge tests established that silencing VP9 gene increased survival rate. To our knowledge, this might be the first report on the efficiency of a non-structural gene utilizing RNAi in 3 shrimp species. Several studies proved that RNAi targeting non-structural genes conferred a higher degree of anti-viral protection (Lima et al., 2013). These findings support the result of VP9-dsRNA treatment sustaining an antiviral protection even after re-infection. Viral clearance observed starting on 1 dpi with WSSV points out that the knockdown of VP9 gene, predicted to be involved in the transcription and regulation of WSSV (Liu et al., 2006a,b), might have disturbed the viral transcription process. In summary, in vivo gene silencing of VP9 resulted to viral clearance as indicated by the suppression of wsv168 gene, and significantly increased shrimp survival even upon re-infection. This significant increase is confirmed in at least 3 shrimp species, highlighting its potential application in mitigating WSSV infection. Lastly, VP9 definitely play a key role in viral replication. Taken altogether, this study proved that VP9 gene is a potential target for viral treatment and plays a major role in the infectivity of WSSV. Acknowledgments This study was supported in part by the Department of Science and Technology Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development—Japan Society for the Promotion of Science (DOST-JSPS) bilateral program Philippines, the Department of Science and Technology Science Education Institute (DOST-SEI) through the scholarship grant, Commission on Higher Education Philippine Higher Education Research Network (CHED-PHERNet), and the Research Center for the Natural and Applied Sciences of University of Santo Tomas by housing the experiment. We also thank Dr. Christopher Marlowe Caipang for his valuable comments and suggestions in the manuscript. References Bartholomay, L.C., Loy, D.S., Loy, J.D., Harris, D.L., 2012. Nucleic-acid based antivirals: augmenting RNA interference to ‘vaccinate’ Litopenaeus vannamei. J. Invertebr. Pathol. 110, 261–266. Bateman, K.S., Tew, I., French, C., Hicks, R.J., Martin, P., Munro, J., Stentiford, G.D., 2012. Susceptibility to infection and pathogenicity of white spot disease (WSD) in non-model crustacean host taxa from temperate regions. J. Invertebr. Pathol. 110 (3), 340–351. Caipang, C.M.A., Sibonga, M.F.J., Geduspan, J.S., Amar, M.J.A., 2012. An optimized loop-mediated isothermal amplification (lamp) assay for the detection of white spot syndrome virus (wssv) among cultured shrimps in the philippines. J. Anim. Plant Sci. 22 (4), 927–932. Carpp, L., Galler, R., Bonaldo, M., 2011. Interaction between the yellow fever virus nonstructural protein NS3 and the host protein Alix contributes to the release of infectious particles. Microbes Infect. 13, 85–95.
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Peinado-Guevara, L.I., Lopez-Meyer, M., 2006. Detailed monitoring of white spot syndrome virus (WSSV) in shrimp commercial ponds in Sinaloa, Mexico by nested PCR. Aquaculture 251, 33–45. Robalino, J., Browdy, S., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G., 2004. Induction of antivaral immunity by double-stranded RNA in a marine invertebrate. J. Virol. 78 (19), 10442–10448. Robalino, J., Bartlett, T., Shepard, E., Prior, S., Jaramillo, G., Scura, E., Chapman, R.W., Gross, P.S., Browdy, C.L., Warr, G.W., 2005. Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response? J. Virol. 79 (21), 13561–13571. Robalino, J., Bartlett, T.C., Chapman, R.W., Gross, P.S., Browdy, C.L., Warr, G.W., 2007. Double-stranded RNA and anti-viral immunity in marine shrimp: inducible host mechanism and evidence for the evolution of viral counter-responses. Dev. Comp. Immunol. 31, 539–547. Rout, N., Kumar, S., Jaganmohan, S., Murugan, V., 2007. DNA vaccines encoding viral envelope proteins confer protective effect immunity against WSSV in black tiger shrimp. Vaccine 25, 2778–2786. Sanchez-Paz, A., 2010. White spot syndrome virus: an overview on an immediate concern. Vet. Res. 41, 43. Sarathi, M., Nazeer Basha, A., Ravi, M., Venkatesan, C., Senthil Kumar, B., Sahul Hameed, A.S., 2008. Clearance of white spot syndrome virus (WSSV) and immunological changes in experimentally WSSV-injected Macrobrachium rosenbergii. Fish Shellfish Immunol. 25 (3), 222–230. Tirasophon, W., Yodmuang, S., Chinnirunvong, W., Chinnirunvong, W., Plongthongkum, N., Panyim, S., 2007. Therapeutic inhibition of yellow head virus multiplication in infected shrimps by YHV-protease dsRNA. Antiviral Res. 74 (2), 150–155. Tonganunt, M., Saelee, N., Chotigeat, W., Phongdara, A., 2009. Identification of a receptor for activated protein kinase C1 (Pm-RACK1) a cellular gene product from black tiger shrimp (Penaeus monodon) interacts with a protein, VP9 from the white spot syndrome virus. Fish Shellfish Immunol. 26, 509–514. Tsai, M.F., Lo, C.F., van Hulten, M.C.W., Tzeng, H.F., Chou, C.M., Huang, C.J., Wang, C.H., Lin, J.W., Vlak, J.M., Kou, G.H., 2000. Transcriptional analysis of the ribonucleotide reductase genes of shrimp white spot syndrome virus. Virology 277 (1), 92–99. van Hulten, M.C., Tsai, M.F., Schipper, C.A., Lo, C.F., Kou, G.H., Vlak, J.M., 2000. Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions. J. Gen. Virol. 81 (2), 307–316. Wang, H.C., Wang, H.C., Ko, T.P., Lee, Y.M., Leu, J.H., Ho, C.H., Huang, W.P., Lo, C.F., Wang, A., 2008. White spot syndrome virus protein ICP11: a histone-binding DNA mimic that disrupts nucleosome assembly. Proc. Natl. Acad. Sci. U. S. A. 105 (52), 20758–20763. Wu, Y., Ling, L., Yang, L.-S., Weng, S.-P., Chan, S.-M., He, J.-G., 2007. Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence-specific siRNA. Aquaculture 271, 21–30. Yodmuang, S.W., Tirasophon, Y., Roshorn, W., Chinnirunvong, W., Panyim, S., 2006. YHV-protease dsRNA inhibits YHV replication in Penaeu smonodon and prevents mortality. Biochem. Biophys. Res. Commun. 341, 351–356. Yoganandhan, K., Hameed, A.S.S., 2007. Tolerance to white spot syndrome virus (WSSV) in the freshwater prawn Macrobrachium rosenbergii is associated with low VP28 envelope protein expression. Dis. Aquat. Organ. 73, 193–199.
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Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii Rod Russel R. Alenton a , Hidehiro Kondo d , Ikuo Hirono d , Mary Beth B. Maningas a,b,c,∗ a
The Graduate School, University of Santo Tomas, Espa˜ na, 1015 Manila, Philippines Department of Biological Sciences, College of Science, University of Santo Tomas, Espa˜ na, 1015 Manila, Philippines Molecular Biology and Biotechnology Laboratory, Research Cluster for Natural and Applied Sciences, University of Santo Tomas, Espa˜ na, 1015 Manila, Philippines d Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, Japan b c
a r t i c l e
i n f o
Article history: Received 6 October 2015 Received in revised form 19 January 2016 Accepted 20 January 2016 Available online 23 January 2016 Keywords: VP9 RNA interference Macrobrachium rosenbergii Gene silencing WSSV
a b s t r a c t White Spot Syndrome Virus (WSSV) remains the most widespread and devastating infectious agent that hit the shrimp aquaculture industry worldwide. To date, there are no known effective strategies yet to combat WSSV infection. Hence, functional studies on genes critical for viral infection is essential in elucidating shrimp–virus interaction. Here we report the function of a gene from WSSV coding for a non-structural protein, VP9, utilizing RNA interference. Silencing of VP9 gene also effectively suppressed other gene region in the WSSV genome (wsv168 gene) as early as day 1 post infection (dpi). Three setups using Macrobrachium rosenbergii shrimp were prepared for treatment using VP9-dsRNA, GFP-dsRNA, and PBS. Each shrimp was challenge with WSSV, and survival rate was recorded. VP9- and GFP-dsRNA injected shrimps showed a significant survival rate of 80% and 70%, respectively, in contrast to 0% of the PBS injected shrimps at 25 dpi. Re-infection of shrimp survivors using a higher viral titer concentration, concurrent with the infection of new shrimp samples for the PBS control group, resulted in a significant 67% survival rate for VP9-dsRNA compared to 0% with that of GFP-dsRNA and PBS group. Challenge test on two more species, Penaeus monodon and Marsupenaeus japonicus, also significantly increased survival after VP9-dsRNA treatment. Our results provided evidence that VP9 gene plays an essential role in WSSV replication and it can be a potent target gene in the development of RNAi therapeutics for shrimps. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Shrimp is an indispensable source of revenue, as it accounts for almost 15% of the total aquaculture commodity in trade worldwide (FAO, 2014). However, the inflicting diseases on today’s aquaculture hampered the sustainable growth of the shrimp culture industry worldwide. In Asia-Pacific countries, the industry reported annual losses of about 4 billion US dollars (FAO, 2008). A large fraction of the damage to the industry is undoubtedly caused by viruses, which were accounted to have over 20 strains that of which affected both penaeid shrimp wild stocks and commercial production (Flegel, 2006). Among the known viruses to affect shrimp culture, the White Spot Syndrome Virus (WSSV) is one of the most potent and
∗ Corresponding author at: Molecular Biology and Biotechnology Laboratory, Research Cluster for Natural and Applied Sciences, University of Santo Tomas, ˜ 1015 Manila, Philippines. Fax: +63 2 7409730/7314031. Espana, E-mail addresses: [email protected], [email protected] (M.B.B. Maningas). http://dx.doi.org/10.1016/j.virusres.2016.01.013 0168-1702/© 2016 Elsevier B.V. All rights reserved.
widespread pathogen, as this virus can spread the disease rapidly in a span of 2–10 days post-infection and can bring 100% mortality (Flegel, 2006; Peinado-Guevara and Lopez-Meyer, 2006). Although there are some methods that displayed efficacy against the virus under experimental conditions, no effective treatments have been available to address WSSV problem in the field (Gitterle et al., 2006a,b; Dang et al., 2010; Cock and Gitterle, 2009). Understanding the underlying molecular interaction between the host and pathogen is critical in creating strategies to prevent diseases. A novel approach to understand host–pathogen interaction is the utilization of RNA interference (RNAi) technology. RNAi is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) triggers the silencing of a cognate gene. Unequivocal evidences pointed out the efficiency of RNAi in studying gene function and its implication in mounting antiviral responses in eukaryotes. In the shrimp system, a number of studies have demonstrated the effectiveness of RNAi in studying shrimp–pathogen interaction. Injection of WSSV genespecific dsRNA efficiently suppressed viral replication in penaeid shrimp (Kim et al., 2007; Robalino et al., 2007) and suppression of yellow head virus (YHV) replication by cognate-dsRNA, signifi-
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cantly reduced mortality in the black tiger shrimp (Tirasophon et al., 2007; Yodmuang et al., 2006). VP9, also known as ICP11, is a WSSV nonstructural protein encoded by ORF wssv230, which is suspected to be involved in the replication of viral genome, production of viral particles, and inhibition of host cell functions (Liu et al., 2006b; Wang et al., 2008; Tonganunt et al., 2009; Carpp et al., 2011). VP9 was identified and characterized to have DNA recognition folding and specific metal binding sites (Liu et al., 2006a,b; Chen et al., 2009). This DNA binding sites suggests that VP9 acts as DNA mimic that specifically binds to histone proteins H2A, H2B, and H3 (Wang et al., 2008). These studies showed that VP9 of WSSV plays an important role in the early infection of the virus. However the function of VP9 in vivo is yet to be elucidated (Liu et al., 2006a,b). Understanding the function of VP9 in WSSV infectivity will contribute to the existing knowledge on the virus and for drug development and prevention strategies. Here, we report the function of VP9 gene in vivo by utilizing RNA-interference technology. We further demonstrate that silencing VP9 gene impairs WSSV infectivity and significantly increase survival rate upon challenge with WSSV, at least in three shrimp species.
2. Materials and methods 2.1. Laboratory set-up and shrimp acclimatization Macrobrachium rosenbergii shrimp were purchased from Southeast Asian Fisheries Development Center (SEAFDEC) Binangonan, Rizal, Philippines, where the shrimps were hatched and reared in a closed system free from any history of infection. The samples were then brought to the laboratory placed in sealed polyethylene bags inflated with oxygen, followed by a week-long acclimatization period before the actual experiment. One hundred (100) juvenile shrimp, weighing 3–5 g were used in this study. The shrimps were maintained in filtered re-circulating de-chlorinated tap water tanks system maintained at 22–25 ◦ C and 0 ppt salinity. Water was replaced twice a week, however only during acclimatization period, and same water was used throughout the experiment starting from viral infection. The amount of feeds given was 1% of the total body weight (g) of the shrimp samples per tank on a daily basis.
2.2. PCR and RT-PCR primers synthesis For the optimization of PCR conditions, wsv168 primers (Flegel, 2006) were used for the confirmation of WSSV infection on shrimp samples. The elongation factor-1␣ (EF-1␣) used as an internal control (Table 1).
The primers used in the production of double stranded RNA using T7 RiboMAXTM Express Large Scale RNA Production System require a T7 promoter. Hence, T7 promoter was incorporated to gene specific primers (WSSV, GFP, & VP9) (Table 1). All primer sets were synthesized by 1st Base company (Singapore). RT-PCR analysis made use of primers specific for VP9 gene. Likewise, wsv168, EF-1␣ and primers were used as mentioned above (Table 1). 2.3. In vitro production of double-stranded (ds)RNA using T7 polymerase Double stranded RNA (dsRNA) was generated in vitro using T7 RiboMAXTM Express (Promega, USA) following the manufacturer’s instructions. The cDNA template used was synthesized from extracted total RNA using TRIzol Reagent (Invitrogen Life Technologies, USA) from WSSV-infected shrimp. The target genes, VP9 and GFP, were amplified using T7GFP and T7VP9 primers sets (Table 1), producing sense and anti-sense strand separately. Thermal cycling conditions were: Initial denaturation at 95 ◦ C for 5 min, followed by 30 cycles of denaturation at 95 ◦ C for 30 s, annealing at 50 ◦ C for 30 s, and extension at 72 ◦ C for 30 s, then a final extension of 72 ◦ C for 10 min. Products were then viewed through 1% Agarose gel electrophoresis prior to purification using ethanol precipitation, and then were quantified through UV–vis spectrophotometer. Purified PCR products with 1 g/L concentration were transcribed to yield single stranded RNAs. Equal volume of single stranded RNAs were allowed to anneal by incubation at 70 ◦ C for 10 min to produce double stranded RNA. dsRNA were further purified following the protocol provided by RiboMAX Express (Promega, USA) and then quantified through spectrophotomer. dsRNA stocks were prepared to a final concentration of 1 g/L. 2.4. Preparation of viral stock inocula and median lethal dosage (LD50 ) WSSV stock was isolated from WSSV-infected shrimp obtained from SEAFDEC, Iloilo, Philippines. WSSV infection was confirmed through Polymerase Chain Reaction (PCR) using wsv168 (Table 1). WSSV isolation was done as previously described by Rout et al. (2007). Viral isolates were obtained from gills of WSSV infected shrimp. 10–20 mg of tissues were homogenized in a 1.5 microcentrifuge tube with 600 L of PBS previously stored in 4 ◦ C freezer, and then the homogenate was centrifuged at 8000 rpm. The supernatant was then transferred to a new tube and filtered through 0.2 m Nylon Filter Media (Whatman® ) to a new tube. 100 L of the viral isolate was injected to 5 healthy M. rosenbergii shrimps, from which a new viral stockwas isolated for the challenge test,
Table 1 Primer sequences for PCR and RT-PCR. Primer name
Nucleotide sequence (5 -3 )
VP9
F-CCAGACTGACGCCGATTTCTT R-CGATGCCTCCATTGAGGACAAA
T7VP9
Liu et al. (2006a,b) Liu et al. (2006a,b)
F-TAATACGACTCACTATAGGCCAGACTGACGCCGATTTCTTRTAATACGACTCACTATAGGCGATGCCTCCATTGAGGACAAA
GFP Maningas et al. (2008)
F-ATGGTGAGCAAGGGCGAGGA R-TTACTTGTACAGCTCGTCCA
T7GFP Maningas et al. (2008)
F-TAATACGACTCACTATAGGATGGTGAGCAAGGGCGAGGA R-TAATACGACTCACTATAGGTTACTTGTACA GCTCGTCCA
WSSV/wsv168 Flegel (2006)
F-GTACGGCAATACTGGAGGAGGT R-GGAGATGTGTAAGATGGACAAG
EF-1␣ Maningas et al. (2008)
F-ATGGTTGTCAACTTTGCCCC R-TTGACCTCCTTGATCACACC
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Fig. 1. (A–C) RT-PCR analysis of shrimp samples treated with VP9-dsRNA, GFP-dsRNA, and PBS from representative tissue gills (g) and hemocyte (hm) sampled on 0, 1, 3, 7 dpi; using primers for VP9 and control gene EF-1␣, together with DNA marker (M) and blank control reaction (B).
following the same procedure mentioned above. The WSSV viral stockwas stored at −80 ◦ C freezer until the commencement of the challenge test. Presence of the virus was confirmed through PCR using DNA of the infected shrimp samples as template. The virus concentration used in the challenge test was determined based on the median lethal dosage (LD50 ). To determine the concentration, a serial dilution (10−1 up to 10−7 ) of the viral stock was performed by diluting 100 L of the viral isolate to 900 L PBS. 100 L of virus solution with the said dosages were intramuscularly injected to four shrimps per concentration, between the 2nd and 3rd tail segment. To confirm WSSV infection on dead shrimps, PCR was done using wsv168 primers (Table 1). Results of LD50 indicated 10−2 was the optimum concentration for the challenge test. Aliquots of 10−2 viral isolates were prepared and 100 L of the viral inoculum was injected to each shrimp. 2.5. RNAi & challenge test with WSSV 5 g of both VP9- and GFP-dsRNA were separately suspended in 100 L PBS, and were intra-muscularly injected between the 2nd and 3rd tail segment of each shrimp. PBS was also injected to serve as control. Twenty-two (22) shrimps were injected per treatment (VP9-, GFP-dsRNA, and PBS) were divided and placed in three separate tanks, 12 shrimps (for gene expression samples) on one tank and 10 shrimps (for mortality data) on the other. Twenty-four (24) hours after treatment, the viral challenge test followed by injecting in the same manner mentioned above 100 L of 10−2 concentration of WSSV viral stock. Shrimp mortality was recorded daily up to the 10th day-post-infection (dpi). For the determination of gene expression, 3 shrimp samples were randomly taken on 0, 1, 3, and 7 dpi. After the first challenge test, survivors from VP9- and GFPdsRNA treatment remained healthy until 30 dpi and thus subjected to re-infection using a higher (10−1 ) concentration of the viral stock. The control set-up (for PBS) was replenished with naïve M. rosenbergii samples and were injected with 100 L of PBS. Survival data was recorded until 19 dpi. To confirm the anti-viral property of VP9-dsRNA treatment, challenge test was likewise done on two other shrimp species Penaeus monodon and Marsupenaeus japonicus. 2.6. cDNA synthesis and RT-PCR analysis Relevant tissues (gills, heart, hepatopancreas, muscles, intestine lymphoid, and hemocyte) were dissected out from three individual shrimp samples of each treatment groups. Total RNA was extracted using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s protocol. Extracted RNA was then viewed and quantified
using UV–vis spectrophotometer. Extracted RNA was then converted to cDNA by adding 1 L of oligo(dT), 10 mMdNTP Mix, then the solution was incubated at 65 ◦ C for 5 min and chilled on ice for 1 min and centrifuged briefly using table-top centrifuge. To each mixture, 4 L of 5× First-Strand Buffer, and 1 L of 200 Units/L SuperScript III reverse transcriptase and 1 L RNase-Out (Invitrogen, USA). The solutions were mixed and allowed to stand for 5 min at room temperature, then incubated at 50 ◦ C for 60 min to stop the reaction. Viral clearance was tested using RT-PCR analysis from cDNA templates of the infected shrimp samples from VP9-, GFP-dsRNA, and PBS on 0, 1, 3, and 7 dpi. Primers specific for WSSV (wsv168) detection were used for RT-PCR analysis (Table 1). Likewise, expression of gene transcripts of EF-1␣ and VP9 were observed through RT-PCR with 10 L reactions containing 1X Buffer (with MgCl2 ), 2 mM dNTP’s and 1 g cDNA template, 2 M of sense and ant-sense primers, 1 unit of Taq polymerase, 5.2 L RNase-free water. Thermal cycling conditions were: initial denaturation at 95 ◦ C for 5 min, followed by 28 cycles of denaturation at 95 ◦ C for 30 s, annealing at 50 ◦ C for 30 s, and extension at 72 ◦ C then a final extension of 72 ◦ C for 10 min. Products were then viewed through Agarose gel electrophoresis, with 100 bp DNA marker. 2.7. Statistical analysis Kaplan–Meier survival curve with a chi-square test with 99% confidence interval using GraphPad Prism software 5 was used to analyze the survival data. 3. Results 3.1. In vivo gene silencing of VP9 RT-PCR analysis showed that the VP9 gene is expressed more in the gills and hemocyte using WSSV infected shrimps. Hence, hemocyte and gill tissues were utilized for the RT-PCR experiments. On both control groups, GFP-dsRNA and PBS treated shrimp VP9 gene transcript was consistently expressed in gills and hemocyte on 0–7 dpi (Fig. 1A and B). Expression of housekeeping gene EF-1␣ transcript was also consistent as shown in Fig. 1A–C. However for VP9-dsRNA treated shrimps, VP9 gene was silenced on both tissues at 0 (3 hpi) up to 7 dpi. To further investigate the effect of silencing the VP9 gene on the virus’ infection on shrimp, wsv168 primers (Table 1) were used to detect WSSV on the tissue samples. This set of primer is routinely used for WSSV detection (Flegel, 2006). For VP9dsRNA (bottom row), wsv-168 transcript was detected on 0 dpi on gills and hemocyte (Fig. 2). However, on 1 dpi wsv168 transcript was only detected on gills (g) samples and no longer on hemocytes
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Fig. 2. Gene expression using WSSV primers from tissues gills (g) and hemocyte (hm) of VP9-dsRNA, GFP-dsRNA, and PBS treatment groups sampled on 0, 1, 3, and 7 dpi.
(hm). wsv-168 transcript was no longer detected on 3 and 7 dpi. For the control groups, PBS and GFP-dsRNA treated shrimp wsv168 transcript was consistently expressed from 0 to 7 dpi. 3.2. Effects of silencing VP9 gene on shrimp survival upon WSSV infection To determine the involvement of the viral protein (VP9) gene in the interaction of WSSV and its shrimp hosts, a viral challenge test was conducted using WSSV. Challenge test data showed that VP9and GFP-dsRNA treated shrimps had a higher survival rate of 80%, and 70% respectively compared to 0% in the PBS treated shrimp at 25 dpi. Survival rates were maintained at 15 dpi for both VP9- and GPF-dsRNA treatment, while the PBS reached 0% (Fig. 3A). After 31 days, re-infection of shrimp survivors from challenge test with WSSV using ten times higher viral concentration (10−1 ), shrimps injected with VP9-dsRNA sustained 67% survival, while GFP-dsRNA and PBS treated reached 0% survival rate at 18 dpi (Fig. 3B). Challenge test using P. monodon and M. japonicus displayed increase in survival of 70% and 65%, respectively at 7 dpi, compared to that of the control groups with 10% and 20% at 7 dpi for PBS and GFP, respectively (Fig. 4A and B). Statistical analysis, Kaplan–Meier survival curve using chisquare test, revealed a significant difference on survival curves. A significant protective effect against the virus was observed for VP9-dsRNA and GFP-dsRNA treated shrimps compared with the PBS control group. 4. Discussion VP9 was identified as a non-structural protein having DNA recognition folding and specific metal binding sites which allows it to bind to nucleosomes acting as a DNA mimic which may enable the virus to unwind host DNA making it vulnerable that may lead to cell death (Liu et al., 2006a,b; Chen et al., 2009; Wang et al., 2008). Another implication of the previous results would be that host DNA is left unwounded by VP9 for WSSV to incorporate its
genome to the infected host. Taken together, it can be inferred that VP9 is involved in the early stage of WSSV infection and to confirm this and the previous results, we hypothesize that an in vivo knockdown of the gene would impair or inhibit WSSV infection. In this study, gene silencing through RNA interference was successfully utilized to elucidate the function of VP9 gene in vivo, confirming its involvement in WSSV infectivity as demonstrated by RT-PCR and its effect on shrimp survival after challenge test with WSSV. Results of RT-PCR (Fig. 1A) demonstrated that VP9 transcripts could be suppressed as early day 0 post-infection in WSSV infected shrimp samples (taken 3 hpi). This confirms the transcriptional analysis by real-time RT-PCR which showed that VP9 transcript is expressed during the early (2 hpi) up to the late stage of the virus’ infection which may account for it is early silencing at 3 hpi (2006b). Silencing of VP9 gene caused inhibition of wsv168 gene expression at 1 dpi (Fig. 2). In this study, we used wsv168 primers for the detection of viral clearance as this gene has been widely used as a target for detection methods for shrimp farms in countries like Thailand and the Philippines to screen broodstock and postlarva for WSSV infection (Flegel, 2006; Caipang et al., 2012; Muegue et al., 2013). Interestingly, blast results of wsv168 (AF536176) primer target sequence (Flegel, 2006), using NCBI BLAST program (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi), showed 99% identity to WSSV ribonucleotide reductase (RR1) gene (AF099142), which is an enzyme involved in nucleic acid metabolism, and was found to be involved in WSSV infection. Also, similar to VP9, wsv168/RR1 is one of WSSV “early genes” (van Hulten et al., 2000; Tsai et al., 2000; Lin et al., 2002). This result implies that silencing of VP9 also inhibit the expression of another gene (wsv168/RR1) needed by the virus for its DNA metabolism and synthesis. Using wsv168 as indicator of viral clearance, results show that whenVP9 was silenced at 3 hpi, it was not enough for the dsRNA to completely eliminate viral load, as viral clearance started at 1 dpi for hemocyte samples (Fig. 2). In another study involving silencing of viral genes, the earliest reported viral clearance started at 1 dpi (Dang et al., 2010). This suggests that VP9-dsRNA caused an early interruption in the viral infection mechanism. These findings confirms previous reports that early-transcribed viral genes are most likely to be involved in the viral replication and modification of the host’s cellular mechanism at the beginning of the infection, leading to viral inhibition (Wang et al., 2008; Wu et al., 2007). The molecular events corroborate the results of the challenge tests. Silencing of VP9 and WSSV clearance is confirmed by the challenge test data where survival of M. rosenbergii increased for VP9and GFP-dsRNA treated shrimps while the PBS treated shrimps decreased to 0% at 15 dpi. The VP9-dsRNA treated shrimp maintained 60% survival until 31 dpi (Fig. 3A). Treatment with GFP-dsRNA, although no viral genes were suppressed, displayed a protective effect. Antiviral protection by
Fig. 3. Challenge test with WSSV on M. rosenbergii shrimps treated with VP9-dsRNA, GFP-dsRNA, and PBS (A). Re-infection of shrimp survivors from the 1st challenge test from VP9-& GFP-dsRNA treated. Naïve shrimps were added for the PBS treatment (B).
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Fig. 4. Challenge test on Penaeus monodon (A) Marsupenaeus japonicus (B) shrimps treated with VP9-dsRNA, GFP-dsRNA, and PBS.
GFP-dsRNA demonstrates the non-specific anti-viral protection by arbitrary-sequence dsRNA against viruses (White spot syndrome and Taura syndrome virus) observed in shrimp (Maningas et al., 2008; Robalino et al., 2004). This phenomenon therefore illustrates that the shrimp’s anti-viral system recognizes dsRNA as a virusassociated molecular pattern present among shrimp (Robalino et al., 2004, 2007; Labreuche et al., 2010). However, during the first infection (Fig. 3A) the non-specific protection displayed by GFP-dsRNA in M. rosenbergii appeared to be as effective as VP9-dsRNA, making it difficult to discriminate the effectiveness of protection obtained from silencing VP9 from the non-specific antiviral protection. Thus, a re-infection experiment was done to clarify which treatment could withstand another challenge test (with 10 times higher viral concentration). Reinfection data showed that only VP9-dsRNA sustained its antiviral activity confirming the long-term protection sequence-specific antiviral immunity (Fig. 3B). These results support the other sequencespecific dsRNA treatment targeting viral proteins where efficiency of antiviral protection brought by RNAi lies in the genes targeted for silencing (Mejía-Ruíz et al., 2011). A recent review by Lima et al. (2013) mentioned the possibility of a dual-stimulation of innate immunity and antiviral silencing by dsRNA among invertebrates, which may therefore make specific RNAi-mediated anti-viral protection more effective and have a long-term effect. In contrast, GFP-dsRNA only triggered innate immune response, which did not persist upon re-infection. This is in accord with the study of Bartholomay et al. (2012) and Robalino et al. (2005) where nonspecific dsRNA triggers solely the innate immunity against viruses, which can only delay the onset of infection or provide protection in a short-term manner, which was also observed in the challenge test with P. monodon and M. japonicus (Fig. 4A and B). The protection rendered by silencing VP9 gene was evident in at least three other shrimp species thriving in different environment conditions namely; the freshwater—M. rosenbergii, the brackish water—P. monodon (Fig. 4A) and the marine species—M. japonicus (Fig. 4B). However, the results of GFP-dsRNA treatment were seemingly inconsistent among M. rosenbergii and the two other species. Survival was sustained by GFP-dsRNA at 50% until 31 dpi for M. rosenbergii, as compared to brackish water—P. monodon and the marine species—M. japonicus where only 20% survived at 7 dpi for both species. This difference in the protective effect of GFP-dsRNA treatment may be associated with the variability in susceptibility to WSSV infection among shrimp species (Bateman et al., 2012). Freshwater shrimp, in general, are known to be less susceptible compared to species thriving in waters with higher salinity as this make the optimal condition for WSSV infection (SanchezPaz, 2010; Jiravanichpaisal et al., 2004; Gao et al., 2011). Moreover, M. rosenbergii was specifically reported to possess tolerance to WSSV infection confirming the species-dependent susceptibility to WSSV (Hameed et al., 2000; Yoganandhan and Hameed, 2007;
Sarathi et al., 2008). These accounts for the increased protection rendered by GFP-dsRNA during the first challenge test (Fig. 3A). Taken together, all challenge tests established that silencing VP9 gene increased survival rate. To our knowledge, this might be the first report on the efficiency of a non-structural gene utilizing RNAi in 3 shrimp species. Several studies proved that RNAi targeting non-structural genes conferred a higher degree of anti-viral protection (Lima et al., 2013). These findings support the result of VP9-dsRNA treatment sustaining an antiviral protection even after re-infection. Viral clearance observed starting on 1 dpi with WSSV points out that the knockdown of VP9 gene, predicted to be involved in the transcription and regulation of WSSV (Liu et al., 2006a,b), might have disturbed the viral transcription process. In summary, in vivo gene silencing of VP9 resulted to viral clearance as indicated by the suppression of wsv168 gene, and significantly increased shrimp survival even upon re-infection. This significant increase is confirmed in at least 3 shrimp species, highlighting its potential application in mitigating WSSV infection. Lastly, VP9 definitely play a key role in viral replication. Taken altogether, this study proved that VP9 gene is a potential target for viral treatment and plays a major role in the infectivity of WSSV. Acknowledgments This study was supported in part by the Department of Science and Technology Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development—Japan Society for the Promotion of Science (DOST-JSPS) bilateral program Philippines, the Department of Science and Technology Science Education Institute (DOST-SEI) through the scholarship grant, Commission on Higher Education Philippine Higher Education Research Network (CHED-PHERNet), and the Research Center for the Natural and Applied Sciences of University of Santo Tomas by housing the experiment. We also thank Dr. Christopher Marlowe Caipang for his valuable comments and suggestions in the manuscript. References Bartholomay, L.C., Loy, D.S., Loy, J.D., Harris, D.L., 2012. Nucleic-acid based antivirals: augmenting RNA interference to ‘vaccinate’ Litopenaeus vannamei. J. Invertebr. Pathol. 110, 261–266. Bateman, K.S., Tew, I., French, C., Hicks, R.J., Martin, P., Munro, J., Stentiford, G.D., 2012. Susceptibility to infection and pathogenicity of white spot disease (WSD) in non-model crustacean host taxa from temperate regions. J. Invertebr. Pathol. 110 (3), 340–351. Caipang, C.M.A., Sibonga, M.F.J., Geduspan, J.S., Amar, M.J.A., 2012. An optimized loop-mediated isothermal amplification (lamp) assay for the detection of white spot syndrome virus (wssv) among cultured shrimps in the philippines. J. Anim. Plant Sci. 22 (4), 927–932. Carpp, L., Galler, R., Bonaldo, M., 2011. Interaction between the yellow fever virus nonstructural protein NS3 and the host protein Alix contributes to the release of infectious particles. Microbes Infect. 13, 85–95.
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