Effective inhibition of Japanese encephalitis virus replication by shRNAs targeting various viral genes in vitro and in vivo

Virology 454-455 (2014) 48–59

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Effective inhibition of Japanese encephalitis virus replication by shRNAs targeting various viral genes in vitro and in vivo Ting Shen a, Ke Liu a,b, Denian Miao c, Ruibing Cao a, Puyan Chen a,n a Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China b Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, PR China c Institute of Animal Husbandry & Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 22 October 2013 Returned to author for revisions 26 November 2013 Accepted 26 January 2014 Available online 22 February 2014

Japanese encephalitis virus (JEV) is a serious mosquito-borne flavivirus that causes acute encephalitis in humans and many animals, with a high fatality rate. RNA interference (RNAi) is an evolutionarily conserved mechanism for the specific suppression of gene expression, which can be used as a reasonable antiviral strategy. In this study, 10 shRNAs targeting different regions of the JEV genome were designed, and their inhibition of viral replication in vitro and in vivo was determined. Treatment with these shRNAs significantly inhibited JEV replication in BHK-21 and SK-N-SH cells. An immunohistochemical analysis of suckling mice showed that brain sections pretreated with pGP-JE-1, pGP-JE-2 or pGP-JE-3 lacked viral particles. The survival of BALB/c mice challenged with 20 LD50 of the JEV NJ2008 strain at 24 h postinjection or simultaneously with pGP-JE-2 was 83.3% and 66.7%, respectively. The results demonstrated that the efficiency of gene silencing and virus inhibition varied between shRNAs to different target genes and sites. Meanwhile, the shRNA-mediated antiviral effect was dose- and time-dependent, including prophylactic and therapeutic effect on virus infection both in vitro and in vivo. The whole results indicate that these shRNAs can inhibit JEV infection sufficiently in vitro and in vivo and might be a potential new tool for controlling JEV infection. & 2014 Elsevier Inc. All rights reserved.

Keywords: JEV RNAi ShRNA In vitro and in vivo

Introduction Japanese encephalitis virus (JEV) belongs to the Flavivirus genus of the Flaviviridae family and causes infection of the central nervous system in humans and stillbirths in swine (Burke et al., 2001). JEV has a zoonotic transmission cycle between birds and mosquitoes, with swine serving as intermediated amplifier hosts and the virus can spread from swine to humans via mosquito bites (van de Hurk et al., 2009; Weaver and Barrett, 2004). According to the latest report of the World Health Organization, Japanese encephalitis (JE) is endemic in 24 Asian and Oceanian countries, with an estimated 68,000 JE cases annually. More than 3 billion people live in JE-endemic countries (Campbell et al., 2011; Mackenzie and Williams, 2009). Furthermore, JE infections are lethal in about 25–30% of cases, mostly in infants, and lead to permanent sequelae in about 50% of cases (Ghosh and Basu, 2009; Flohic et al., 2013). JE is thus a continuing public health threat. Accordingly, the development of new treatments and antiviral drugs against JEV are expected. JEV contains a single-stranded, plus-sense RNA genome of approximately 11 kb. It consists of a single open reading frame n

Corresponding author. Fax: þ86 25 84396335. E-mail address: [email protected] (T. Shen).

http://dx.doi.org/10.1016/j.virol.2014.01.025 0042-6822 & 2014 Elsevier Inc. All rights reserved.

that codes for a large polyprotein of 3432 amino acids that is coand post-translationally cleaved into three structural proteins (capsid, C; premembrane, prM; and envelop, E) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) (Lindenbach et al., 2007; Sumiyoshi et al., 1987). RNAi is a post-transcriptional mechanism of gene silencing first described as an innate response to viral infections in plants and subsequently in all higher-order eukaryotes (Bosher and Labouesse, 2000; Hannon, 2002). In this process, dsRNA is cleaved into siRNAs of 21–25 nt by the RNase III-like enzyme dicer. These siRNAs are associated with a multiprotein complex known as the RNA-induced silencing complex (RISC), and they ultimately degrade the homologous mRNA and prevent protein translation (Dykxhoorn et al., 2003). A major advance in the field occurred with the discovery that synthetic short dsRNAs resembling the dicer-processed product could mediate specific gene silencing in mammalian cells without evoking an interferon (IFN) response (Dahlgren et al., 2006; Elbashir et al., 2001). These findings have led to the emergence of a new field of drug discovery with RNAi therapeutics that target a wide variety of human diseases, ranging from cancer to metabolic diseases and viral infections (de Fougerolles et al., 2007). Recent studies have demonstrated the efficacy of RNAiinduced inhibition of viruses in vitro and in vivo, including hepatitis

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Fig. 1. shRNA-induced inhibition of JEV replication was detected by indirect immunofluorescence. JEV was stained with red fluorescence and nucleus was stained with DAPI. Stained cells were observed by confocal laser scanning microscopy. Magnification, 200  . Data are from a representative experiment.

C virus (Arbuthnot et al., 2007; Pan et al., 2012; Suhy et al., 2012), hepatitis B virus (Pichard et al., 2012; Wang et al., 2013; Wooddell et al., 2013), West Nile virus (Anthony et al., 2009; Krishnan et al., 2008; Schnettler et al., 2012), and influenza virus (Langlois et al., 2013; Meliopoulos et al., 2012). In this study, 10 different regions of the major JEV genome containing the C, E, NS1, NS3, NS4b and NS5 genes were selected as the silencing targets. Ten corresponding shRNA-expressing plasmids were constructed. Using a shRNA, we established definitive proof that the replication of JEV can be inhibited effectively in BHK-21 and SK-N-SH cells. Furthermore, we first analyzed and compared the antiviral activity among the shRNAs targeting JEV various genes. Moreover, we explored the feasibility of shRNAbased intervention to suppress lethal JE in mice. Our results provide novel insights into the inhibition of JEV replication in cells and animals, and highlight the antiviral potential of RNAi.

Results Confocal fluorescence analysis of shRNA-induced inhibition of JEV in BHK-21 cells To substantiate the inhibitory levels of different shRNAs intuitively, an indirect immunofluorescence assay was performed using an anti-JEV-E protein monoclonal antibody and the Alexa Fluors 594 s second antibody at 24 h post-challenge (hpc). JEV was stained with red fluorescence and nucleus was stained with 40',6-diamidino2-phenylindole (DAPI). The results showed that all ten shRNAs suppressed JEV replication significantly compared with pGP-NC. Notably, the red fluorescence was barely detectable in the cells treated with pGP-JE-2, pGP-JE-1, pGP-JE-3, pGP-JNS4b-1. In conclusion, the ten shRNAs we designed played a role of down-regulation in JEV replication in BHK-21 cells successfully (Fig. 1).

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Fig. 1. (continued)

shRNAs reduce JEV titer in BHK-21 cells On one hand, the results of TCID50 showed that the shRNAmediated antiviral effect was dose-dependent. The inhibitory efficacy reduced slightly with the increased injection of JEV (Fig. 2A). The high concentrations of shRNAs inhibited JEV replication more effectively than low concentrations of shRNAs (Fig. 2B). On the other hand, the results of TCID50 showed that the shRNAmediated antiviral effect was time-dependent. No matter whether

shRNAs transfection prior or posterior to JEV infection, the antiviral effect was slightly reduced with increasing hours of observation, however shRNAs inhibited JEV replication effectively and stably in BHK-21 cells during at least 72 h post-challenge/transfection (Fig. 2C and E). Remarkably, the JEV titer was reduced about 104-fold in cells pretreated with pGP-JE-2, pGP-JE-1 or pGP-JE-3, compared with pGP-NC. The JEV titer of the samples pretreated with pGP-JNS4b-1, pGP-JNS3, pGP-JC or pGP-JNS1-1 was reduced by 103-fold compared with pGP-NC. The JEV titer of the samples treated with pGP-JNS1-2,

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Fig. 2. Decrease of virus yield in BHK-21 cells transfected with shRNAs. (A) Cells were pretransfected with 0.8 μg shRNA, then challenged with JEV at MOIs of 0.05, 0.5 and 1. Culture supernatants were collected at 24 hpc. (B) Cells were pretransfected with 0.4, 0.8 and 1.2 μg shRNA, then challenged with JEV at 0.1 MOI. Culture supernatants were collected at 24 hpc. (C) Cells were pretransfected with 0.8 μg shRNA, then challenged with JEV at 0.1 MOI. Culture supernatants were collected at 24/48/72 hpc. (D) Cells were challenged with 0.1 MOI of JEV for 1.5/3/6 h, then transfected with 0.8 μg shRNA. Culture supernatants were collected at 24 hpt. (E) Cells were challenged with 0.1 MOI of JEV for 1.5 h, then transfected with 0.8 μg shRNA. Culture supernatants were collected at 24/48/72 hpt. All the culture supernatants were measured by TCID50 (Po 0.05).

pGP-JNS4b-2 or pGP-JNS5 was decreased 10 times compared with the negative control (Fig. 2C). Furthermore, even if virus infection 6 h prior to shRNAs transfection, the TCID50 values were reduced significantly compared with pGP-NC (Fig. 2D). shRNAs inhibit JEV replication at the mRNA level To determine the relative expression of JEV mRNA by treatment with the shRNAs, total RNA was extracted from BHK-21 and SK-NSH cells at 24, 48 and 72 hpc for quantitative RT-PCR analysis. Primers targeting the JEV 3'-untranslated region, GAPDH (as the internal reference for BHK-21 cells) and beta-actin (as the internal

reference for SK-N-SH cells) were used. The data showed that all ten shRNAs suppressed the expression of JEV mRNA notably in cells compared with pGP-NC group, however the inhibitory effect varied among these shRNAs. In BHK-21 cells, the shRNAs inhibited JEV replication by 450% except for pGP-JNS1-2 and pGP-JNS5, especially pGP-JE-1, pGP-JE-2, pGP-JE-3 and pGP-JNS4b-1 suppressed JEV outstandingly all by 490% (Fig. 3A). Meanwhile, we determined the antiviral activity of the shRNAs in human neuroblast cells. The relative expression of JEV 3'-untranslated mRNA to beta-actin mRNA was o50% in SK-N-SH cells treated with pGP-JC, pGP-JE-1, pGP-JE-2, pGP-JE-3, pGP-JNS-1, pGP-JNS3 or pGP-JNS4b1. In particular, more than 90% of viral mRNA expression was

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Fig. 3. Quantitative RT-PCR analysis of the inhibition efficiency of ten shRNAs to JEV. (A) JEV RNA extracted from BHK-21 or SK-N-SH cells pretreated with different shRNAs at intervals post-challenge (24/48/72 h), GAPDH as the internal reference for BHK-21, beta-actin as the internal reference for SK-N-SH cells. (B) JEV RNA were extracted from BHK-21 or SK-N-SH cells at 72 hpt, which were first challenged with JEV for 1.5, 3 and 6 h followed with transfection of each shRNA. (C) BHK-21 cells were pretransfected with different concentrations of shRNAs. At 72 hpc, relative expression of JEV mRNA was determined (Po 0.05).

powerfully silenced by pGP-JE-1, pGP-JE-2 and pGP-JE-3 in SK-NSH cells (Fig. 3A). Besides, the data indicated that the level of JEV transcripts were reduced stably in both cell types at intervals postchallenge, in other words, the shRNAs can inhibit JEV replication effectively through at least 72 hpc in BHK-21 and SK-N-SH cells. To further research the inhibitory effect–time relationship, cells were infected with JEV for 1.5, 3 and 6 h, and then viral RNA was extracted at 72 h post-transfection (hpt) from BHK-21 and SK-NSH cells. The inhibitory effect of pGP-JE-1, pGP-JE-2 and pGP-JE-3 was still 485% in both cell types (Fig. 3B). It demonstrates that the RNAi-mediated inhibition of virus remains effectively even after an infection has been established in cells. However, the overall therapeutic effect of shRNAs was relatively weaker than their prophylactic effect against viral replication. The inhibition of JEV replication in BHK-21 and SK-N-SH cells is time dependent by shRNAs. Moreover, due to the outstanding antiviral performance of shRNAs in human nerve cells (SK-N-SH cells), the clinical

application of shRNA-mediated RNAi is potential to suppress JEV-induced encephalitis. Besides, the high concentrations of shRNAs suppressed JEV mRNA expression more effectively than low concentrations of shRNAs (Fig. 3C). Assay of shRNA inhibition of JEV replication in BHK-21 cells by flow cytometry The inhibitory effect of the various shRNAs on the expression of the E protein were quantitatively determined by flow cytometry in BHK-21 cells at 48 hpc. The extent of E protein down-regulation was quantified by assessing the numbers of FITC-positive cells. The percentage of FITC-positive cells was o50% except in the pGPJNS5 treated wells (58.7%). The inhibitory efficiency of pGP-JE-2, pGP-JE-1, pGP-JE-3, pGP-JNS4b-1, pGP-JC and pGP-JNS3 was 97.4%, 92.3%, 87.7%, 81.5%, 79.4% and 72.5%, respectively, relative to the

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Fig. 4. Flow cytometry analysis of JEV positive cells. The percent of JEV positive cells in BHK-21 cells were measured by assessing the number of FITC-positive cells. Data are from a representative experiment.

Fig. 5. Western blot analysis of gene silencing at the protein level. Cells were harvested and separate by 12% SDS-PAGE. Beta-actin protein served as the loading control: reduction of JEV-E protein levels in BHK-21 cells. Mock is non-infected cell control. Data are from a representative experiment.

negative control pGP-NC (Fig. 4). Apparently, the expression of JEV E protein was strongly reduced by the transfection with these 6 shRNAs above, whereas the transfection with the remaining 4 shRNAs resulted in a somewhat less robust reduction. shRNAs inhibit JEV replication at the protein level by western blotting The E protein is one of the most important JEV proteins and has an approximate molecular mass of 54 kDa. To further analyze the inhibitory effect of shRNAs at protein level, BHK-21 cells were assayed by western blotting at 48 hpc. All the BHK-21 cells expressed the 42-kDa β-actin protein detected by an anti-beta-actin mAb, and only the infected cells expressed the expected 54-kDa E protein as detected by an anti-JEV-E mAb. No significant band was seen in the cells transfected with pGP-JE-2. Only a faint band of the E protein was observed in the transfections with pGP-JE-1, pGP-JE-3, pGP-JNS4b-1 and pGP-JC compared with the pGP-NC transfection. The pGP-NS3, pGP-JNS1-1, pGP-JNS4b-2, pGP-JNS1-2 and pGP-JNS5 bands were thinner and lighter than pGP-NC band. No E protein was observed in the normal cells (Mock) (Fig. 5). These results indicated that 3 shRNAs targeting the E gene suppressed the translation of the JEV E protein especially well.

Fig. 6. (A) Absence of IFN-β induction in BHK-21 and SK-N-SH cells cultures following shRNA transfection. IFN-β production levels were determined by ELISA for supernatants of nontransfected cells (mock) and cells transfected with each shRNA (100 nM) or with poly(I-C) (0.5 μg/ml). Samples differ significantly from poly(I-C) (Po0.05). (B) Absence of TNF-α induction in SK-N-SH cells cultures following shRNA transfection. TNF-α production levels were determined by ELISA for supernatants of nontransfected cells (mock) and cells transfected with each shRNA (100 nM) or with LPS (1 μg/ml). Samples differ significantly from LPS (Po0.05).

shRNAs were able to induce IFN-β production in uninfected BHK-21 and SK-N-SH cells. Supernatants were collected at 24 hpt from shRNA-treated BHK-21 or SK-N-SH cells, and analyzed by ELISA for IFN-β induction. The results showed that the supernatants revealed no apparent differences at the level of IFN-β between shRNA-treated and nontransfected cells (mock) in two type cells, whereas poly(I-C) induced a strong IFN response (Fig. 6A). Furthermore, to substantiate the potential of clinical application of shRNA, we determined whether the simulation of shRNA were possible to induce some side-effects in SK-N-SH cells, such as inflammatory response. Supernatants were collected at 24 hpt from shRNA-treated SK-N-SH cells, and analyzed by ELISA for TNF-α induction. The results showed that the level of TNF-α in shRNAtreated cells remained stable, relative to nontransfected cells (mock), whereas LPS induced TNF-α secretion to a high degree (Fig. 6B). Thus, in our study, the antiviral effect of shRNA was not due to the induction of an IFN response. shRNA effectively inhibits JEV replication by RNAi-mediated degradation of viral RNA in BHK-21 and SK-N-SH cells.

Cytokines analysis in BHK-21 and SK-N-SH cells with shRNAs simulation

shRNAs protect the suckling mice against JEV infection

Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. It has been reported that siRNAs can stimulate the IFN pathway under certain circumstances (Malathi et al., 2007; Sledz et al., 2003). Therefore, we examined whether the

Brain sections from suckling mice challenged with JEV were examined for pathological changes 4 days post-challenge (dpc). Brains from the mice of virus control and negative control (pGPNC) showed the typical histopathological features of a diffuse, disseminated viral encephalitis with hemorrhage, extensive

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pGP-JC

pGP-JE-1

pGP-JE-2

pGP-JE-3

pGP-JNS1-1

pGP-JNS1-2

pGP-JNS3

pGP-JNS4b-1

pGP-JNS4b-2

pGP-JNS5

Virus control

Mock

pGP-NC Fig. 7. Immunohistochemistry analysis of brain sections from shRNAs-treated suckling mice. The control groups were negative control (pGP-NC), virus control (JEV) and mock (PBS). The arrows in figure staining dark brown are the sites of virus particles, which were stained with DAB. Magnification, 400  . Data are from a representative experiment.

perivascular leukocyte infiltration, and neuronal apoptosis. Many virus particles were present in the brain sections, staining dark brown. The virus particles were barely detectable in the brains sections from the mice pretreated with pGP-JE-1, pGP-JE-2, pGP-JE3 or pGP-JNS4b-1, similar to the mock control (PBS). The numbers of virus particles in the brain sections from the mice pretreated with pGP-JC, pGP-JNS3, pGP-JNS1-1, pGP-JNS1-2, pGP-JNS4b-2 and pGPJNS5 was significantly lower than in the virus control group (Fig. 7).

Moreover, the brain tissue were measured by plaque assays on BHK-21 on 4 dpc, for quantification of JEV in brains of suckling mice. The results showed that brain homogenates from the virus control and negative control mice revealed extremely high levels of viral replication, whereas the homogenates from the shRNAtreated mice remained at a low level of JEV. Remarkably, the JEV titer was reduced about 104-fold in the mice treated with pGP-JE2, pGP-JE-1 or pGP-JE-3, compared with the virus control and

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negative control. And the treatment of pGP-NS3, pGP-NS4b-1 or pGP-JC decreased the JEV replication in suckling mice to the degree of 103-fold, relative to the virus control and negative control (Fig. 8). Thus, shRNAs depressed the viral load of JEV in brain of mice significantly.

shRNAs protect BALB/c mice against JEV challenge We evaluated the potential of the shRNAs to protect mice against a lethal dose of a JEV virulent strain challenge. BALB/c mice were injected i.e. with each different shRNAs including pGP-NC. Subsequently, the mice were challenged with 20 LD50 of NJ2008 at the same site 24 hpt and were observed for 15 days. Four shRNAs targeting the structure domains of the JEV genome protected 50% of the mice; notably, 83.3% of the mice pretreated with pGP-JE1 or pGP-JE2 survived (Fig. 9A). The survival of the mice treated with pGP-JNS1-1, pGP-JNS3 or pGP-JNS4b-1 was, respectively, 50%, 50% and 66.7% at 15 days post-challenge (Fig. 9B). Furthermore, we

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tested the ability of the shRNAs to protect against increasing doses of JEV (100 LD50). Up to 50% of the mice that were pretreated with pGP-JE-1, pGP-JE-2, pGP-JE-3 or pGP-JNS4b-1 survived to the end of the observation period; notably, the prophylactic ability of the three shRNAs targeted to the E gene resulted in a survival rate of 66.7% (Fig. 9C and D). Moreover, we simultaneously challenged mice with 20 LD50 of NJ2008 and injected them with shRNAs. pGP-JE-1, pGP-JE-2 or pGPJE-3 all protected 66.7% of the mice to survive (Fig. 9E); pGP-JNS3 or pGP-JNS4b-1 conferred 50% protection (Fig. 9F). When the mice were challenged with 100 LD50 of NJ2008, the survival rate was 450% in those treated with pGP-JE-1 or pGP-JE-2 through 15 dpc (Fig. 9G). The prophylactic effect of the six shRNAs targeting the JEV nonstructural domains was relatively weaker than that of the four shRNAs targeting the JEV structural domains, especially the JEV E gene. The survival of the mice treated with the same shRNA challenged with 100 LD50 of NJ2008 decreased slightly compared

Table 1 RNAi target genes and sequences.

Fig. 8. The viral load in the brain tissues from the suckling mice treated with shRNA were plaque-titrated on BHK-21 cell monolayers. Each symbol represents an individual mouse. The horizontal line corresponds to the median viral titers of the group (P o0.05).

Target gene

Site

Target sequences

C E-1 E-2 E-3 NS1-1 NS1-2 NS3 NS4b-1 NS4b-2 NS5 NC

423-441 1287-1305 1555-1573 1681-1699 2946-2964 4112-4130 6085-6103 7352-7370 8010-8028 10732-10750 Nonsense

5'-GGCTCAATCATGTGGCTCG-3'a 5'-GGATGTGGACTTTTCGGGA-3'b 5'-GGAGTGGACTGAACACTGA-3' 5'-ACAGAGAACTCCTCATGGA-3' 5'-GACTTCGGCTTTGGCATCA-3'c 5'-GAAAGGAGCTGTACTCTTG-3' 5'-AGATCATGTTAGACAACAT-3' 5'-GAATGCCGTTGTTGACGGA-3' 5'-GAACCGATGCTCATGCAGA-3' 5'-GGACTAGAGGTTAGAGGAG-3' 5'-GTTCTCCGAACGTGTCACGT-3'

A nonsense sequence was designed as the negative control (NC), which of the 56-nt oligonucleotide shRNA was 5'-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACG TGACACGTTCGGAGAATTTTTTG-3'(sense). a b c

Murakami et al. (2005). Kumar et al. (2006). Liu et al. (2006).

Fig. 9. shRNAs protect BALB/c mice from JEV challenge. (A and C) Survival rate of mice pre-injected i.c. with pGP-JC/pGP-JE-1/pGP-JE-2/pGP-JE3 were challenged with 20/100 LD50 JEV. (B and D) Survival rate of mice pre-injected i.c. with pGP-JNS1-1/pGP-JNS1-2/pGP-JNS3 /pGP-JNS4b-1/pGP-JNS4b-2/pGP-JNS5 were challenged with 20/100 LD50 JEV. (E and G) Survival rate of mice were injected i.c. with pGP-JC/pGP-JE-1/pGP-JE-2/pGP-JE3 and 20/100 LD50 JEV simultaneous. (F and H) Survival rate of mice were injected i.c. with pGP-JNS1-1/pGP-JNS1-2 /pGP-JNS3/pGP-JNS4b-1/pGP-JNS4b-2/pGP-JNS5 and 20/100 LD50 JEV simultaneous.

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with those challenged with 20 LD50 of NJ2008. Remarkably, the inhibitory efficiency of RNAi was dose-dependent to a point; however, the protection failed to last stably when the titer or quantity of the virus challenge reached a threshold. Similarly, the inhibitory efficiency of RNAi was time-dependent; the survival of the mice that were treated with the shRNAs first was relatively higher than in those that were simultaneously challenged with the virus and the shRNAs.

Discussion JE is an important public health problem worldwide. An estimated 3 billion people are at risk, and JE has recently spread to new territories (Campbell et al., 2011). Vaccine inoculation is the major strategy to control JE. But inoculation failure and emerging infection often leaded to diseases. Until now, no specific remedy for JEV is obtained. Our study is focus on the treating of JEV infection. Although some RNAi sequences were reported in JEV inhibition (Kumar et al., 2006; Liu et al., 2006; Murakami et al., 2005; Qi et al., 2008), the overall screening of RNAi in vitro and in vivo is not determined. In this study, we designed 10 shRNAs targeting various JEV genes to assay the antiviral effect in BHK-21 and SK-N-SH cells. Furthermore, we determined the antiviral effect of shRNAs in mouse. JEV genome encoded three structural proteins (C, M and E) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) (Lindenbach et al., 2007). We designed 10 RNAi sequences target these genes, and all of these showed markedly antiviral activity in vitro and in vivo. In contrast, shRNA targeting structural genes showed higher antiviral activity than nonstructural genes. On one hand, nonstructural proteins are enzymes for viral replication and hydrolysis. These enzymes are important to virus replication, but little enzymes are needed in this process (Bollati et al., 2010). On the other hand, nonstructural proteins showed inhibitory activity against host immunity respond. NS5 was reported as an inhibitor of interferon pathway, and other NS proteins in West Nile virus (WNV) or Dengue virus (DENV) also showed inhibitory activity against immunity respond (Mazzon et al., 2009). To sum up, the results inferred that shRNA targeting nonstructural gene reduced the amount of NS proteins and released the host antiviral immunity system, but the viral enzyme activity is not affected (Bollati et al., 2010). Structural genes influence the adsorption, entry, assemble and release of JEV. Antiviral results indicated shRNA targeting structural genes is the best way to inhibit virus. The highest antiviral activity was determined by shRNAs targeting E gene, this results infer the important role of E protein in JEV replication. E protein is an envelope glycoprotein which determines the virus adsorption, entry, assemble and other course. It is the key protein in JEV life (Luca et al., 2012). In addition to E gene, shRNA target C and NS4b1 gene showed high antiviral activity too. C protein influences the nucleic localization of JEV (Pan et al., 2009) and NS4b influence the replication and hydrolysis of JEV. These two proteins are important to JEV infection, and the results showed that JEV replication was reduced significantly after C and NS4b gene silencing. Human neuroblastoma cells showed the same characteristics like nerve cells, we used SK-N-SH cells as the nerve cells infection model in this study. In SK-N-SH cells, shRNAs showed high antiviral activity against JEV. In contrast to BHK-21 cells, shRNAs targeting E, NS4b-1 and C also indicated the robust antiviral effect. Inflammation is an important feature of encephalitis virus infection and excessive inflammation respond would induce brain injuries and sequelae in JEV patients. In our study, TNF-α levels were detected in shRNA-transfected SK-N-SH cells. Results indicated that there are no TNF-α induction by shRNA-expressing plasmid. Thus, shRNA-expressing plasmid is a safe antiviral agent

in encephalitis treatment. Besides, IFN-β was detected in this study. Results indicated that shRNA-expressing plasmid is a steady antiviral agent without evoking interferon response. These results also inferred that our shRNAs inhibited JEV replication directly. In vivo, the results of immunohistochemistry showed that brains sections from suckling mice pretreated with pGP-JE-1, pGP-JE-2, pGPJE-3 or pGP-JNS4b-1 lacked virus particles, similar to the Mock controls. The number of virus particles in brain sections from mice pretreated with pGP-JC, pGP-JNS3, pGP-JNS1-1, pGP-JNS1-2, pGPJNS4b-2 and pGP-JNS5 was significantly lower than that in the virus control and negative control. The prophylactic effect of the shRNAs targeting JEV structural domains was generally stronger than that of the shRNAs targeting JEV nonstructural domains in vivo. These results strongly indicated that shRNA is an potential antiviral agent for the excellent antiviral activity in vivo. The overall survival rate by the prophylactic effect of shRNAs was relatively higher than their therapeutic effect. Furthermore, the protection rate of shRNA exists the dose-dependence, as the survival rates of mice challenged with 100 LD50 JEV lower than those with 20 LD50 JEV. And from all the results in vivo, shRNAs targeting E gene have the best antiviral effect. These results in vivo demonstrated that shRNA-expressing plasmid could be used as an antiviral drug in vivo. In summary, we constructed 10 shRNA plasmids targeting the genome of JEV, which could inhibit JEV replication in vitro and in vivo to various degrees. shRNA target E, NS4b and C gene showed the inhibition outstandingly. It is possible that RNAi will be able to be used as inhibitors of flavivirus replication. However, for the development of a potential and useful antiviral drug, further clinical experiments using RNAi are necessary.

Materials and methods Cells and virus Baby hamster kidney cells (BHK-21) and human neuroblastoma cell line (SK-N-SH) were maintained at 37 1C in an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. JEV strain NJ2008 (GQ918133) was used for viral challenge. Animals Suckling mice and female BALB/c mice aged 4–6 weeks (purchased from Model Animal Research Center of Nanjing University) were used in vivo experiments. All mice were maintained in sterile cages in specific-pathogen-free environments. All animal experiments were performed in accordance with the Animal Ethics and Experimental Committee Guidelines of Nanjing Agricultural University. Construction of shRNA expressing plasmids Ten target sequences in the coding region of C, M, E, NS1, NS3, NS4b and NS5 genes were selected according to a web-based siRNA design tool (siRNA target-finder, http://www.ambion.com/) (Table 1). The specificity of these sequences was verified by a BLAST search. A nonsense siRNA sequence was also designed as the negative control. The target sequences were used to design two complementary siRNA template oligonucleotides. All oligonucleotides were synthesized by Genepharma. After annealing the oligonucleotides, they were inserted into the BamHI and BbsI sites of the siRNA expression vector pGPU6/Neo (abbreviation: pGP; Shanghai Genepharma., Ltd.). These recombinant plasmids transformed into Escherichia coli DH5α, then, were extracted, digested and sequenced ( by BGI-Shenzhen Corporation) to identify the

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positive plasmids, which were named pGP-JC, pGP-JE-1, pGPJE-2, pGP-JE-3, pGP-JNS1-1, pGP-JNS1-2, pGP-JNS3, pGP-JNS4b-1, pGP-JNS4b-2, pGP-JNS5 and pGP-NC. The recombinant plasmids were purified with the QIAGEN Plasmid Purification Max Kits (QIAGEN) and quantified by the Eppendorf Biophotometer. Cell transfection and viral infection Growth and titration of JEV were conducted using cultured BHK-21 cells, and TCID50 values were calculated using the Reed– Muench method. Viral suspensions of 106–107 TCID50/ml were used for the experiments. BHK-21 cells were plated in 24 well plates at 2  105 cells per well in antibiotic-free medium overnight. When the cell layer reached 70% confluence, 0.8 μg of 10 shRNAs and negative control (pGP-NC) were individually transfected into cells in 100 μl OPTIMEM medium (Invitrogen) mixed with Lipofectamine 2000 (Invitrogen). Non-transfected BHK-21 cells were used as a blank control (Mock). After 6 h of shRNA treatment, the culture medium was immediately replaced with fresh medium containing 2% FBS. Twenty-four hours post-transfection, the cells were infected with the JEV strain NJ2008 at 0.1 MOI. The cultures were incubated for 1.5 h, and then the culture medium was replaced with fresh medium containing 2% FBS. SK-N-SH cells were treated the same with BHK-21 cells as described above. Moreover, to normalize the transfection efficiency of shRNAs, the relative expression of neomycin resistance gene of pGPU6/Neo was determined by quantitative RT-PCR. The specific primers were as follows: forward primer: 5'-TGATATTGCTGAAGAACT-3'; reverse primer: 5'-TGATATTGCTGAAGAA CT-3'. GAPDH and beta-actin served as an internal reference for BHK-21 and SK-N-SH cells. The results were in Supplementary data (Fig. 1S). Confocal fluorescence analysis BHK-21 cells were pretransfected and infected as described above. 24 hpc, the infected cells were fixed in 80% ice-cold acetone at  20 1C for 10 min and blocked in 2% BSA at room temperature for 2 h. After washing, the cells were incubated with a mouse monoclonal antibody against the E protein of JEV (kindly provided by Pro. Shengbo Cao, Huazhong Agricultural University, Wuhan, China) and the Alexa Fluors 594 s antibody (Invitrogen). The cells were stained with 40',6-diamidino-2-phenylindole (DAPI) for 10 min. After washing, the cells were examined and photographed with confocal fluorescence microscopy (Nikon). TCID50 measurement BHK-21 cells were transfected with different concentrations (0.4, 0.8 and 1.2 μg) of each shRNA 24 h before JEV infection at various MOIs (0.05, 0.5 and 1). Supernatant fluids were harvested at various times (24, 48 and 72 h) post-challenge. Serial 10-fold dilutions were made from the supernatant stocks, and 100 μl samples of each dilution were added to duplicated wells of a 96well plate containing a confluent monolayer of BHK-21 cells. Seven days were allowed for the appearance of cytopathology. The dilution causing cytopathology in half of the cultures (the median tissue culture infective dose, TCID50) was then calculated by the Reed and Muench method. In addition, cells were infected with JEV for 1.5, 3 and 6 h, then transfected with shRNAs. At 24, 48 and 72 hpt, supernatant fluids were measured by TCID50. Quantitative RT-PCR To analyze the inhibitory effect at mRNA level, SYBR Green I real-time RT-PCR was performed using specific primers based on the sequence of the JEV 3' noncoding region as follows: forward

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primer: 5'-CCC TCA GAA CCG TCT CGG AA-3', reverse primer: 5'CTA TTC CCA GGT GTC AAT ATG CTG-3'. GAPDH served as an internal reference for BHK-21 cells and the specific primers were as follows: forward primer: 5'-CCC CAA TGT GTC CGT CGT G-3'; reverse primer: 5'-GCC TGC TTC ACC ACC TTC T-3'. beta-actin served as an internal reference for SK-N-SH cells and the specific primers were as follows: forward primer: 5'-GGA CTT CGA GCA AGA GAT GG-3'; reverse primer: 5'-AGC ACT GTG TTG GCG TAC AG3'. At 24/48/72 hpc, the cells pretreated with the shRNAs with different concentrations in 24-well plates were lysed by TRIzol Reagent (Invitrogen); the cells challenged with JEV for 1.5/3/6 h were transfected with shRNAs and lysed by TRIzol Reagent (Invitrogen) at 72 hpt. Total RNA was isolated according to the manufacturer's instructions. Reverse transcription was carried out by using Moloney murine leukemia virus reverse transcriptase (Takara) in a 10 μl reaction mixture containing 3 μl RNA, according to the manufacturer's recommendation. Amplification was carried out in a 50-μl reaction volume containing 4 μl cDNA, 25 μl Power SYBR Green PCR Master Mix (Takara), 1 μl ROX Reference Dye, 0.5 μl of each primer and 19 μl ddH2O. The reaction protocol was 95 1C for 30 s, followed by 40 cycles of 95 1C for 5 s and 60 1C for 31 s. Real-time RT-PCR was performed in an ABI PRISM 7300 sequence detection system and analyzed with ABI PRISM 7300SDS software (Applied Biosystems). Cycle times of internal reference that varied by 4 1.0 U in triplicate were discarded. Cycle times of the target gene were averaged and normalized to the averaged cycle time of GAPDH or β-actin accordingly. Flow cytometry assay BHK-21 cells were transfected and infected as describe above. At 48 hpc the cells were washed gently in PBS, trypsinized, and resuspended in PBS, and then incubated with anti-JEV-E protein monoclonal antibody, followed by incubation with FITC–goat antimouse IgG (Boster). The FITC-positive cells were evaluated by flow cytometry using the FACS Calibur Flow Cytometry System (BD Biosciences), assisted by the Cell Engineering Center of Nanjing Drum Tower Hospital. Western blotting analysis BHK-21 cells were transfected and infected as describe above. At 48 hpc the cells were harvested from six-well plates, washed twice with PBS, and boiled for 10 min after mixing with 5  loading buffer. Proteins were analyzed by 12% SDS-PAGE followed by blotting onto two polyvinylidene difluoride membranes, which were then blocked in PBS containing 5% non-fat dry milk overnight at 4 1C. One membrane was incubated with anti-JEV-E protein monoclonal antibody and the other with anti-beta-actin monoclonal antibody (Boster) for 2 h at room temperature. The membranes were washed three times with PBS, followed by incubation with peroxidase-conjugated goat anti-mouse antibody or peroxidase-conjugated SPA (Staphylococal Protein A, Boster) for 2 h at room temperature. Membranes were washed three times with TBS (100 mmol/L Tris  HCl pH7.5, 150 mmol/L NaCl). The proteins were visualized by enhanced chemiluminescence. E protein and beta-actin served as the target protein and internal protein reference, respectively. ELISA for beta interferon (IFN-β) and alpha TNF (TNF-α) analysis BHK-21 and SK-N-SH cells were seeded into 24-well plates 24 h before transfection. Cells were transfected with each shRNA (100 nM) or with poly(I-C) (0.5 μg/ml, Sigma), each of which was mixed with Lipofectamine 2000 (Vigne et al., 2009). At 24 hpt, cell culture supernatants were collected, and mouse/human IFN-β was

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measured by Mouse or Human IFN-beta ELISA Kit (R&D Systems, USA) following the manufacture's instructions. SK-N-SH cells were transfected with each shRNA (100 nM) mixed with Lipofectamine 2000. The positive control is the cells treated with LPS (1 μg/ml, Sigma)for 6 h (Hoareau et al., 2010). At 24 hpt, cell culture supernatants were collected, and human TNF-α was measured by Human TNF-alpha Quantikine ELISA Kit (R&D Systems, USA) following the manufacture's instructions. Immunohistochemistry analysis Suckling mice were into 11 groups of four mice, and then inoculated intracranially (i.c.) with 2 μg/g each different shRNA every group, through the bregma (4 mm deep vertically into the brain using a Hamilton syringe fitted with a 30-gauge needle) (Kumar et al., 2006). The mice were challenged with 5 LD50 JEV by i.c. inoculation through the bregma at the same spot as described above. After 96 h, brains were removed to 10% formalin, embedded in paraffin, processed with anti-JEV-E protein monoclonal antibody, followed with peroxidase-conjugated SPA (Boster), and finally, stained with DAB (3, 3N-Diaminobenzidine, Boster). Sections were stained with hematoxylin for pathological analysis. The mice injected with pGP-NC were taken as the negative control. The mice only challenged with PBS or JEV were taken as mock group and virus control. Plaque formation assay Suckling mice were treated by each shRNA and challenged with JEV the same as described above. On 4 dpc, the brain homogenates were inoculated for 1.5 h on BHK-21 cells in six-well plates. The culture fluid was removed, 2 ml agarose was added per well to overlay the BHK-21 cell monolayer, and culture was continued at 37 1C in an atmosphere of 5% CO2 until there was a significant number of plaques in the cells. Cells were stained with crystal violet for 15 min, and then the staining solution was discarded. The number of plaques was counted. The control groups were negative control, virus control and mock. Immunoprotection test BABL/c mice (6 per group) were inoculated i.c. with 5 μg/g each various shRNA every group, through the bregma. After 24 h, the mice were challenge with 20 or 100 LD50 of JEV, and then continuously observed for survival over time with negative control (pGP-NC) and mock groups. In addition, each group of six mice were treated i.c. with 5 μg/g one shRNA and 20 or 100 LD50 JEV simultaneously, and then observed for survival. Negative control (pGP-NC) and mock group were treated and observed as described above. Statistical analysis All assays described herein were repeated at least three times, and all of the measurements were made in triplicate. Mean values 7 standard deviation (SD) were calculated using Microsoft Excel. Statistical analysis was performed by one-way analysis of variance, and values were considered significant when p o0,05. Figures were created using the GraphPadTM Prism 5.0 software.

Acknowledgments This work was supported by the National Special Research Programs for Non-Profit Trades, Ministry of Agriculture (No. 200803015) and the National Special Research Programs for

Cultivating New Transgenic Organisms, Ministry of Agriculture (No. 2009ZX08006-007B).

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