Significant inhibition of Tembusu virus envelope and NS5 gene using an adenovirus-mediated short hairpin RNA delivery system

Infection, Genetics and Evolution 54 (2017) 387–396

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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid

Research paper

Significant inhibition of Tembusu virus envelope and NS5 gene using an adenovirus-mediated short hairpin RNA delivery system

MARK

Hongzhi Wanga,b,c, Qiang Fengd, Lei Weid, Liling Zhuoe, Hao Chena,b,c, Youxiang Diaoa,b,c,⁎, Yi Tanga,b,c,⁎ a

College of Animal Science and Veterinary Medicine, Shandong Agriculture University, #61 Dai Zong Avenue, Tai'an, Shandong 271018, China Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agriculture University, #61 Dai Zong Avenue, Tai'an, Shandong 271018, China c Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agriculture University, #61 Dai Zong Avenue, Tai'an, Shandong 271018, China d Taian City Central Hospital, #29 Long Tan Road, Tai'an, Shandong 271000, China e Zaozhuang University, College of Life Science, Bei An Road, Zaozhuang, Shandong 277160, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Tembusu virus RNAi Duck Adenovirus vector In vitro

Tembusu virus (TMUV) is a mosquito-borne flavivirus, which was first isolated in the tropics during the 1970s. Recently, a disease characterized by ovarian haemorrhage and neurological symptoms was observed in ducks in China, which threatens poultry production. However, there is no suitable vaccination strategy or effective antiviral drugs to combat TMUV infections. Consequently, there is an urgent need to develop a new anti-TMUV therapy. In this study, we report an efficient short hairpin RNA (shRNA) delivery strategy for the inhibition of TMUV production using an adenovirus vector system. Using specifically designed shRNAs based on the E and NS5 protein genes of TMUV, the vector-expressed viral genes, TMUV RNA replication and infectious virus production were downregulated at different levels in Vero cells, where the shRNA (NS52) was highly effective in inhibiting TMUV. Using the human adenovirus type 5 shRNA delivery system, the recombinant adenovirus (rAdNS52) inhibited TMUV multiplication with high efficiency. Furthermore, the significant dose-dependent inhibition of viral RNA copies induced by rAd-NS52 was found in TMUV-infected cells, which could last for at least 96 h post infection. Our results indicated that the adenovirus-mediated delivery of shRNAs could play an active role in future TMUV antiviral therapeutics.

1. Introduction Tembusu virus (TUMV) is a member of the Ntaya virus group in the genus Flavivirus and the family Flaviviridae, which includes highly pathogenic human pathogens, such as the dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus (Mukhopadhyay et al., 2005). Similar to most flaviviruses, the TMUV virion is approximately 50–60 nm in diameter, and it comprises a single, positivestrand RNA genome, which is packaged by the virus capsid protein in a lipid bilayer derived from host cells (Su et al., 2011; Tang et al., 2013a). TMUV has an approximately 11-kb-long 5′-capped genome, which contains one open reading frame (ORF) flanked by approximately 145 nucleotide (nt) 5′ and approximately 618 nt 3′ non-translated regions (Tang et al., 2012; Wan et al., 2012). After the infection of susceptible cells by TMUV, the ORF is translated into a single 3410-amino acid (aa) residue polyprotein precursor, which matures into three structural

⁎

proteins and seven non-structural (NS) proteins (Chen et al., 2013). The structural proteins comprise the virus particle, and they are incorporated into the progeny TMUVs, including capsid, envelope (E) and precursor of membrane (prM) proteins. The NS proteins are encoded within the viral genome, but they are not observed in the mature TMUV virion, including NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Tang et al., 2012). Among these proteins, the E proteins of TMUVs play important roles in cellular receptor binding, virus assembly, fusion modulation and potentially the induction of immunity (Yu et al., 2013; Zhao et al., 2015). The 3′-terminal of NS5 protein is an RNA-dependent RNA polymerase (RdRp) domain, which is responsible for the replication of the positive-strand RNA genome in an asymmetric and semi-conservative process (Liu et al., 2013). Both the E protein and NS5 RdRp domain are important targets for antiviral therapy development in many other flaviviruses by inhibiting viral entry and viral genome replication, respectively (Julander et al., 2011; Ray and Shi, 2006).

Corresponding authors at: College of Animal Science and Veterinary Medicine, Shandong Agriculture University, #61 Dai Zong Avenue, Tai'an, Shandong 271018, China. E-mail addresses: [email protected] (Y. Diao), [email protected] (Y. Tang).

http://dx.doi.org/10.1016/j.meegid.2017.08.001 Received 15 May 2017; Received in revised form 28 July 2017; Accepted 1 August 2017 Available online 03 August 2017 1567-1348/ © 2017 Published by Elsevier B.V.

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Fig. 1. Schematic description of target viral genome, shRNA expression cassette and vector, AdV 5 shRNA delivery system, and EGFP reporter vector. (A) The position of target shRNA on TMUV genome and the construction of shRNA expression plasmid and rAd. The designed 55-mer shRNA template oligonucleotides were marked with three-color-boxes which included the BamH I and Hind III restriction site at 5′ and 3′ end, respectively. The pSilencer™ 4.1-CMV neo vector is a shRNA expression plasmid and used for shRNA template cloning. The pDC312 is a AdV 5 shuttle plasmid which can retrieve the shRNA expression cassette from pSilencer™ 4.1-CMV vector. The pBHGloxΔE1,3Cre is a packaging plasmid, together with pDC312 shuttle plasmid, were used for the construction of rAd. (B) The construction of EGFP reporter vector. The partial of E gene and NS5 gene were amplified using RT-PCR and cloned into pEGFP-N1 vector under the control of CMV promoter which fused to the N-terminal of the EGFP protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

In the 1970s, TMUV was first isolated from Culex mosquitoes in Malaysia during the surveillance of arbovirus infections (Platt et al., 1975), but humans and birds appeared to be infrequently infected with TMUV at that time (Bowen et al., 1975). Subsequently, a pathogenic TMUV strain, designated as Sitiawan virus, was isolated from broiler chickens during the late 1990s in Malaysia, which caused encephalitis

and retarded growth in infected chickens (Kono et al., 2000). In 2010, a highly contagious disease emerged in breeder, layer and meat-type duck farms in Eastern China (Cao et al., 2011). The infected ducks and ducklings typically exhibited severely decreased egg production and/or severe neurological disorders, which caused huge economic losses in the Chinese duck industry (Su et al., 2011). The causative agent of this 388

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2.2. shRNA expression plasmid construction

emerging disease was identified as the new type of TMUV, and it has continually affected duck production in China since 2010(Zhou et al., 2015). In addition to China, the new TMUV was recently detected among ducks in Malaysia (Homonnay et al., 2014) and Thailand (Thontiravong et al., 2015). The TMUV has not been reported to cause illness in humans, but TMUV-specific antibodies and genomic RNA were detected in duck farm workers in China (Tang et al., 2013b). Promising live attenuated TMUV vaccine candidates are currently being studied (Chen et al., 2014; Li et al., 2014), but the immune response against TMUV is not completely understood. Furthermore, no antiviral therapy is currently available to treat TMUV infections. The application of gene silencing RNA interference (RNAi) has provided a novel strategy for antiviral therapy (Bagasra, 2005). RNAi already serves as an antiviral defence tool in animal cells (van Rij and Andino, 2006), and it comprises a biological process where RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules through sequence complementarity (Hu, 2005). The RNAi process occurs in eukaryotic cells where it is induced by 21–23 nt small/short interfering RNA (siRNA), which originates from double-stranded RNA (dsRNA) cut by the RNAase III-like enzyme (Dicer) (Agrawal et al., 2003). The siRNA is incorporated into a multiprotein complex known as the RNA-induced silencing complex (RISC), which degrades or inactivates complementary mRNA and viral RNA (Filipowicz et al., 2008). siRNA can be synthesized directly and transfected into cells (Elbashir et al., 2001), but it is also possible to create a short hairpin RNA (shRNA)-expressing plasmid (Brummelkamp et al., 2002) or viral vector (Xia et al., 2002) for subsequent transfection. The vector-expressed shRNA can be processed by Dicer in the cytoplasm into siRNA (Cullen, 2006). Many studies have shown that RNAi is a useful tool for inhibiting the replication of flaviviruses such as West Nile virus (Bai et al., 2005), Japanese encephalitis virus (Wu et al., 2011), dengue virus (Korrapati et al., 2012), yellow fever virus (Pacca et al., 2009) and tick-borne encephalitis virus (Achazi et al., 2012). However, studies of the effects of RNAi on TMUV infection have not been reported. In the present study, we designed and screened a number of shRNAs to target the TMUV genome using different evaluation systems. Using the most effective shRNA for TMUV inhibition (NS52), we constructed a replication-defective recombinant adenovirus (rAd) vector, designated as rAd-NS52, to deliver shRNA that targeted the NS5 RdRp domain. We found that rAd-NS52 can inhibit the replication of TMUV with high efficiency in Vero cells. Interestingly, our results showed that this inhibition was dose-dependent, and it could last for at least 96 h post infection.

The E gene and RdRp domain of the NS5 gene are preferred for antiviral therapy development in flaviviruses (Ray and Shi, 2006) and these two genes is highly conserved among different TMUV field strains (Supplementary figure), so we selected the sequences of these two regions for online RNAi target site searches and design (http://www. ambion.com/techlib/misc/siRNA_tools.html). Three target sequences (E1, E2 and E3) in the coding region of the E gene and three target sequences in NS5 (NS51, NS52 and NS53) were selected according to the shRNA online design criteria (Fig. 1A). The sequences of the target sites were subjected to BLASTN online search in GenBank (http://blast. ncbi.nlm.nih.gov/Blast.cgi) to confirm their specificity. To construct the shRNA expression plasmids, 55-mer shRNA template oligonucleotides were chemically synthesized for each target sequence and annealed into dsDNA fragments as shRNA expression templates. Using the BamHI and HindIII cohesive ends of each fragment, the shRNA expression templates were inserted into the cloning sites of the shRNA expression vector pSilencer4.1-CMV neo (Ambion, Foster City, CA, USA) under the transcriptional control of the CMV promoter. The pSilencer4.1-CMV control plasmid was used as a negative control in the RNAi experiment (pSilencer-control), which contained non-TMUV targeted shRNA under the control of the CMV promoter. Successful shRNA expression plasmid construction was confirmed by sequencing using the forward primer (5′-AGGCGATTAAGTTGGGTA-3′) and reverse primer (5′-CGGTAGGCGTGTACGGTG-3′). 2.3. GFP reporter plasmid construction The 923-bp and 1133-bp coding regions of the E gene and the RdRp domain of the NS5 gene, respectively, were amplified from the total RNA extracted from a TMUV-positive cell culture supernatant using the primers described in our previous study (Tang et al., 2015) (Fig. 1B). For recombination-based cloning, 15 homologous base sequences on either side of the EcoR I restriction site used to linearize the vector were added to the original primers, according to the manufacturer's introductions for the CloneEZ® PCR Cloning Kit (GenScript, Piscataway Township, NJ, USA). The E and NS5 RdRp domain RT-PCR products were cloned into the EcoR I restriction site of pEGFP-N1 (Clontech, Mountain View, CA, USA) using the CloneEZ® PCR cloning kit, as described above, fused to the N-terminal of the EGFP under the CMV immediate early promoter (Fig. 1B). The constructed eukaryotic expression vectors, designated as pEGFP-N1-E and pEGFP-N1-NS5, were used as EGFP reporter vectors to evaluate the effects of the designed shRNAs in inhibiting the target genes.

2. Material and methods

2.4. Plasmid transfection

2.1. Cells and viruses

Vero cells were seeded onto six-well plates 24 h before transfection. When the cell monolayer reached about 70%–80% confluence, 3 μg each of pSilencer-E1, pSilencer-E2 and pSilencer-E3 were co-transfected with 3 μg of pEGFP-E, while 3 μg each of pSilencer-NS51, pSilencerNS52 and pSilencer-NS53 were co-transfected with 3 μg of pEGFP-NS5, both using Lipofectamine™ 2000 (Qiagen, Valencia, CA, USA) transfection reagent according to the manufacturer's instructions. The negative controls comprised co-transfection with 3 μg each of pEGFP-E and pEGFP-NS5, respectively, with 3 μg of pSilencer-control. The Lipofectamine™ 2000-treated Vero cells were used as the mock transfection control.

Competent DH5α Escherichia coli cells (Invitrogen, Carlsbad, CA, USA) were used for plasmid proliferation and routine cloning. Human adenovirus type 5(AdV5)-transformed HEK293 cells (CRL-3216, ATCC, Manassas, VA, USA) and African green monkey kidney cells (Vero) (CCL-81, ATCC, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Gibco, Gaithersburg, MD,USA) at 37 °C in an incubator containing 5% CO2. The TMUV strain from a layer duck (zc-1 strain, GenBank accession No. KF557894) was used for RNAi evaluation. The virus propagation and test were carried out according to the approved guidelines by the Institutional Biosafety Committee, Shandong Agricultural University (http://web01.sdau.edu.cn/s/242/t/2113/a/71046/info. jspy).

2.5. Analysis of EGFP fusion protein expression in Vero cells At 24 h post transfection, E-EGFP and NS5-EGFP fusion protein expression were examined under a fluorescence microscope (Olympus, Tokyo, Japan) at 100× magnification. After microscopic observation, the monolayers of transfected cells and mock cells were washed gently 389

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pDC312 (Microbix, Hamilton, Ontario, Canada) (Fig. 1A). Next, 3 μg of each of the recombinant AdV 5 shuttle vectors, designated as pDC312NS52 and pDC312-control, was co-transfected with 1 μg of the packaging plasmid pBHGΔE1,3Cre (Microbix, Hamilton, Ontario, Canada) into HEK293 cells, as described previously (Ng et al., 1999). The cells were harvested 9–12 days after transfection and then passaged three times in HEK293 cells to generate a high-titer viral stock. The successful construction of the shRNA expression rAds, designated as rAd-NS52 and rAd-control, was verified using PCR by amplifying the 880-bp E2B region of the viral genome (adv1: 5′-TCGTTTCTCAGCAGCTGTTG-3′; adv2: 5′-CATCTGAACTCAAAGCGTGG-3′) and titrated using an AdenoX Rapid Titer Kit (BD, Franklin Lakes, NJ, USA).

using phosphate-buffered saline (PBS), trypsinized and resuspended in cold PBS before the flow cytometry analysis. The percentages of EGFPpositive cells were calculated using a Guava EasyCyte Mini Cytometer (Millipore, Billerica, MA, USA) at a flow rate of 800 cells per minute. The cell counting results were analysed using the Guava Express Plus method. 2.6. Analyses of the TMUV E and NS5 genes in Vero cells by real-time RTPCR Relative real-time RT-PCR (rRT-PCR) assays, as described in our previous study (Yu et al., 2012), were used to confirm the specific silencing of the target genes. Briefly, total RNA was extracted from the transfected cells or control cells using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA), according to the manufacturer's instructions. Reverse transcription was performed using an MLV-RT kit (Promega, Madison, WI, USA) with a reaction mixture of 10 μL, which contained 2 μL of the RNA extracted according to the manufacturer's instructions. Next, 2 μL of cDNA from the previous step was subjected to rRT-PCR analysis using specific primers for the E gene, NS5 gene and β-actin (Supplementary Table S1) and SYBR® Green Realtime PCR Master Mix (Thermo Scientific, Waltham, MA, USA), according to the manufacturer's instructions. rRT-PCR was performed using a 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) where the thermal cycling profile was as follows: 94 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing and elongation at 60 °C for 60 s. The fluorescence signal of each sample was collected at the end of each annealing and elongation step.

2.9. Inhibition of TMUV in vitro by shRNA expression using rAd To assess the capacity of rAd to inhibit TMUV replication in vitro, Vero cells susceptible to rAd infection but not permissive of productive replication were seeded onto six-well cell culture plates 24 h before infection. When the cell monolayers reached about 70%–80% confluence, they were inoculated with rAd-NS52 and rAd-control at MOIs of 10, 50, 100, 250, 500 and 1000 in maintenance DMEM, as described above. At 24 h post inoculation, the cells were challenged with TMUV at a MOI of 100 without removing the rAd-NS52 and rAd-control suspension. The Vero cells were also firstly inoculated with TMUV at a MOI of 100. At 24 h post inoculation, the TMUV infected cells were inoculated with rAd-NS52 and rAd-control at MOIs of 10, 50, 100, 250, 500 and 1000 in maintenance DMEM without removing TMUV suspension. The relative NS5 gene expression levels in these samples were measured using the rRT-PCR assay described above at 24 h post-infection with TMUV or rAds. The investigation of the inhibition of TMUV replication in Vero cells due to rAd-NS52 at different time points after TMUV infection was carried out by seeding Vero cells in six-well plates and individual infecting with the rAd-control and rAd-NS52 (500 MOI). At 24 h post inoculation with rAd, the cells were challenged with TMUV (100 MOI), and the TMUV-infected cells were harvested at 24, 48, 72 and 96 h post-infection with TMUV to determine the suppressive effects of rAd-NS5 on TMUV replication in Vero cells.

2.7. TMUV infection, titration and western blot analysis using Vero cells To determine whether the plasmid expressed by the shRNAs inhibited TMUV replication in Vero cells, we first transfected Vero cells with different shRNA expression plasmids. Briefly, the Vero cells were seeded onto six-well cell culture plates at 24 h before transfection. When the cell monolayer reached about 70%–80% confluence, the Vero cells were transfected with 3 μg each of pSilencer-E2, pSilencer-E2, pSilencer-E3, pSilencer-NS51, pSilencer-NS52 and pSilencer-NS53. The pSilencer-control and mock-transfected Vero cells were used as controls. At 24 h post transfection, virus infection was carried out with TMUV at a MOI of 100. Briefly, after removing the culture medium, 200 μL of TMUV in maintenance DMEM (2% FBS) was added to each well. After incubating for 2 h at 37 °C, the non-absorbed virions were removed by washing the cells twice with PBS, and 3 mL of maintenance DMEM was then added directly to each well. After culture for 48 h at 37 °C under 5% CO2, all of the infected cells were harvested for rRTPCR, to determine the TCID50, (Li et al., 2014) and for western blot analysis. For western blotting, the harvested cells were mixed with an equal volume of sodium dodecyl sulphate (SDS) lysis buffer (100 mM Tris–HCl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol). After boiling for 10 min, the samples mixed with SDS lysis buffer were subjected to SDS-polyacrylamide gel electrophoresis, followed by blotting onto nitrocellulose membranes (Millipore, Bedford, MA, USA). The membrane was incubated with anti-E/ NS5 monoclonal antibody and mouse anti-β-actin antibody (Abcam, Shanghai, China), before incubating with horseradish peroxidase-conjugated goat anti-mouse antibody. The proteins were visualized using an enhanced HRP-DAB Substrate Kit (TIANGEN, Beijing, China).

2.10. Statistical analysis The statistical significance of differences between treated samples and control samples were assessed using the two-tailed Student's t-test with Microsoft Excel and visualized by OriginPro 8.5 software (OriginLab, Northampton, MA, USA). Differences were considered statistically significant at P < 0.05 and highly significant at P < 0.01. 3. Results 3.1. shRNAs downregulates the expression of green fluorescent protein (GFP) reporter proteins Enhanced GFP (EGFP) reporter plasmids (pEGFP-N1-E and pEGFPN1-NS5) were co-transfected with the corresponding specific shRNA expression plasmids for screening (Fig. 1). The EGFP reporter plasmids were also co-transfected with the pSilencer-control plasmid and were used as the non-interference control. Compared with the non-interference control samples (Fig. 2A), the E gene- and NS5 gene-specific shRNA expression plasmids efficiently decreased the expression of the corresponding EGFP fusion proteins, thereby resulting in lower numbers of cells with EGFP fluorescence (Fig. 2A). The most significant decrease in EGFP expression was observed with pSilencer-E1 and pSilencer-NS52 for the E gene and NS5 gene, respectively (Fig. 2A). The EGFP-positive cells in different tests were quantitatively validated by flow cytometry assays after microscopic observations. Thus, the average percentages (n = 3) of E-EGFP positive cells with the pSilencer-control, pSilencer-E1, pSilencer-E2 and pSilencer-E3 treated samples were

2.8. Construction of rAd to express the shRNAs To construct the rAd shuttle vectors, the human CMV promoterdriven shRNA expression cassette was retrieved from the selected pSilencer-NS52 and pSilencer-control plasmids described above by EcoRI and HindIII restriction enzyme digestion and then inserted into the corresponding position in the linearized AdV 5 shuttle vector of 390

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(caption on next page)

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Fig. 2. Transient expression of shRNAs conferred the sequence-specific inhibition of expression of TMUV E and NS5 in Vero cells. (A) Fluorescence observation of co-transfection of pEGFP-N1-E and pEGFP-N1-NS5, respectively, with their corresponding shRNA expression plasmids at 24 h post-transfection (100× magnification). (B) and (C) Flow cytometry counting of EGPF positive cells and interference efficiency calculating of pEGFP-N1-E and E gene specific shRNA expression plasmids co-transfected Vero cells at 24 h post-transfection. (D) and (E) Flow cytometry counting of EGPF positive cells and interference efficiency calculating of pEGFP-N1-NS5 and NS5gene specific shRNA expression plasmids co-transfected Vero cells at 24 h post-transfection. The interference efficiency was calculated relative to the EGFP positive cell numbers in pSliencer-control and E/NS5 EGFP reporter vectors co-transfected cells as 100%. The values shown are the means of three replicates. Error bars indicated standard deviation. Ninety five percent confidence intervals are shown (*P < 0.05 and **P < 0.01).

cells, the most effective shRNA sequence for TMUV inhibition, i.e. NS52, was selected to create the shRNA for expression by the rAd vector, which is well documented as being successful in shRNA delivery (Feng et al., 2008; Long et al., 2012; Ma et al., 2012) as well as significantly affecting the endogenous RNAi and interferon pathways in infected cells (Narvaiza et al., 2006). The pSilencer-control vector with the non-specific shRNA sequence was also used to construct the rAdcontrol. After co-transfection with the corresponding shuttle plasmids and packaging plasmid for 8 days, the typical adenovirus cytopathic effect (CPE) was observed in transfected cells (Fig. 4A) with detachment of the monolayer and round cell formation, whereas the CPE was not observed in mock-transfected cells (Fig. 4B). The CPE cells were harvested at 10 days post-transfection when the CPE was absolutely complete (all cells were rounded and 80% were floating). After plaque purification, rAd-NS52 and the rAd-control were passaged continually in human embryonic kidney (HEK293) cells and complete CPE appearance was observed within 7 days post-transfection at the third passage (rAd-NS52 titer = 2.85 × 109 TCID50/mL; rAd-control titer = 3 × 109 TCID50/mL). The presence of rAd-NS52 and the rAdcontrol in the CPE cells was further confirmed by E2B region-based PCR amplification. Thus, the specific primers generated the predicted 880bp amplicons from the DNA extracted from rAd-NS52 and rAd-control but not from the mock-transfected cells (Fig. 4C).

26.04%, 6.39%, 18.99% and 11.18%, respectively (Fig. 2B). The interference efficiencies of pSilencer-E1, pSilencer-E2 and pSilencer-E3 were calculated as 75.50%, 27.28% and 56.84%, respectively, compared with the pSilencer-control (Fig. 2C). The average percentages (n = 3) of NS5-EGFP positive cells with pSilencer-control, pSilencerNS51, pSilencer-NS52 and pSilencer-NS53 were 18.92%, 12.75%, 5.23% and 7.84%, respectively (Fig. 2D). The interference efficiencies of pSilencer-NS51, pSilencer-NS52 and pSilencer-NS53 were calculated as 32.54%, 72.37% and 58.46%, respectively, compared with pSilencercontrol (Fig. 2E). The average percentages of EGFP-positive cells with the mock treatment were < 0.1%, which can be considered as instrument noise. 3.2. shRNAs inhibits the replication of TMUV in Vero cells After confirming the efficiency of the specific shRNA expression plasmids at induced EGFP fusion gene silencing, we quantified the replication of TMUV in Vero cells to determine whether the designed shRNAs affected the progress of TMUV multiplication. Compared with the TMUV mock-infected transfection cells and pSilencer-control transfected cells, the shRNA expression plasmids pSilencer-E1, pSilencer-E2 and pSilencer-E3 significantly inhibited the replication of TMUV with relative E gene expression intensities of 19.67%, 50.88% and 37.40%, respectively (mock = 100% and pSilencer-control = 97.88%) (Fig. 3A, blue bar). When we used the E-gene-specific shRNA expression plasmids to inhibit TMUV replication, there was also significant suppression of the NS5 gene with relative gene expression intensities of 26.37%, 45.01% and 45.36%, respectively (mock = eff and pSilencer-control = 98.50%) (Fig. 3A, red bar). In addition, the shRNA expression plasmids pSilencer-NS51, pSilencer-NS52 and pSilencer-NS53 significantly inhibited the replication of TMUV with relative gene expression intensities of 48.57%, 20.33% and 48.10% for the E gene (mock = 100% and pSilencer-control = 98.27%), respectively, and 52.25%, 16.90% and 45.86% for the NS5 gene (mock = 100% and pSilencer-control = 96.7%) (Fig. 3B). Similar inhibitory effects of the specific shRNAs were also confirmed by titrating the TMUV progeny in the shRNA expression plasmidtransfected Vero cells. Compared with the TMUV mock-infected transfection cells and pSilencer-control transfected cells, all of the tested specific shRNA expression plasmids could inhibit the replication of TMUV causing 100.86 to 102.02 50% tissue culture infective dose (TCID50) decreases in the viral titers in Vero cells (mock titer = 105.37 TCID50 and pSilencer-control titer = 105.42 TCID50) with the E genespecific shRNAs (Fig. 3C) and 100.69 to 102.17 TCID50 decreases in the viral titers in Vero cells (mock titer = 105.28 TCID50 and pSilencercontrol titer = 105.36 TCID50) with the NS5 gene-specific shRNAs (Fig. 3D). Western blot analysis demonstrated that E protein (Fig. 3E) and NS5 protein (Fig. 3F) expression were effectively inhibited by their specific shRNA expression plasmids, but there were no significant differences in the expression of β-actin among different groups (Fig. 3E and F). In the pSilencer-control transfected cells, no significant inhibition was observed at relevant gene expression intensities, as well as the protein and viral titer levels, thereby indicating that the non-specific shRNA did not interfere with TMUV production.

3.4. rAd-mediated dose- and time-dependent suppression of TMUV replication in Vero cells To examine the suppressive effects of shRNA expression by rAd induced RNAi on TMUV replication, two independent tests were carried out. For the test 1, the Vero cells were first inoculated with rAd-NS52 and the rAd-control at increasing multiplicities of infection (MOIs) of 10, 50, 100, 250, 500 and 1000. At 24 h post inoculation with rAd, the cells were challenged with a fixed amount of TMUV (100 MOI) to determine the relative degree of NS5 gene suppression at 24 h post-infection. The results showed that the inhibitory effect was clearly correlated with the dose of rAd-NS52, i.e. 56.27% inhibition with 10 MOI, 63.97% inhibition with 50 MOI, 72.80% with 100 MOI, 78.87% inhibition with 250 MOI, 80.63% inhibition with 500 MOI and 81.50% inhibition with 1000 MOI (Fig. 4D). For the test 2, The Vero cells were first inoculated with TMUV (100 MOI), and the rAd-NS52 and the rAdcontrol at MOIs of 10, 50, 100, 250, 500 and 1000 were subsequently inoculated at 24 h post inoculation with TMUV. The results showed that the inhibitory effect was clearly correlated with the dose of rAd-NS52, i.e. 50.12% inhibition with 10 MOI, 58.34% inhibition with 50 MOI, 69.15% with 100 MOI, 72.83% inhibition with 250 MOI, 78.35% inhibition with 500 MOI and 79.03% inhibition with 1000 MOI (Fig. 4E). The inhibitory effects of rAd-NS52 on TMUV in test 1 are higher than those of test 2. This is mainly because of first inoculation of TMUV may lead to more virus progenies release into culture supernatant and these progenies cannot be inhibited by the current strategy. Compared with the TMUV-infected Vero cells, there was no significant antiviral activity against TMUV from the rAd-control in both tests, and the minor inhibitory effects (1.4%–4.2%) were considered to be non-specific (Fig. 4D and E). To further investigate the inhibition of TMUV replication in Vero cells due to rAd-NS52 at different time points after TMUV infection, Vero cells were seeded in six-well plates and infected individually with the rAd-control and rAd-NS52 (500 MOI). At 24 h post inoculation with rAd, the cells were challenged with TMUV (100 MOI), and the TMUV-

3.3. Construction of the shRNA for expression by rAd After screening the six constructed shRNA expression plasmids using the EGFP reporter system and testing TMUV replication in Vero 392

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Fig. 3. Inhibition of TMUV replication induced by shRNA expression plasmids in Vero cells. Vero cells were transfected with pSliencer-control, pSilencer-E2, pSilencer-E2, pSilencer-E3, pSilencer-NS51, pSilencer-NS52 and pSilencer-NS53, respectively. The pSilencer-control and mock transfection Vero cells were working as control. At 24 h post-transfection, the transfected cells were infected with TMUV (100 MOI), respectively. (A) and (B) Effects on TMUV RNA replication induced by E gene and NS5 gene specific shRNAs in Vero cells at 48 h post TMUV infection. The relative intensities of E gene and NS5 gene was quantified by rRT-PCR and normalized to that of β-actin mRNA in the same sample. The E gene relative intensities were shown in blue bars and the NS5 gene relative intensities were shown in red bars. The relative viral gene intensity in cells transfected with pSliencer-control was defined as 100%. (C) and (D) Effects on TMUV titer induced by E gene and NS5 gene specific shRNAs in Vero cells at 48 h post TMUV infection. The method to determine the TMUV titer was as described in Method section. The value of -Log TCID50 of TMUV titer in cells transfected with the in pSliencer-control was defined as 100%. All of above values were the means of three independent experiments. Error bars indicated standard deviation. Ninety five percent confidence intervals are shown (*P < 0.05 and **P < 0.01). (E) and (F) Western blot analysis of specific siRNAs plasmids inhibited TMUV E protein and NS5 protein expression in Vreo cells at 48 h post TMUV-infection. The E, NS5 and β-actin proteins were detected using specific antibodies. Lane Vero, the cells neither transfected with shRNA expression plasmid nor infected with TMUV. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Fig. 4G).

infected cells were harvested at 24, 48, 72 and 96 h post-infection with TMUV to determine the suppressive effects of rAd-NS5 on TMUV replication in Vero cells. Marked suppression of NS5 gene expression was observed in the rAd-NS52 group from 24 h to 96 h post inoculation with TMUV compared with the TMUV mono-infection group and rAd-control group (Fig. 4F). The inhibitory effects of rAd-NS52 on TMUV replication at different time points were calculated as 82.5%, 70.03%, 69.70% and 67.14% at 24, 48, 72 and 96 h post-infection with TMUV, thereby indicating that the inhibitory effect induced by rAd-NS52 could last for at least 96 h, but it weakened over time post-inoculation with rAd-NS52

4. Discussion TMUV was first identified as a tropical pathogen during the 1970s, and birds were considered to be its maintenance hosts (Simpson et al., 1975). Subsequent reports then indicated that birds became sick or died from TMUV infections. However, since 2010, extensive TMUV infections in duck farms have continuously been diagnosed in different Asian countries, mainly in China, which have severely affected duck 393

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Fig. 4. The characterization of the rAd-NS52 and inhibition of TMUV replication by rAd-NS52 in Vero cells. (A) The HEK293 cells at 8 day post co-transfection of shuttle plasmid and packaging plasmid with the typical adenovirus CPE of the detachment of the monolayer and round cell formation. (B) Mock transfected HEK293 cells at 8 day post-treatment. (C) Agarose gel electrophoresis results of PCR products using primers to amplify the Ad5-E2B region. The 880 bp product is specific for the Ad5-E2B region. Lane M, a 1-kb DNA marker; Lane HEK293, DNA from uninfected HEK293 cells; Lane rAd-control, DNA from rAd-control infected HEK293 cells; Lane rAd-NS52, DNA from rAd-NS52 infected HEK293 cells. (D) The dosedependent inhibition of TMUV replication. Vero cells were infected with TMUV at 100 MOI at 24 h after inoculation with those rAd-control and rAd-NS52 at 10, 50,100, 250, 500 and 1000 MOI, respectively. The NS5 gene relative intensities were determined by rRT-PCR after 24 h post TMUV infection. Blue bars: rAd-control infected cells. Red bars: rAd-NS52 infected cells. The relative NS5 gene intensity in TMUV mono-infected cells was defined as 100%. (E) The dose-dependent inhibition of TMUV replication. Vero cells were infected with TMUV at 100 MOI. At 24 h post inoculation, the TMUV infected cells were inoculated with rAd-control or rAd-NS52 at 10, 50,100, 250, 500 and 1000 MOI, respectively. The NS5 gene relative intensities were determined by rRT-PCR after 24 h post rAds infection. Blue bars: rAd-control infected cells. Red bars: rAd-NS52 infected cells. The relative NS5 gene intensity in TMUV mono-infected cells was defined as 100%. (F) and (G) The time-dependent inhibition of TMUV replication. Vero cells were infected with TMUV at 100 MOI at 24 h after inoculation with those rAd-control and rAd-NS52 at 500 MOI. The relative expression/intensities of NS5 gene were determined by rRT-PCR at 24, 48, 72, 96 h post TMUV infection. The relative NS5 gene intensity in TMUV mono-infected cells was defined as 100%. Blue bars: rAd-control infected cells. Red bars: rAd-NS52 infected cells. The values above were the means of three independent experiments. Error bars indicated standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

or cells (Huelsmann et al., 2006). In this study, for the first time, we demonstrated the successful application of RNAi to suppress the expression of the E and NS5 genes of TMUV in Vero cells. The transcription of six specifically designed shRNAs was driven by the pSilencer 4.1-CMV neo vector. We then screened the inhibitory effects of

production (Cao et al., 2011). However, there is no approved vaccine or an effective antiviral therapy to combat TMUV infections. RNA silencing is an important antiviral response in mammalian, bird, insect and plant cells (Obbard et al., 2009), where a 21–23 nt dsRNA is capable of mediating gene-specific silencing without adverse effects on organisms 394

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destroyed (Tijsterman et al., 2002). Application of RNAi to flavivirus antiviral strategies has also need to notice the shRNA-induced toxicity which includes off-target effect, activation of the interferon response, and saturation of the cellular silencing machinery (Narvaiza et al., 2006). For in vitro system, the off-target effect was questioned in some RNAi off-target studies about the specificity of shRNA (Jackson and Linsley, 2004). In this study, we designed each 3 specific shRNAs for E gene and NS5 gene and observed specific downregulation of target genes; whereas no downregulations were observed in the non-specific shRNA control. Such result indicated the specific shRNA induced viral gene downregulation is not due to the shRNA off-target effect. For the off-target effect on Vero cells, we have not conducted detailed experiments to rule out it in our system. However, the Vero cells transfected with different shRNA expression plasmids didn't show significant CPE. The usual method for inducing sustained RNAi in flavivirus antiviral strategies is to introduce plasmids that synthesize shRNAs using RNA Pol III promoters (Achazi et al., 2012; Anthony et al., 2009; Korrapati et al., 2012). These promoters synthesize shRNAs and elicit RNAi in an efficient manner, but they lack cell specificity; it is difficult to monitor the shRNA expression levels (Zhou et al., 2005). In the current study, we employed a modified human cytomegalovirus (CMV) promoter, a typical RNA Pol II promoter, which can direct shRNA synthesis and mediate gene silencing more efficiently than RNA Pol III promoters (Weiwei et al., 2009). Our shRNA expression plasmids could suppress TMUV replication in Vero cells, but the inhibitory effect may be limited by the plasmid concentration, transfection efficiency and toxicity of the transfection reagents. To readily deliver shRNA into cells with high efficiency and induce steady interference with a long duration, we transferred the CMV-driven shRNA expression cassette from the most effective shRNA expression plasmid (pSilencer-NS52) into the AdV 5 vector shuttle plasmid, and we constructed rAd-NS52 to express the specific shRNA. The shRNA-expressing rAd-NS52 vector was found to be an effective inhibitor in the presence of a high titer of TMUV in Vero cells, where the inhibitory effect was dose-dependent. When the infectious dose of rAd-NS52 reached 250 MOI, the RNAi efficiency of rAdNS52 was significantly higher than that of the corresponding plasmid vector, thereby indicating that rAd could deliver the shRNA more efficiently into the infected cells than the plasmid. However, the RNAi effect decreased over time post challenge. This is mainly because rAdNS52 is a replication-defective virus, which can only replicate in HEK293 cells. The rAd-NS52 vector constructed in this study was based on a commercialized human adenovirus system for biosafety purposes, but it still has the potential to infect bird cells/hosts (Olah et al., 1990; Toro et al., 2007), which facilitates further in vivo RNAi study in a duck host. In the present study, we detected the suppression of TMUV replication in Vero cells based on multiple parameters, including the EGFP fluorescence intensity, relative viral gene expression levels, viral antigens and viral titers, which together demonstrated the utility of the plasmid and the rAd-mediated delivery of shRNAs as an effective tool for inhibiting TMUV replication. The efficacy of the shRNAs should be studied further using a duck adenovirus vector based shRNA expression system for inhibiting the replication of TMUV since the most TMUV infections were found on ducks.

the designed shRNAs by co-transfecting the specific shRNA expression plasmids with the eukaryotic expression plasmids pEGFP-N1-E and pEGFP-N1-NS5 in Vero cells before evaluating the relative E and NS5 gene expression levels as well as the viral titers of shRNA expression plasmid-transfected cells after inoculation with TMUV. The RNAi anti-TMUV strategy employed in the present study targets the E and NS5 genes for gene silencing, which are well-documented in many other flavivirus RNAi studies (Kumar et al., 2006; McCown et al., 2003; Qi et al., 2008). The E gene encodes the glycoprotein in flaviviruses, and it is a class II fusion protein, which mainly comprises βsheets (domain I), a short transmembrane domain (domain II) and an immunoglobulin-like carboxy terminal domain (domain III) (De La Guardia and Lleonart, 2014). This protein is by far the most important molecule in TMUV during the viral entry process because it appears to be responsible for receptor recognition and attachment to the cell, clathrin-mediated endocytosis and the subsequent fusion of the viral and cellular membranes (Yu et al., 2013). Thus, the E gene is considered to be an ideal target in RNAi antiviral strategies. The three E genespecific shRNAs designed in this study were targeted at the sequences encoding domains I, II and III to interfere with the entry/fusion process in TMUV progeny after infection. The E protein is encoded by one of the most variable genes in different TMUV strains (Yu et al., 2013), so selecting the most conserved region with a high interference efficiency also needed to be considered during the design of the shRNA. In contrast to the E protein-encoding gene, the NS5 protein-encoding gene is the most conserved gene among different TMUV strains, especially twothirds of the C-terminal where the conserved RdRp domain is located (Tang et al., 2012). The NS5 RdRp in flaviviruses plays a vital role during the viral life cycle via the replication of viral RNA, and it is also suitable for exploitation in anti-TMUV strategies (Lim et al., 2015). Thus, three NS5 gene-specific shRNAs were designed according to the different sub-domains, as described in other flaviviruses (fingers, palm and thumb) (Lim et al., 2015), thereby exerting RNAi effects during the early events in the TMUV life cycle. We used the EGFP reporter system to monitor the effects of the designed shRNAs on the E and NS5 genes. By fusing the E and NS5 proteins to the N-terminal of the EGFP proteins, the inhibitory effects of the specific shRNAs on E or NS5 gene expression could be evaluated directly by EGFP induced green fluorescence in the transfected Vero cells. N-terminal tagging with GFP has adversely affected protein localization in some studies compared with the C-terminal tagging method (Palmer and Freeman, 2004), but it is the preferred RNAi reporting system for guaranteeing the expression of the proteins that are tagged (Ji et al., 2008; Zhou et al., 2007). The results showed that all of the E- and NS5-specific shRNAs effectively downregulated the expression of their corresponding genes. These findings were supported by EGFP fluorescence observations, flow cytometry, relative RT-PCR, TMUV titration and western blot analysis, which confirmed the inhibitory effects on both the gene and protein expression levels. Our analysis of the inhibitory effects of the designed shRNAs on the EGFP-tagged E and NS5 proteins showed that pSilencerE1 (interference efficiency = approximately 75%) and pSilencer-NS52 (interference efficiency = approximately 67%) could inhibit the expression of the targeted TMUV gene with the highest efficiency. After analyzing the performance of the shRNA expression plasmids on the inhibition of TMUV replication, almost identical results were obtained compared to the EGFP reporter system, but the inhibitory effect of pSilencer-NS52 was higher than that of pSilencer-E1. Interestingly, according to the TMUV replication inhibition test, suppression of the E or NS5 gene downregulated the expression of the other gene (NS5 or E). Using the pSilencer-E2 and pSilencer-NS51 plasmids, the inhibitory effect on the non-targeted gene was higher than that on the targeted gene at the gene expression level. The reasons for this are unclear, but it may be associated with the characteristics of linear non-segmented TMUV genomes, which include a single ORF that encodes all of the viral proteins. Thus, when one siRNA combining site on the TMUV ORF was recognized by RISC, the whole target viral RNA could be completely

Acknowledgments This study was funded through The China Agriculture Research System (CARS-42-04B); National Natural Science Foundation of China (31272583, 31472199, 31602047); Science and Technology Development Plan of Shandong Province (2014GNC111023); Funds of Shandong “Double Tops” Program. Competing financial interests The authors declare no competing financial interests. 395

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