CORD-19_Custom_license_subset  

CORD-19:dd0159ba60fc4458dc4a2c632fe9072d8cc7ff36 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 0-125 Sentence denotes Host miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type I interferons
T2 127-135 Sentence denotes Abstract
T3 136-336 Sentence denotes MicroRNAs (miRNAs) play important roles in viral infections, especially by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection.
T4 337-553 Sentence denotes To identify host miRNAs important to controlling porcine reproductive and respiratory syndrome virus (PRRSV) infection, we screened 15 miRNAs that were previously implicated in innate immunity or antiviral functions.
T5 554-707 Sentence denotes Over-expression of the miR-26 family strongly inhibited PRRSV replication in vitro, as shown by virus titer assays, Western blotting, and qRT-PCR assays.
T6 708-782 Sentence denotes MiR-26a inhibited the replication of both type 1 and type 2 PRRSV strains.
T7 783-840 Sentence denotes Mutating the seed region of miR-26 restored viral titers.
T8 841-1046 Sentence denotes Luciferase reporters showed that miR-26a does not target the PRRSV genome directly but instead affects the expression of type I interferon and the IFN-stimulated genes MX1 and ISG15 during PRRSV infection.
T9 1047-1246 Sentence denotes These results demonstrate the important role of miR-26a in modulating PRRSV infection and also support the possibility of using host miR-26a to achieve RNAi-mediated antiviral therapeutic strategies.
T10 1248-1539 Sentence denotes MicroRNAs (miRNAs) are small (∼22 nucleotides) non-coding RNAs that bind to complementary sequences in the untranslated regions of target mRNAs and contribute to gene regulation by reducing mRNA translation or destabilizing transcripts (Grassmann and Jeang, 2008; Skalsky and Cullen, 2010) .
T11 1540-1838 Sentence denotes The commonly accepted mechanism of miRNA regulation is that the seed region (2 ∼ 8 nucleotides at the 5 end) of an miRNA is complementary to the 5 or 3 untranslated region (5 -or 3 -UTR) of an mRNA, leading to mRNA degradation or translational inhibition (Bartel, 2009; Gottwein and Cullen, 2008) .
T12 1839-2007 Sentence denotes Recent work has shown the importance of miRNAs in regulating host-pathogen interactions and innate immunity (Lodish et al., 2008; Scaria et al., 2007; tenOever, 2013) .
T13 2008-2237 Sentence denotes Host miRNAs can affect viral replication by binding directly to viral RNA (Lecellier et al., 2005) or by indirectly modulating host factors to provide a less permissive environment for virus replication (Triboulet et al., 2007) .
T14 2238-2383 Sentence denotes As miRNAs are small molecules without antigenic properties, they are considered to have potential efficacy in antiviral therapeutic applications.
T15 2384-2636 Sentence denotes For example, human miR-122 is an essential component of the biology of hepatitis C virus replication (Jopling, 2010; Jopling et al., 2005) and therapeutic blocking of miR-122 suppresses hepatitis C viremia in non-human primates (Lanford et al., 2010) .
T16 2637-2836 Sentence denotes Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a member of the arterivirus family, is the causative agent of Porcine Reproductive and Respiratory Syndrome [PRRS; (Chand et al., 2012) ].
T17 2837-3107 Sentence denotes Based on their genetic and antigenic differences, PRRSV strains are classified into two distinct genotypes, North American (type 2) and European (type 1), represented by the VR-2332 (Benfield et al., 1992) and Lelystad virus (LV) (Wensvoort et al., 1991) , respectively.
T18 3108-3253 Sentence denotes These two genotypes on two different continents share only approximately 60% nucleotide sequence identity (Forsberg, 2005; Hanada et al., 2005) .
T19 3254-3440 Sentence denotes Many strategies for controlling PRRSV transmission have been proposed but have generally shown little success, which has stimulated the search for new ways to control PRRSV transmission.
T20 3441-3534 Sentence denotes PRRSV can escape from innate immunity and cause persistent infections (Miller et al., 2004) .
T21 3535-3664 Sentence denotes In mammalian cells, viral infection is a potent trigger of the interferon (IFN) response (Sadler and Williams, 2008; Sen, 2001) .
T22 3665-3921 Sentence denotes Type I interferons can initiate the activation of JAK/STAT signaling to induce the expression of hundreds of IFN-stimulated genes (ISGs), which play an important role in antiviral activities (Albina et al., 1998; Katze et al., 2002; Overend et al., 2007) .
T23 3922-4032 Sentence denotes However, in contrast to porcine respiratory coronavirus, PRRSV is a poor IFN-inducer (Buddaert et al., 1998) .
T24 4033-4253 Sentence denotes Many miRNAs regulate IFN production Pedersen et al., 2007) , maintain mRNA stability (Li et al., 2012) , and regulate signals downstream of IFN to modulate antiviral immunity (Wang et al., 2010; Yoshikawa et al., 2012) .
T25 4254-4413 Sentence denotes While most miRNAs characterized to date decrease the production of IFNs (Alam and O'Neill, 2011) , a few miRNAs that upregulate type I IFNs have been reported.
T26 4414-4554 Sentence denotes Recent research has revealed that miR-23 may play a positive modulatory role in IFN production during PRRSV infection (Zhang et al., 2014) .
T27 4555-4724 Sentence denotes Given the breadth of miRNA-mediated regulation of mammalian immunity (Grassmann and Jeang, 2008) , the role of host miRNAs in PRRSV infection is of significant interest.
T28 4725-4782 Sentence denotes Here, we found that miR-26a is an anti-PRRSV host factor.
T29 4783-4893 Sentence denotes Over-expression of miR-26a inhibited infection by both of the major PRRSV genotypes in a dosedependent manner.
T30 4894-5080 Sentence denotes We found that miR-26a does not target the PRRSV genome directly, but rather affects the expression of type I interferon and the IFN-stimulated genes MX1 and ISG15 during PRRSV infection.
T31 5081-5244 Sentence denotes Our study reveals an example of a miRNA that affects viral propagation and highlights a host factor that may be important for future control measures against PRRS.
T32 5245-5482 Sentence denotes MARC-145 cells were grown in MEM (Invitrogen) with 10% fetal bovine serum (FBS, Gibco-BRL, Gaithersburg, MD, USA) and were maintained with 2% FBS at 37 • C in a humidified 5% CO 2 atmosphere as described previously (Yuan and Wei, 2008) .
T33 5483-5604 Sentence denotes Baby hamster kidney (BHK-21, ATCC CCL10) cells were cultured in EMEM (ATCC, Manassas, VA, USA) supplemented with 10% FBS.
T34 5605-5880 Sentence denotes Porcine alveolar macrophages (PAMs) were harvested from the lungs of 6-week-old PRRSV-negative piglets as described previously (Wensvoort et al., 1991) and maintained at 37 • C in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS). vAPRRS (GenBank accession No.
T35 5881-5943 Sentence denotes GQ330474) (Yuan and Wei, 2008) and vSHE (GenBank accession No.
T36 5944-6197 Sentence denotes GQ461593) (Tian et al., 2011) were rescued from pAPRRS and pSHE, respectively. vJX143 (at passage 3) was isolated from the serum of a dying piglet displaying the clinical sings of porcine high fever disease (PHFD) in 2006. vJXM100 (GenBank accession No.
T37 6198-6344 Sentence denotes GQ475526) was obtained through 100 serial passages of the highly pathogenic PRRSV vJX143 strain (EU708726) in MARC-145 cells (Wang et al., 2013) .
T38 6345-6421 Sentence denotes The infectious cDNA clone pJX143 was derived from vJX143 (Lv et al., 2008) .
T39 6422-6530 Sentence denotes High-titer virus stocks were obtained by infecting MARC-145 cells at low multiplicities of infection (MOIs).
T40 6531-6686 Sentence denotes Infected cell supernatants were harvested after an 80% cytopathic effect (CPE) appeared, then the viruses were stored at −80 • C as stocks for further use.
T41 6687-6761 Sentence denotes Virus titer was determined by standard TCID 50 assay using MARC-145 cells.
T42 6762-6950 Sentence denotes miRNA mimics (Table 1) , which are double-stranded 2 -Omethyl-modified RNA oligonucleotides with sequence complementarity to mature miRNAs were synthesized by GenePharma (Shanghai, China).
T43 6951-7257 Sentence denotes The sense sequences of the miR-26 mimics were: miR-26a-5 -uucaaguaauccaggauaggcu-3 ; miR-26b-5 -uucaaguaauucaggauaggu-3 ; corresponding non-seed-mutated miR-26 mimics (26-1A, 26-9U, and 26-1A9U) or seed-mutated miR-26 mimics (26a-m, 26b-m, c-m) are listed in Table 1 (underlined letters are mutated bases).
T44 7258-7331 Sentence denotes The negative-control (NC) mimic sequence was 5 -uucuccgaacgugucacgutt-3 .
T45 7332-7336 Sentence denotes 2.3.
T46 7337-7587 Sentence denotes Transfection of miRNA mimic and viral multi-step growth kinetics miRNA or NC mimics were transfected into PAMs or MARC-145 cells at a concentration of 80 nM (except for dosedependence experiments) using X-tremeGENE siRNA Transfection Reagent (Roche).
T47 7588-7657 Sentence denotes Twenty-four hours after transfection, cells were infected with PRRSV.
T48 7658-7811 Sentence denotes For analysis of PRRSV growth, supernatants (0.2 ml/well) from cell cultures were collected at different time points post-infection and frozen at −80 • C.
T49 7812-7989 Sentence denotes For virus quantification at each time point, a viral titer was measured in MARC-145 cells by standard TCID 50 assay using the method of Reed and Muench (Reed and Muench, 1938) .
T50 7990-8236 Sentence denotes Indirect immunofluorescence assays (IFA) were performed as described previously (Tian et al., 2011) for the detection of nucleocapsid (N) protein in PRRSV vJX143 infected MARC-145 cells or PAMs pre-transfected with miR-26 family or mutant mimics.
T51 8237-8464 Sentence denotes Cells were fixed with cold methanol followed by blocking with 1% bovine serum albumin (BSA) and then incubated for 2 h with a monoclonal antibody (SR30A, Rural Technologies) that specifically recognizes type 2 PRRSV N proteins.
T52 8465-8627 Sentence denotes After washing with phosphate-buffered saline (PBS), the cells were incubated for 1 h with Alexa Fluor 568-labeled goat anti-mouse secondary antibody (Invitrogen).
T53 8628-8723 Sentence denotes Cell nuclei were counterstained with 1 g/ml of 4 , 6 -diamidino-2phenylindole (DAPI) for 5 min.
T54 8724-8832 Sentence denotes After a final PBS wash step, cells were visually analyzed using an Olympus inverted fluorescence microscope.
T55 8833-8916 Sentence denotes MARC-145 cells were transfected with miR-26a or NC mimics prior to PRRSV infection.
T56 8917-9048 Sentence denotes Cells were washed twice with PBS at 48 h post-infection and lysed with lysis buffer in the presence of 1 mM N-ethylmaleimide (NEM).
T57 9049-9185 Sentence denotes After incubation for 10 min on ice, cell lysates were centrifuged at 12,000 × g for 15 min at 4 • C and the supernatants were collected.
T58 9186-9325 Sentence denotes Protein samples were prepared in reducing buffer (50 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 0.02% [wt./vol.] bromophenol blue, 100 mM DTT).
T59 9326-9473 Sentence denotes Samples then were heated at 95 • C for 5 min, resolved on 15% SDS polyacrylamide gels, and transferred to Hybond-Pmembranes (Amersham Biosciences).
T60 9474-9606 Sentence denotes Membranes were blocked with 5% nonfat dry milk in TBST (100 mM NaCl, 10 mM Tris, pH 7.6, 0.1% Tween 20) for 2 h at room temperature.
T61 9607-9799 Sentence denotes Membranes were incubated overnight at 4 • C with primary antibody (1AG11) that specifically recognizes both type 2 and type 1 PRRSV N proteins (kindly provided by Ingenasa Co., Madrid, Spain).
T62 9800-10141 Sentence denotes After washing with TBST, blots were incubated with horseradish peroxidase (HRP)conjugated goat anti-mouse secondary antibody (Santa Cruz) for 1 h at room temperature, washed again with TBST, and developed using SuperSignal West Pico or Femto chemiluminescent substrate according to the manufacturer's instructions (Thermo Fisher Scientific).
T63 10142-10244 Sentence denotes Total RNA and miRNA were extracted with TRIzol (Invitrogen) following the manufacturer's instructions.
T64 10245-10334 Sentence denotes PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara) was used for reverse transcription.
T65 10335-10486 Sentence denotes Quantitative RT-PCR (qPCR) analysis was performed using a Step-one Plus real-time PCR system (Applied Biosystems) and a SYBR Premix Ex Taq TM (Takara).
T66 10487-10685 Sentence denotes For detection of endogenous miRNAs, a commercial miRcute miRNA First-Strand cDNA Synthesis was purchased from TIANGEN BIOTECH (Beijing, China) and used for polyadenylation and reverse transcription.
T67 10686-10814 Sentence denotes A commercial miRcute miRNA qPCR detection kit was purchased from TIANGEN BIOTECH (Beijing, China) for measuring miRNA abundance.
T68 10815-10973 Sentence denotes MARC-145 cells infected with PRRSV at a MOI of 0.01 were collected at the indicated time points and total RNA was extracted using TRIzol reagent (Invitrogen).
T69 10974-11056 Sentence denotes One g of this total RNA was then used for reverse transcription with an RT-primer.
T70 11057-11210 Sentence denotes The abundance of the miRNA of interest in the resulting cDNA was determined by qPCR using a universal reverse primer and a miRNA-specific forward primer.
T71 11211-11327 Sentence denotes The PCR procedure comprised pre-denaturation at 94 • C for 2 min, and 40 cycles of 94 • C for 20 s, 60 • C for 15 s.
T72 11328-11421 Sentence denotes The ubiquitously expressed U6 small nuclear RNA (TIANGEN) was used for normalization purpose.
T73 11422-11490 Sentence denotes All primers used for miRNA qPCR were included in the commercial kit.
T74 11491-11595 Sentence denotes The levels of ORF7 RNA, IFN-␣/␤, MX1, ISG15 mRNA were quantified using a SYBR Premix Ex Taq TM (Takara).
T75 11596-11774 Sentence denotes Relative expression levels were analyzed using the Ct method (Bookout et al., 2006) , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an endogenous control.
T76 11775-11843 Sentence denotes Universal type I interferon was purchased from PBL InterferonSource.
T77 11844-11893 Sentence denotes All PCR experiments were performed in triplicate.
T78 11894-11931 Sentence denotes Other primers are listed in Table 2 .
T79 11932-12103 Sentence denotes The pGL3-Control luciferase reporter vector (Promega) was used as the cloning vector for luciferase assays to analyze potential miR-26a target regions in the PRRSV genome.
T80 12104-12272 Sentence denotes Twenty cDNA fragments encompassing the PRRSV genome were amplified by PCR from PRRSV pJX143 and subcloned into the pGL3-Control vector downstream of the luciferase ORF.
T81 12273-12364 Sentence denotes The primers used are listed Table 2 Sequence of oligonucleotide primers used in this study.
T82 12365-12383 Sentence denotes Sequence Table 2 .
T83 12384-12436 Sentence denotes All cDNA constructs were verified by DNA sequencing.
T84 12437-12570 Sentence denotes Plasmids and miRNA mimics were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer s protocol.
T85 12571-12874 Sentence denotes For luciferase reporter assays, subconfluent BHK-21 cells cultured in 12-well plates were co-transfected with 500 ng/well of the indicated reporter plasmid and 100 ng/well of pRL-CMV (as an internal control to normalize transfection efficiency, Promega) along with the indicated amount of miR-26a mimic.
T86 12875-12998 Sentence denotes Cells were lysed 24 h later for determination of firefly luciferase activities using the Luciferase assay system (Promega).
T87 12999-13189 Sentence denotes Data are presented as the relative luciferase activities in miR-26a mimic-transfected cells relative to NC mimic-transfected controls and are representative of three independent experiments.
T88 13191-13601 Sentence denotes To screen potential miRNAs for their ability to inhibit PRRSV replication, mimics of 15 miRNAs that are well-conserved among different species and have been previously implicated in innate immunity and/or antiviral functions (Banerjee et al., 2013; Foley and O'Neill, 2012; Huang et al., 2014; Yoo and Liu, 2013; Pauley and Chan, 2008; Schulte et al., 2013; Selvamani et al., 2014) were synthesized (Table 1) .
T89 13602-13727 Sentence denotes MARC-145 cells were transfected with individual miRNA mimics (80 nM) and then infected with PRRSV (vAPRRS) at an MOI of 0.01.
T90 13728-13832 Sentence denotes Supernatants from infected cells were collected at 24 and 48 h post-infection to determine viral titers.
T91 13833-13936 Sentence denotes Among the miRNAs tested, over-expression of the miR-26a mimic strongly reduced PRRSV titers (Fig. 1A) .
T92 13937-14155 Sentence denotes Transfection of miR-26a/26b inhibitors demonstrated the opposite effects (Fig. 1B) , indicating that miR-26 has antiviral activity against PRRSV replication and that miR-26a is a more efficient suppressor than miR-26b.
T93 14156-14262 Sentence denotes All the other miRNA mimics tested had no demonstrable impact on PRRSV titers in MARC-145 cells (Fig. 1A) .
T94 14263-14423 Sentence denotes Furthermore, immunofluorescence assays using a FITC-conjugated monoclonal antibody against the PRRSV N protein were consistent with viral titer data (Fig. 1C) .
T95 14424-14705 Sentence denotes To rule out the possibility that this antiviral effect of miR-26a was specific to an individual PRRSV strain, we analyzed the viral growth curves of two type 2 PRRSV strains (vJX143, vJXM100) and a type 1 PRRSV strain (vSHE) in MARC-145 cells transfected with NC or miR-26a mimics.
T96 14706-14853 Sentence denotes Over-expression of the miR-26a mimic, but not the NC mimic, reduced PRRSV replication in multiple PRRSV strains of differing genotypes ( Fig. 2A) .
T97 14854-15033 Sentence denotes To corroborate our findings with miR-26a further, MARC-145 cells were transfected with increasing concentrations of miR-26a mimic (5, 20, 40, 80 nM) and then infected with vAPRRS.
T98 15034-15162 Sentence denotes Both PRRSV growth and the amount of ORF7 mRNA level were inhibited as a function of the dose of miR-26a mimic ( Fig. 2B and C) .
T99 15163-15322 Sentence denotes Consistent with this, transfecting the miR-26a mimic also reduced the accumulation of the PRRSV nucleocapsid (N) protein in a dose-dependent manner (Fig. 2D) .
T100 15323-15534 Sentence denotes To exclude the possibility that reduced PRRSV replication was due to potential toxicity of the miR-26a mimic, MARC-145 cells were transfected with the miR-26a mimic at different doses (40 nM, 80 nM, and 160 nM).
T101 15535-15663 Sentence denotes No appreciable effect of the miR-26a mimic (at up to 160 nM) on cellular viability and morphology was observed (data not shown).
T102 15664-15788 Sentence denotes Collectively, these data show that miR-26a reduces PRRSV replication in multiple PRRSV genotypes in a dose-dependent manner.
T103 15790-15877 Sentence denotes We next investigated the effect of other miR-26 miRNA and mutants on PRRSV replication.
T104 15878-16046 Sentence denotes Because miR-26a is highly conserved between monkeys and pigs, we conducted the subsequent investigations in PAMs, which are the target cells of PRRSV infection in vivo.
T105 16047-16161 Sentence denotes As previously reported, miRNA-mRNA interactions may require seed-matched sites at nucleotides 2-8 (Bartel, 2009 ).
T106 16162-16286 Sentence denotes Thus, we mutated miR-26a mimic at non-seed nucleotides 1, 9, or 1 and 9 and miR-26 at seed nucleotides 2-6 (26a-m and 26bm).
T107 16287-16344 Sentence denotes Both miR-26a and miR-26b had anti-PRRSV activity in PAMs.
T108 16345-16527 Sentence denotes PAMs transfected with miR-26a or miR-26b mimics yielded significantly lower PRRSV titers and ORF7 gene expression compared with cells transfected with the NC mimic ( Fig. 3A and B) .
T109 16528-16670 Sentence denotes Three miR-26a mutants with non-seed mutations retained their ability to inhibit PRRSV progeny production and gene expression ( Fig. 3A and B).
T110 16671-16900 Sentence denotes By contrast, seed mutations at nts 2-6 abrogated the ability of miR-26 family members to repress PRRSV replication and gene expression (Fig. 3A and B) , showing that the seed region was essential for inhibiting PRRSV replication.
T111 16901-17065 Sentence denotes To investigate further inhibition effect of miR-26 family and mutants on PRRSV infection, we used an immunofluorescence assay to detect the PRRSV N protein in PAMs.
T112 17066-17268 Sentence denotes N protein expression in PAMs was suppressed by both miR-26a and miR-26b (Fig. 3C , top) and by miR-26 non-seed mutants (Fig. 3C, middle) , but was not affected by miR-26 seed mutants (Fig. 3C, bottom) .
T113 17269-17385 Sentence denotes We then analyzed the growth dynamics of HP-PRRSV isolate vJX143 in PAMs transfected with miR-26 family or NC mimics.
T114 17386-17548 Sentence denotes Viral growth was suppressed about 1000-fold in PAMs transfected with miR-26a and about 100-fold in PAMs transfected with miR-26b at 24 h postinfection (Fig. 3D) .
T115 17549-17623 Sentence denotes Notably, miR-26a was more efficient suppressing viral growth than miR-26b.
T116 17624-17815 Sentence denotes These results indicated that miR-26 family members, especially miR-26a, can inhibit vJX143 replication in PAMs. We then analyzed the kinetics of miR-26 expression in PRRSV infected PAM cells.
T117 17816-17915 Sentence denotes The relative expression of miR-26 was upregulated as a function of PRRSV infection time (Fig. 3E) .
T118 17916-18072 Sentence denotes Targeting a specific viral sequence represents an efficient strategy by which miRNAs can inhibit viral replication (Jopling, 2010; Lecellier et al., 2005) .
T119 18073-18260 Sentence denotes In recent studies, miR-181 and miR-23 were confirmed to reduce viral gene expression and viral growth due to direct targeting of PRRSV genomic RNA (Guo et al., 2013; Zhang et al., 2014) .
T120 18261-18654 Sentence denotes We determined whether miR-26a specifically targets the PRRSV genome to exert its antiviral effect by constructing a range of firefly luciferase reporter pGL3-Control based plasmids, which contained the cDNA fragments representing the 5 UTR, nsp1-nsp12, ORF2-ORF7, and the 3 UTR of the PRRSV genome Statistical significance was analyzed using t-tests; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
T121 18655-18657 Sentence denotes C.
T122 18658-18770 Sentence denotes PAMs were transfected with the indicated miRNA mimics and then infected with PRRSV vJX143 (MOI = 0.01) for 24 h.
T123 18771-18915 Sentence denotes Cells were fixed and immunostained with the mouse monoclonal SR30A antibody against the viral N protein and FITC-conjugated goat anti mouse IgG.
T124 18916-18972 Sentence denotes Cellular nuclei were counterstained with DAPI (1 mg/ml).
T125 18973-18975 Sentence denotes D.
T126 18976-19035 Sentence denotes PRRSV growth in PAMs transfected with miR-26 family mimics.
T127 19036-19153 Sentence denotes PAMs were transfected with miR-26 family or NC mimics for 24 h and then infected with PRRSV vJX143 at an MOI of 0.01.
T128 19154-19226 Sentence denotes Culture supernatants were collected at the indicated times and titrated.
T129 19227-19229 Sentence denotes E.
T130 19230-19290 Sentence denotes Time-course of miR-26a/26b expression after PRRSV infection.
T131 19291-19445 Sentence denotes PAM cells infected with vJX143 at a MOI of 0.01 were collected at the indicated times and qRT-PCR analysis was performed to detect miR-26a/26b expression.
T132 19446-19627 Sentence denotes Relative miR-26a/b expression refers to the change in miR-26a/b expression levels in PRRSV-infected PAMs relative to mock PAMs. downstream of the firefly luciferase gene (Fig. 4A ).
T133 19628-19770 Sentence denotes If the PRRSV cDNA insert contains a miR-26a target sequence, luciferase reporter expression is expected to be subjected to miR-26a-regulation.
T134 19771-19914 Sentence denotes MiR-26a or NC mimics were co-transfected with the individual reporter vectors into BHK-21 cells, along with an internal control vector pRL-CMV.
T135 19915-19985 Sentence denotes Relative luciferase activities were quantified 24 h post-transfection.
T136 19986-20214 Sentence denotes The relative luciferase activities for different vectors containing various PRRSV cDNA fragments were not significantly different between cells transfected with miR-26a mimic as compared with cells transfected with the NC mimic.
T137 20215-20281 Sentence denotes Thus, miR-26a does not appear to target directly the PRRSV genome.
T138 20282-20455 Sentence denotes We found that over-expression of miR-26a increased IFN-␣/␤ expression during vJX143 infection (MOI = 0.01) at 36 h in PAMs as compared with over-expression of NC (Fig. 5A ).
T139 20456-20542 Sentence denotes The IFN-stimulated genes MX1 and ISG15 were also significantly upregulated (Fig. 5B) .
T140 20543-20672 Sentence denotes Transfecting the miR-26a mimic into PAMs in the absence of PRRSV infection also enhanced type I IFN expression ( Fig. 5A and B) .
T141 20673-20802 Sentence denotes IFN-␣ and IFN-␤ were induced about 1.6-fold in un-infected PAMs, and about 4.9-and 2.8-fold in PRRSV infected PAMs, respectively.
T142 20803-20940 Sentence denotes ISG15 and MX1 were increased about 3.5-and 2.2-fold in un-infected PAMs, and about 3.6-and 2.4-fold in PRRSV infected PAMs, respectively.
T143 20941-21041 Sentence denotes In MARC-145 cells, over-expression of miR-26a up-regulated IFN-␣ and ISG15 more strongly (Fig. 5C ).
T144 21042-21200 Sentence denotes IFN-␣ and ISG15 were induced about 4.4-and 9.8-fold in un-infected MARC-145 cells, and about 9.1-and 20.4-fold in PRRSV infected MARC-145 cells, respectively.
T145 21201-21394 Sentence denotes Transfection of miR-26a inhibitors did not increase the expression of IFN-␣ or ISG15 (Fig. 5C) , confirming that the induction of the innate immune response is specifically mediated by miR-26a.
T146 21396-21575 Sentence denotes There is a growing body of evidence that cellular miRNAs are important regulators of innate and adaptive immune responses and the intricate networks of host-pathogen interactions.
T147 21576-21697 Sentence denotes Herein, we identified miR-26a as an inhibitor of PRRSV replication that does not directly target the PRRSV genome ( Figs.
T148 21698-21708 Sentence denotes 1 and 4) .
T149 21709-21954 Sentence denotes Overexpressed miR-26a reduced PRRSV replication and viral gene expression (Fig. 2) , in not only MARC-145 cells, but also in PAMs (Fig. 3) , the main target cell for PRRSV replication in vivo, confirming the biological relevance of this finding.
T150 21955-22092 Sentence denotes MiR-26a belongs to a broadly conserved miRNA family with perfectly identical sequences among vertebrates (Griffiths-Jones et al., 2006) .
T151 22093-22400 Sentence denotes Previous studies of miR-26a have shown that this miRNA is an important regulator of cell proliferation and differentiation that targets the SMAD1 transcription factor (Ezh2), a suppressor of skeletal muscle cell differentiation (Lu et al., 2011; Luzi et al., 2008; Sander et al., 2008; Zhang et al., 2011) .
T152 22401-22618 Sentence denotes By infecting PAMs with PRRSV strain VR-2332, Liu et al. generated small RNA expression profiles at 12, 24 and 48 h post-infection to identify alterations in miRNA expression associated with PRRSV (Yoo and Liu, 2013) .
T153 22619-22803 Sentence denotes Overall, 40 cellular miRNAs were differentially expressed during at least one time point in PRRSV-infected PAMs. However, in this study, miR-26a was not mentioned (Yoo and Liu, 2013) .
T154 22804-22940 Sentence denotes Contrary to the previous study, we found that the expression of miR-26a was up-regulated about 2-fold at 48 h post-infection (Fig. 3E) .
T155 22941-23087 Sentence denotes One mechanism by which host miRNAs regulate viral replication is the direct targeting of viral sequences (Jopling, 2010; Lecellier et al., 2005) .
T156 23088-23270 Sentence denotes However, PRRSV is a fast-evolving RNA virus (Prieto et al., 2009) and the relatively high mutation rate may limit the application of this kind of RNAi-mediated antiviral therapeutic.
T157 23271-23368 Sentence denotes Cellular miRNAs can also indirectly modulate cellular pathways that perturb the viral life cycle.
T158 23369-23579 Sentence denotes In particular, the activation or enhancement of innate antiviral immune pathways has been suggested to be responsible for the antiviral effect of certain miRNAs (Lecellier et al., 2005; Pedersen et al., 2007) .
T159 23580-23726 Sentence denotes In the current study, the reduction of PRRSV replication by miR-26a did not appear to involve direct targeting of the PRRSV genomic RNA (Fig. 4) .
T160 23727-23899 Sentence denotes Moreover, this reduction occurred in both type 1 and type 2 PRRSV strains ( Fig. 2A) although these two genotypes share only approximately 60% nucleotide sequence identity.
T161 23900-24005 Sentence denotes These data led us to hypothesize that miR-26a might act on a cellular factor to reduce PRRSV replication.
T162 24006-24164 Sentence denotes The results presented here support a link between PRRSV replication and the altered expression of miR-26a in targeting host innate immune responses (Fig. 5) .
T163 24165-24363 Sentence denotes Type I interferons (IFNs) are potent antiviral cytokines whose expression is triggered through recognition of viral components by pattern recognition receptors via a cascade of signaling molecules .
T164 24364-24500 Sentence denotes PAMs are the main target cells for PRRSV infection, and many gene expression studies have explored the immune response of PAMs to PRRSV.
T165 24501-24686 Sentence denotes Such studies have shown that the expression levels of MX1, USP, IFN-␤, IL-10, and TNF-␣ are affected by PRRSV infection (Albina et al., 1998; Luo et al., 2008; Van Reeth et al., 1999) .
T166 24687-24879 Sentence denotes Overall, these analyses suggest that PRRSV subverts host defenses by inhibiting the expression of pro-inflammatory cytokines (Van Reeth et al., 1999) and stimulating weak production of IFN-␣ .
T167 24880-25128 Sentence denotes Our results showed that over-expression of miR-26a was capable of inducing expression of IFN-␣/␤ and the IFN-stimulated genes ISG15 and MX1, which might result in activation of the IFN response and further lead to the inhibition of virus infection.
T168 25129-25300 Sentence denotes The restoration of innate immune responses to produce type I IFNs in PAMs seems to be miRNA specific, because another miRNA (miR-181b) had no such effect (data not shown).
T169 25301-25430 Sentence denotes Thus, it is possible that miR-26a-induced type I IFN expression can overcome PRRSV interference, contributing to viral clearance.
T170 25431-25760 Sentence denotes This mechanism provides a higher genetic barrier to the emergence of viral escape mutants, so the identification and characterization of miR-26a as an inhibitor of PRRSV replication may open new ways to control Fig. 5 . miR-26a increases type I IFN expression during PRRSV infection. qRT-PCR analysis of (A. type I IFN ␣/␤ and B.
T171 25761-25940 Sentence denotes MX1/ISG15) expression in PAMs transfected with NC or miR-26a mimics or left untreated (mock) for 24 h, and then infected with vJX143 for 36 h at an MOI of 0.01, or left untreated.
T172 25941-26053 Sentence denotes Data were normalized to GAPDH expression and are the mean ± standard deviation of three independent experiments.
T173 26054-26230 Sentence denotes C. qRT-PCR analysis of IFN-␣ and ISG15 expression in Marc-145 cells transfected with NC, miR-26a mimics or inhibitors, and then infected with vJX143 for 36 h at an MOI of 0.01.
T174 26231-26274 Sentence denotes Data were normalized to ␤-actin expression.
T175 26275-26444 Sentence denotes Statistical significance was analyzed using t-tests; *, P < 0.05; **, P < 0.01; ***, P < 0.001. future PRRS outbreaks, for which effective control measures remain scant.
T176 26445-26561 Sentence denotes Our results showed that miR-26a also can mediate the activation of IFNs in the absence of PRRSV infection (Fig. 5 ).
T177 26562-26775 Sentence denotes The possible causes may relate to recent studies about a new function of miRNAs, which is independent of their conventional role in post-transcriptional gene regulation Fabbri et al., 2012; Lehmann et al., 2012) .
T178 26776-27013 Sentence denotes MiR-21, miR-29a, and let-7b have dual functions; on one hand, they bind to Argonaute proteins and guide the silencing of target genes, and on the other hand, they act independently of Argonaute proteins by interacting directly with TLRs.
T179 27014-27114 Sentence denotes Although there is no current evidence, miR-26a may also serve as ligands for TLRs and activate IFNs.
T180 27115-27192 Sentence denotes Future studies will be necessary to unravel the diverse functions of miR-26a.
T181 27193-27277 Sentence denotes Overall, we demonstrated that over-expression of miR-26a inhibits PRRSV replication.
T182 27278-27575 Sentence denotes Although clearly defining the target and physiological role of miR-26a remains an unfinished task, our study provided evidence that over-expression of miR-26a enhances IFN-␣/␤ expression during PRRSV infection, suggesting that miR-26a could be used as a potential target for antiviral development.