[Show abstract][Hide abstract] ABSTRACT: Background
Encephalomyocarditis virus (EMCV) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in China. EMCV VP1 protein was one of the most important structural proteins and played an important role in the protective immunity. In this study, 10 monoclonal antibodies (McAbs) against EMCV VP1 were screened and identified.ResultsEpitope mapping results indicated that McAbs (6E11, 7A7, 7C9) specifically recognized the linear epitopes V(2)ENAEK(7), McAbs (1D1, 2A2, 5A1, 5A11, 5G1) recognized the epitope F(19)VAQPVY(25), and McAbs 1G8 and 3A9 recognized P(42)IGAFTVK(49). Protein sequence alignment of VP1 with 16 EMCV isolates indicated that the epitope F(19)VAQPVY(25) was conserved in all the reference strains. The epitopes P(42)IGAFTVK(49) and V(2)ENAEK(7) only had 1 or 2 variable amino acid among the reference strains. The 3D model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle.Conclusions
In this study, ten McAbs against EMCV VP1 were developed and three B-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in VP1. All the results herein will promote the future investigations into the function of VP1 of EMCV and development of diagnostic methods of EMCV.
[Show abstract][Hide abstract] ABSTRACT: Encephalomyocarditis virus (EMCV) infects animals of various species and causes a variety of clinical symptoms. In this study, an infectious full-length cDNA clone was constructed, and the characteristics of the rescued virus were investigated in vitro and in vivo. Our data demonstrated that the growth kinetics in vitro and plaque morphology of the rescued EMCV rNJ08 strain were similar to those of the parental strain. Although rNJ08 infected BALB/c mice, none of the mice died during the observation period of 14 days post-inoculation. The availability of the infectious cDNA clone provides a genetic platform for studying gene function and for the rational design of vaccines.
Archives of Virology 11/2014; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adhesion protein MSG1 mediating adherence to porcine erythrocytes in Mycoplasma suis (M. suis) invasion has been identified previously. In order to determine the host membrane proteins that interact with MSG1, recombinant His-tagged MSG1 (rMSG1) was used to screen for interacting proteins in the protein extracts of porcine erythrocyte membrane. Potential rMSG1-interacting proteins were initially identified as band 3 and β-actin with molecular weight of 46 and 45 kDa, respectively. Immune fluorescence results showed that rMSG1 can specifically bind with the β-actin of HeLa, BHK-21, and HEK-293A cells, respectively. RNA interference assays further demonstrated that the interaction between β-actin and rMSG1 on HeLa cells was specific and dose dependent. Confocal microscopy showed that both rMSG1 and M. suis can partially co-localize with β-actin on the surface of porcine erythrocytes. Pull-down assays showed that rMSG1 can directly interact with β-actin. Our study is the first to report the interaction of MSG1 with β-actin, which will be of help to understand the pathogenesis of M. suis and develop a cultivation system.
Archives of Microbiology 10/2014; · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A highly virulent and antigenic variant of pseudorabies virus (PRV) broke out in China at the end of 2011 and caused great economic loss in the pig industry. In this study, an infectious bacterial artificial chromosome (BAC) clone containing the full-length genome of the emerged variant PRV ZJ01 strain was generated. The BAC-derived viruses, vZJ01-GFPΔgE/gI (gE/gI deleted strain, and exhibiting green autofluorescence), vZJ01ΔgE/gI (gE/gI deleted strain), and vZJ01gE/gI-R (gE/gI revertant strain), showed similar in vitro growth to their parent strain. In pigs, inactivated vZJ01ΔgE/gI vaccine generated significantly high levels of neutralizing antibodies against ZJ01 compared with Bartha-K61 live vaccine (p<0.05). After fatal ZJ01 challenge, all five animals in the inactivated vZJ01ΔgE/gI vaccine group survived without exhibiting any clinical sings, but two of five animals exhibited central nervous signs in the Bartha-K61 group. Meanwhile, all the non-vaccinated control animals died at 7 days post-challenge. This indicates that the inactivated vZJ01ΔgE/gI vaccine is a promising vaccine candidate for controlling the variant strains of PRV now circulating in China.
[Show abstract][Hide abstract] ABSTRACT: Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals, but goat infection has been rarely reported. Starting in Aug 2013, goats in the Jiangsu and Anhui provinces of eastern China suffered severe respiratory diseases. In order to identify the causative agent, numerous related pathogens were tested with RT-PCR or PCR. A unique PIV3 strain was detected in most of the clinical nasal swabs or serum samples. The virus was isolated on MDBK cells and characterized by RT-PCR, nucleotide sequence analysis and hemagglutination test. The entire M and F gene coding regions, HN, 5′-UTR-N and L gene fragments were amplified using pairs of degenerate primers. Nucleotide, amino acid sequence alignments and phylogenetic analyses based on these genes indicated that the goat-derived PIV3 strain was distinct from previously reported BPIV3 genotypes and HPIV3 strains. The novel isolate, named JS2013, might be a potentially new member of the respirovirus genus. Goats were experimentally infected with JS2013 culture. The virus-inoculated goats displayed coughing and nasal discharges that were related to respiratory diseases. Viremia and virus shedding were detected during 4-10 days post inoculation (dpi). Virus-specific HI antibodies became positive from 14 dpi. This is the first report of the detection of PIV3 from Chinese goat herds and genetic and pathogenetic characterization of the novel goat-derived PIV3.
[Show abstract][Hide abstract] ABSTRACT: Here, we used reverse transcription-PCR (RT-PCR) and western blot to detect protease-activated receptor (PAR) 1, PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma, and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer. The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed. By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells (HEEpiC), we found that among the 28 cases of esophageal squamous cell carcinoma, PAR1 (60%) and PAR2 (71%) were elevated in 17 and 20 cases, respectively, and PAR4 (68%) expression was lowered in 19 cases. Whereas, in human esophageal squamous cells (TE-1 and TE-10), PAR1 and PAR2 expression was increased but PAR4 was decreased. Combined with clinical data, the expression of PAR1 in poorly differentiated (P=0.016) and middle and lower parts of the esophagus (P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower (P=0.049). Regarding TE-1 and TE-10 protein expression, we found that in randomized esophageal carcinoma, PAR1 (P=0.027) and PAR2 (P=0.039) expressions were increased, but lowered for PAR4 (P=0.0001). In HEEpiC, TE-1, TE-10, esophageal and normal esophagus tissue samples (case No. 7), the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%, 95.2%, 83.8%, 62.6% and 48.2%, respectively. Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased. Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.
[Show abstract][Hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of post-weaning multisystemic wasting syndrome, which has spread worldwide. To discover cellular protein responses of PK-15 cells to PCV2 infection, two-dimensional liquid chromatography-tandem mass spectrometry (MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the proteins that were differentially expressed in PK-15 from the PCV2-infected group compared to the uninfected control group. A total of 196 cellular proteins in PK-15 that were significantly altered at different time periods post-infection were identified. These differentially expressed proteins were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc. and their interactions. Moreover, some of these proteins were further confirmed by Western blot. The high number of differentially expressed proteins identified should be very useful in elucidating the mechanism of replication and pathogenesis of PCV2 in the future.
[Show abstract][Hide abstract] ABSTRACT: Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and, to a fewer occurrences, human erysipeloid. It is ubiquitous in nature and commensal in diverse species of animals, wild or domestic, from mammals and birds to reptiles and fish. Mechanisms of its virulence and pathogenicity are poorly understood.
[Show abstract][Hide abstract] ABSTRACT: Since October 2010, porcine diarrhea outbreaks have occurred widely, resulting in major losses in suckling piglets in China. A variant porcine epidemic diarrhea virus (PEDV), characterized by base deletion and insertion in the S gene, compared to classical PEDV CV777, was shown to be responsible for this outbreak. In this study, a multiplex TaqMan probe-based real-time PCR was developed for detecting PEDV and differentiating the variant from classical PEDV, by using two sets of primers and probes based on the S gene of PEDV. The limits of detection of both variant and classical PEDV were 5×10(2) DNA copies. Specificity was determined using eight other viral pathogens of swine. Reproducibility was evaluated using standard dilutions, with coefficients of variation <1.4%. Standard dilutions included in each test allowed quantification of the amount of PEDV. Among 42 intestinal samples from pigs with severe watery diarrhea, 36 variant PEDV and three classical PEDV samples were detected, with viral loads of 10(2)-10(8) copies/μl and 10(3)-10(5) copies/μl, respectively, which suggested that the variant PEDV was prevalent in China. The multiplex TaqMan probe-based real-time PCR should be a useful tool for quantifying viral load, detecting PEDV, and differentiating variant from classical PEDV.
Journal of Virological Methods 06/2014; · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) has been identified as the etiologic agent which causing postweaning multisystemic wasting syndrome in swine farms in the world. Some quantitative proteomic studies showed that many proteins significantly changed in PCV2-infected cells. To explore the role of cellular chaperones during PCV2 infection, cytoprotective chaperone Hsp27 was analyzed in PCV2-infected PK-15 cells in this study. The results showed that Hsp27 could up-regulate and accumulate in phosphorylated forms in the nuclear zone during PCV2 replication. Suppression of Hsp27 phosphorylation with specific chemical inhibitors or downregulation of all forms of Hsp27 via RNA interference significantly reduced the virus replication. Meanwhile, over-expression of Hsp27 enhanced PCV2 genome replication and virion production. It indicated that Hsp27 was required for PCV2 production in PK-15 cells culture. It should be helpful for understanding the mechanism of replication and pathogenesis of PCV2 and development of novel antiviral therapies in the future.
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) causes substantial economic losses to many swine-producing regions. In this study, PRRSV strain NT0801-F80 was derived from its parental isolate NT0801 by 80 passages in Marc-145 cells. Experimental infection of piglets clearly demonstrated that strain NT0801-F80 is less virulent than NT0801. However, whole genome sequencing showed that the genomes of the parental and attenuated strains are highly conserved compared with those of four other pairs of virulent parental/attenuated vaccine strains (VR2332 and RespPRRS MLV, JA142 and Ingelvac(®) ATP MLV, CH-1a and CH-1R, and JXA1 and JXAR). The attenuated strain NT0801-F80 has only 21 nucleotide changes, producing only 14 amino acid changes in NSP2, GP2, GP3, and GP5, compared with those aa sequences of the virulent parental strain. These mutated aa in the attenuated virus may be involved in virulence. These data provide valuable information on the attenuation mechanism of PRRSV that should be useful in future research.
[Show abstract][Hide abstract] ABSTRACT: Trefoil factor 2 (TFF2) plays a protective role in gastric mucosa and may be involved in the progression of gastric cancer, but the detailed functions and underlying molecular mechanisms are not clear. The present study used a combination of clinical observations and molecular methods to investigate the correlation between abnormal expression of TFF2 and gastric cancer progression. TFF2 expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), and western blot and immunohistochemistry analyses. TFF2 methylation levels were analyzed by genomic bisulfite sequencing method. The results showed that TFF2 mRNA and protein expression were decreased in gastric cancer tissues compared with the matched non-cancerous mucosa, and the decreased level was associated with the differentiation and invasion of gastric cancer. Moreover, the average TFF2 methylation level of CpG sites in the promoter region was 70.4% in three gastric cancer tissues, while the level in associated non-neoplastic tissues was 41.0%. Furthermore, the promoter hypermethylation of TFF2 was also found in gastric cancer cell lines, AGS and N87, and gene expression was significantly increased following treatment with a demethylating agent, 5-Aza-2'-deoxycytidine. In conclusion, TFF2 expression was markedly decreased in gastric cancer and promoter hypermethylation was found to regulate the downregulation of TFF2. TFF2 has been suggested as a tumor suppressor in gastric carcinogenesis and metastasis.
[Show abstract][Hide abstract] ABSTRACT: Foot-and-mouth disease virus (FMDV) causes a highly contagious, debilitating disease in cloven-hoofed animals with devastating economic consequences. To survive in the host, FMDV has evolved to antagonize the host type I interferon (IFN) response. Previous studies have reported that L(pro) and 3C(pro) of FMDV are involved in the inhibition of type I IFN production. However, whether the proteins of FMDV can inhibit type I IFN signaling is less well understood. In this study, we first found that 3C(pro) of FMDV functioned to interfere with the JAK-STAT signaling pathway. Expression of 3C(pro) significantly reduced the transcript levels of IFN-stimulated genes (ISGs) and IFN-stimulated response element (ISRE) promoter activity. The protein level, tyrosine phosphorylation of STAT1 and STAT2 and their heterodimerization were not affected. However, the nuclear translocation of STAT1/STAT2 was blocked by the 3C(pro) protein. Further mechanistic studies demonstrated that 3C(pro) induced proteasome- and caspase-independent protein degradation of karyopherin α1 (KPNA1), the nuclear localization signal receptor for tyrosine-phosphorylated STAT1, but not karyopherin α2, α3, or α4. Finally, we showed that the protease activity of 3C(pro) contributed to the degradation of KPNA1 and thus blocked STAT1/STAT2 nuclear translocation. Taken together, results of our experiments described for the first time a novel mechanism by which FMDV evolves to inhibit IFN signaling and counteract host innate antiviral responses.
We show that 3C(pro) of FMDV antagonizes the JAK-STAT signaling pathway by blocking STAT1/STAT2 nuclear translocation. Furthermore, 3C(pro) induces KPNA1 degradation, which is independent of proteasome and caspase pathways. The protease activity of 3C(pro) contributes to the degradation of KPNA1 and governs the ability of 3C(pro) to inhibit the JAK-STAT signaling pathway. This study uncovers a novel mechanism evolved by FMDV to antagonize host innate immune responses.
[Show abstract][Hide abstract] ABSTRACT: PRRSV strain SH1211 was isolated from the lung tissue of a piglet on a large-scale pig farm with approximately 30% morbidity and 50% mortality in mid-eastern China in 2012. The full-length genome of SH1211 was 15 313 nt in size, excluding the polyadenylated sequences, and shared 94.9% nucleotide sequence identity with the HP-PRRSV strain, JXA1. The GP2 and GP5 proteins of SH1211 shared only 91.5% and 85.1% amino acid sequence identities with those of the JXA1, respectively. A deletion at amino acid positions 68 and 69 was identified in the GP3 protein of SH1211, compared with the GP3 of Type-2 PRRSV isolates. A phylogenetic tree based on the nucleotide sequence of the complete genome showed that SH1211 is the most closely related to other HP-PRRSV strains isolated in China. However, phylogenetic analysis based on the GP2 and GP5 proteins showed that SH1211 is the most closely related to the QYYZ strain. A recombination analysis indicated that SH1211 might have been generated through recombination events between the JXA1 and QYYZ in which the GP2 and GP5 coding sequences were exchanged. Thus, SH1211 is a novel PRRSV strain with significant variation. Our analysis of SH1211 provides insight into the role of genetic variation in the antigenicity of PRRSVs in China.
BioMed research international. 01/2014; 2014:306130.
[Show abstract][Hide abstract] ABSTRACT: The Hsp70 chaperone plays a central role in multiple processes within cells. Porcine circovirus type 2 (PCV2) is the essential causal agent of post-weaning multisystemic wasting syndrome (PMWS), which has spread worldwide. But the mechanism of PCV2 replication remains poorly understood. In this study, we firstly found the positive effect of heat stress on the replication of PCV2 in the continuous porcine monocytic cell line 3D4/31. Downregulation of Hsp70 by the specific chaperone inhibitor Quercetin or RNA interference and upregulation of Hsp70 by expression from a recombinant adenovirus showed that Hsp70 enhanced PCV2 genome replication and virion production. A specific interaction between Hsp70 and PCV2 Cap was confirmed by colocalization by confocal microscopy and co-immunoprecipitation. Furthermore, the NF-κB pathway was activated and caspase-3 activity was reduced when Hsp70 was overexpressed in PCV2-infected 3D4/31 cells. These data suggested that Hsp70 positively regulated PCV2 replication, which being helpful for understanding the molecular mechanism of PCV2 infection.
[Show abstract][Hide abstract] ABSTRACT: Haemophilus parasuis is the etiological agent of Glässer's disease in pigs and 15 standard serovars were identified. The widespread disease causes great economic loss in the swine industry worldwide. Aiming to investigate the differences in genome composition and functions among various strains, a highly virulent strain ZJ0906 of H. parasuis serovar 12 from China was analyzed and compared with serovar 5 SH0165. Strain ZJ0906 genome is 2,324,740 base pairs with 40.06% genomic GC content. It contains 2,484 open reading frames (ORF) predicted by Glimmer 3.02, of which 2,352 (∼94.7%) were annotated by NCBI nr blast, 1,745 by COG database and 1,829 by KEGG database. 109 potential virulence factors were annotated in strain ZJ0906 and 3 of which are potentially related to antibiotic resistance. Strain ZJ0906 genome is ∼55 kilobases longer than SH0165 genome, with an extra 211 predicted ORFs. VFDB, ARDB, and PAIDB blast searches showed that ZJ0906 and SH0165 shared a nearly identical panel of potential virulence factors, drug resistant genes and four PAI-like regions which showed high homology to Enterococcus, Escherichia and Salmonella. Synteny analysis showed that gene rearrangements are frequent between the two strains, which may lead to variations in pathogenicity and cross-protection among serovars. KEGG pathway analyses showed strain ZJ0906 shared similar metabolic pathways to strain SH0165. Molecular identification of these genomic elements and potential virulence factors pave the way to the better understanding of mechanisms underlying metabolic capabilities and pathogenicity of H. parasuis and prospective vaccine targets besides the widely used method of inactivated bacteria.
PLoS ONE 09/2013; 8(9):e68350. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of protease-activated receptors (PARs) in lung tumors is controversial. Although PAR4 is preferentially expressed in human lung tissues, its possible significance in lung cancer has not been defined. The studies reported herein used a combination of clinical observations and molecular methods. Surgically resected lung adenocarcinomas and associated adjacent normal lung tissues were collected and BEAS-2B and NCI-H157 cell lines were grown in tissue culture. PAR4 expression was evaluated by RT-PCR, RT-qPCR, Western blotting and immunohistochemistry analysis. The results showed that PAR4 mRNA expression was generally decreased in lung adenocarcinoma tissues as compared with matched noncancerous tissues (67.7%) and was associated with poor differentiation (p=0.017) and metastasis (p=0.04). Western blotting and immunohistochemical analysis also showed that PAR4 protein levels were mostly decreased in lung adenocarcinoma tissues (61.3%), and were also associated with poor differentiation (p=0.035) and clinical stage (p=0.027). Moreover, PAR4 expression was decreased in NCI-H157 cells as compared with BEAS-2B cells. In conclusion, PAR4 expression is significantly decreased in lung adenocarcinoma, and down-regulation of PAR4 is associated with a more clinically aggressive phenotype. PAR4 may acts as a tumor suppressor in lung adenocarcinoma.
Asian Pacific journal of cancer prevention: APJCP 06/2013; 14(6):3793-8. · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of heavy economic losses in many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. Interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) has been characterized as the product of a novel antiviral gene and as an important modulator in innate immunity. However, the role of IFIT3 in PRRSV infection is scarcely understood. In this study, polyinosinic-polycytidylic acid (poly (I:C)) inhibited PRRSV replication in MARC-145 cells, following the appearance of increased IFIT3. Overexpression of porcine IFIT3 resulted in a decrease of PRRSV. Knockdown of IFIT3 in MARC-145 cells increased PRRSV replication and impaired the antiviral activity mediated by poly(I:C). Moreover, in the presence or absence of IFIT3, poly(I:C)-induced IFN-β promoter activity was significantly boosted or crippled, respectively. IFIT3, TBK1 and phosphorylation of IRF3 were activated in poly(I:C)-transfected MARC-145 cells. It demonstrated that IFIT3 plays an important role in IFN-β induction in MARC-145 cells, and, when activated, it can inhibit PRRSV replication.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is largely responsible for heavy economic losses in the swine industry worldwide because of its high mutation rate and subsequent emergence of virulent strains. However, the immunological and pathological responses of pigs to PRRSV strains with different virulence have not been completely elucidated. METHODS: Twenty-four piglets were divided into 4 groups (n = 6 each) and inoculated with highly pathogenic PRRSV isolate BB0907 (HP), low pathogenic PRRSV NT0801 (LP), LP derivative strain NT0801-F70 (LP-der), and DMEM medium (control), respectively. The changes in TLR2, 3, 7, and 8 gene expression and TNF-alpha, IL-1beta, IL-6, IFN-gamma, and IL-10 secretion were evaluated using real-time PCR and ELISA at 6, 9, and 15 days post inoculation (d.p.i.). The cytokine levels were evaluated in the supernatants of porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) following stimulation with LTA, poly(I:C), CL097, and PRRSV individually. RESULTS: HP caused more severe clinical signs and pathological lesions in swine than LP and LP-der had almost no virulence compared with LP. The serum levels of IL-1beta, IL-6, TNF-alpha, and IFN-gamma were increased in HP-infected piglets, which were greater than in those infected with LP or LP-der. The mRNA levels of TLR3, 7, and 8 were significantly up-regulated in PAMs in HP-infected pigs compared to those in groups LP and LP-der. Furthermore, TNF-alpha and IL-1beta secretion in PAMs from group LP was statistically greater than those from the control group after stimulation with either poly(I:C) or CL097. Meanwhile, TNF-alpha, IL-1beta, and IL-6 levels in CL097-stimulated PBMCs from HP-infected pigs were markedly higher than those from the LP- and LP-der-infected groups. CONCLUSIONS: We found that HP was a stronger inducer of TLR 3, 7, and 8 expression and IL-1beta, IL-6, TNF-alpha, and IFN-gamma production compared to LP and LP-der. HP enhanced production of TNF-alpha, IL-1beta, and IL-6 in PBMCs following CL097-stimulation more than LP and LP-der, whereas LP enhanced the secretion of TNF-alpha and IL-1beta in poly(I:C)- and CL097-stimulated PAMs. Our data regarding cellular reactivity to different isolates should be useful in the development of more efficacious vaccines.
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the heavy economic losses in pig industry in the world. Current vaccination strategies provide only a limited protection. Previous studies have demonstrated the immunostimulatory adjuvant effects of Toll-like receptor (TLR) ligands, synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], lipoteichoic acid (LTA) and CL097 in humans and animals. To study the effects of these compounds on the induction of PRRSV-specific immune responses, mice were immunized subcutaneously with killed virus (KV) antigens incorporating pairs of TLR ligands. It was found that poly(I:C) and CL097 induced the higher IFN-γ levels and PRRSV-specific antibodies, comparing with that KV with or without LTA in mice. Piglets were vaccinated with the KV mixed with poly(I:C) or CL097 and the protective effects of the vaccination were evaluated. The results showed that PRRSV-specific antibodies and T lymphocyte proliferation levels in KV mixed with poly(I:C) or CL097 groups were higher than those in KV group. Following challenge with PRRSV, pigs inoculated with KV mixed with poly(I:C) or CL097 showed lighter clinical signs, lower viremia and less pathological lesion of lungs, as compared to those of KV and challenge control groups. It indicated that co-administration of poly(I:C) and CL097 with killed PRRSV vaccine conferred higher protection against PRRSV challenge. TLR3 and TLR7/8 ligands are promising adjuvant candidates for the development of novel vaccines against PRRSV.