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Porcine reproductive and respiratory syndrome is an infectious disease that causes serious economic losses to the swine industry worldwide. To better understand the pathogenesis of the porcine reproductive and respiratory syndrome virus (PRRSV), three PRRSV strains with different molecular markers and virulence were used to infect MARC-145 cells. A total of 1804 proteins were identified, and 233 altered proteins and 72 signaling pathways involved in the proteomic profiling of virus-infected MARC-145 cells increased with the virulence of the PRRSV strain. The three types of viral strains shared a common pathway—the electron transport reaction in mitochondria—in the infected-MARC-145 cells. Moreover, the antisense pathway was the most variable of all significant signaling pathways for the highly virulent SX-1 strain, indicating that this unique pathway may be connected to the high virulence of the SX-1 strain. Our study is the first attempt to provide a proteome profile of MARC-145 cells infected with PRRSV strains with different virulence, and these findings will facilitate a deep understanding of the interactions between this virus and its host.

Isolation and identification of diverse porcine reproductive and respiratory syndrome viruses (PRRSVs) play a fundamental role in PRRSV research and disease management. However, PRRSV has a restricted cell tropism for infection. MARC-145 cells are routinely used for North American genotype PRRSV isolation and vaccine production. But MARC-145 cells have some limitations such as low virus yield. CD163 is a cellular receptor that mediates productive infection of PRRSV in various nonpermissive cell lines. In this study, we established a high and stable porcine CD163- (pCD163-) expressing MARC-145 cell line toward increasing its susceptibility to PRRSV infection. Indirect immunofluorescence assay (IFA) and Western blotting assays showed that pCD163 was expressed higher in pCD163-MARC cell line than MARC-145 cells. Furthermore, the ability of pCD163-MARC cell line to propagate PRRSV was significantly increased as compared with MARC-145 cells. Finally, we found that pCD163-MARC cell line had a higher isolation rate of clinical PRRSV samples and propagated live attenuated PRRS vaccine strains more efficiently than MARC-145 cells. This pCD163-MARC cell line will be a valuable tool for propagation and research of PRRSV.

Porcine circovirus type 2 (PCV2), the essential causative agent of postweaning multisystemic wasting syndrome (PMWS), can be divided into distinct genotypes. Thirty-two PCV2 isolates obtained made from pigs in Shandong Province between 2005 and 2013. Complete genome sequences were obtained in three replicates for each virus isolate, and the sequences were submitted to the NCBI database. The ORF1-encoded amino acid sequences had 98.4 %-100 % identity among the 32 isolates, and there were no significant differences among the three potential glycosylation positions: aa 23-25 (NPS), aa 256-258 (NQT) and aa 286-288 (NAT). The amino acid sequences of ORF2 had 88 %-100 % identity among the 32 isolates and the potential glycosylation position in the cap protein, aa 143-145 (NYS), had no variation. Phylogenetic analysis revealed that the PCV2b/1C genetic lineage was prevalent in swine populations in Shandong Province. It also suggested that selection pressure has made the PCV2 isolates more genetically distant from current vaccine strains.

PRRS is one of the most important diseases in swine industry. Current PRRS inactivated vaccine provides only a limited protection and cannot induce sufficient cell-mediated immune responses. In this study, we first found that the mRNA and protein levels of Th1-type cytokines (IFN-γ, IL-12) and Th2-type cytokines (IL-6, IL-10) were significantly increased through TRIF/MyD88-NF-κB signaling pathway when porcine peripheral blood monocyte-derived dendritic cells (MoDCs) were treated with poly (I: C) of TLR3 ligand and imiquimod of TLR7 ligand, along with inactivated PRRSV antigen. Meanwhile, the ability of catching PRRSV antigen was also significantly enhanced. In mice experiment, it was found that the PRRSV-specific T lymphocyte proliferation, the percentages of CD4+, CD8+ T lymphocytes and PRRSV-specific CD3+ T cells producing IFN-γ and IL-4, the levels of Th1- and Th2-type cytokines and the titers of neutralization antibody were significantly enhanced in poly (I: C), imiquimod along with inactivated PRRSV group. Taken together, results of our experiments described for the first time that synergy of TLR3 and 7 ligands could significantly enhance the function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway and be used as adjuvant candidate for the development of novel PRRS inactivated vaccine.

To improve the efficacy of porcine reproductive and respiratory syndrome virus (PRRSV) subunit vaccine, the immunological enhancement effects of four natural adjuvants were compared, including Astragalus extract, Astragalus/Bacillus production, propolis, and Freund's adjuvant. The results showed that titers of IgG and neutralizing antibody against highly pathogenic PRRSV (HP-PRRSV) glycoprotein 5 (GP5) from the groups of Astragalus/Bacillus adjuvant and Freund's adjuvant co-administered with a recombinant HP-PRRSV GP5 subunit were the highest among all the experimental groups. In cellular immunity, as shown in T lymphocyte proliferation level, Astragalus/Bacillus adjuvant and Freund's adjuvant exhibited the most potent immunological enhancement effects. Exceptionally, pigs inoculated with Freund's adjuvant developed severely delayed humoral immune responses within 28 days post inoculation. The adjuvants of Astragalus extract and propolis also exhibited immunological enhancement effects on humoral immune and cell-mediated immune responses, but which were obviously lower than Astragalus/Bacillus adjuvant and Freund's adjuvant. Following challenge with HP-PRRSV, pigs inoculated with Astragalus/Bacillus adjuvant and Freund's adjuvant co-administered with GP5 subunit showed lower viremia, slighter clinical signs, and less pathological lung lesions compared with the groups inoculated using Astragalus extract and propolis. In conclusion, from immune responses and immunological protection against HP-PRRSV, the Astragalus/Bacillus adjuvant demonstrated the most potent immunological enhancement effect on HP-PRRSV GP5 subunit. Astragalus/Bacillus adjuvant showed promise as a candidate immune adjuvant, to more efficiently prevent and control PRRS.

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 the leader proteinase (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 describe for the first time a novel mechanism by which FMDV evolves to inhibit IFN signaling and counteract host innate antiviral responses. IMPORTANCE 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.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit β-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2) and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections.

Porcine reproductive and respiratory syndrome (PRRS) has become one of the most economically important diseases to the global pork industry. The etiological agent is the PRRS virus (PRRSV). In the spring of 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) appeared in China and caused heavy economic losses. Here we report the complete genomic sequence of a novel PRRSV variant with 174 amino acid deletions in the nonstructural protein 2 ( nsp2 ) gene.

Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most economically important diseases to the global pork industry. Current vaccination strategies only provide a limited protective efficacy. In this study, a DNA vaccine, pVAX1(©)-α-γ-GP35, co-expressing GP3 and GP5 of PRRSV with interferon α/γ was constructed, and its immediate and long-lasting protection against highly pathogenic PRRSV (HP-PRRSV) challenge were examined in pigs. For immediate protection, the results showed that pVAX1(©)-α-γ-GP35 could provide partially protective efficacy, which was similar to the pVAX1(©)-α-γ (expressing interferon α/γ). For long-lasting protection, pigs inoculated with pVAX1(©)-α-γ-GP35 developed significantly higher PRRSV-specific antibody response, T cell proliferation, IFN-γ, and IL-4, than those vaccinated with pVAX1(©)-GP35 (expressing GP3 and GP5 of PRRSV). Following homologous challenge with HP-PRRSV strain SD-JN, pigs inoculated with pVAX1(©)-α-γ-GP35 showed almost no clinical signs, no lung lesions, and significantly lower viremia, as compared to those in pVAX1(©)-GP35 group. It indicated that pVAX1(©)-α-γ-GP35 could induce enhanced immune responses and provide both immediate and long-lasting protection against HP-PRRSV challenge in pigs. The DNA vaccine pVAX1(©)-α-γ-GP35 might be an attractive candidate vaccine for the prevention and control of HP-PRRSV infections.

PORCINE reproductive and respiratory syndrome (prrs) is an economically important disease of pigs, defined by severe respiratory disorders in piglets and reproductive failure in gestating sows and gilts. prrs has been reported in most pig-producing countries in the world, with only a few countries

Porcine circovirus (PCV), belonging to Circoviridae family, is a small, non-enveloped, icosahedral virus containing a circular single-stranded DNA genome (Tischer and others 1982). PCV is divided into two genotypes, PCV type 1 (PCV1) and PCV type 2 (PCV2). PCV1 is a contaminant of PK-15 cells that is non-pathogenic and exists in pigs (Tischer and others 1986, Puvanendiran and others 2011). PCV2 is associated with postweaning multisystemic wasting syndrome (PMWS), which has caused severe losses in the global swine industry in recent decades. Many PK-15 cell lines are contaminated with PCV1, which confers difficulty in using PK-15 cell line to isolate PCV2 for further research. Accordingly, this study aimed to establish a multiplex real-time PCR assay to differentiate and rapidly detect PCV1 and PCV2 in laboratory and clinical samples. PCV2 strain SD2 (GenBank Accession Number DQ478947) was originally isolated from a pig that clinically manifested PMWS (Zhou and others 2009). In the present study, PCV1 was cultured in our laboratory using PK-15 cells. PCV1 and PCV2 viral DNA were extracted by the proteinase K method according to the instructions(Sambrook and others 2001). Primer P15ʹ-GAACCGCGGGCTGGCTGA ACTTTTGAAAGT-3ʹ and …

We report here the genome sequence of a recombinant porcine circovirus type 2 strain SD-3, isolated from a commercial swine farm with an outbreak of postweaning multisystemic wasting syndrome (PMWS) in pigs in Shandong Province of China. The complete circular genome of this isolate is 1,767 nucleotides in length. This recombinant isolate has the ORF1 regions from PCV2a viruses and ORF2 regions from PCV2b. The findings will help us to understand the molecular evolution of porcine circovirus type 2 and the relationship between porcine circovirus type 2 and disease.