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Porcine reproductive and respiratory syndrome virus (PRRSV) is an aetiological agent that can lead to reproductive failure and respiratory diseases of pigs. The replication and pathogenesis of PRRSV, although poorly understood, has been associated with the host factors. DDX18 is a member of DEAD-box RNA helicases (DDXs) family which were proved to participate in viral replication. Previously, we found the DDX18 interacts with both nsp2 and nsp10 of PRRSV by Co-Immunoprecipitation (Co-IP). In the present study, we demonstrated the interactions of DDX18 with nsp2 and nsp10, and located DDX18's binding regions as the N-terminus of nsp2 and both the N-terminus and C-terminus of nsp10. The expression of the nsp2 or nsp10 in MARC-145 cells and primary PAM cells redistributed DDX18 from the nucleus to the cytoplasm, and promoted the viral replication, but silencing of the DDX18 gene in MARC-145 cells down-regulated the replication of PRRSV. These findings proved that the cellular RNA helicase DDX18 plays a role in the replication of PRRSV, and provides insights into the understanding of PRRSV replication.

Pseudorabies virus (PRV) is the causative agent of pseudorabies (PR) which causes large economic losses for Chinese swine industry since breaking out in late 2011. As a member of herpesviruses, PRV is able to escape the host immune elimination and establish latency, resulting in persistent infection. Here, we report that a currently prevalent Chinese PRV variant down-regulated swine leukocyte antigen class I (SLA-I) molecules on the surface of PK-15 cells and targeted them for degradation through lysosome pathway. Viral pUL56 protein, independent of other viral proteins, was associated with this function by inducing degradation of cellular SLA-I heavy chain (HC) in a manner that was dependent on the lysosome machinery. In addition, pUL56 interacted with SLA-I HC and increased its ubiquitination. Further studies demonstrated that the late domains (PPXY motifs) of pUL56 were required for the ubiquitination and degradation of SLA-I HC by pUL56. Together, our findings reveal the mechanisms by which PRV interferes with cytotoxic T lymphocyte (CTL) responses and provide novel insights into the roles of PRV pUL56.

Highlights • The cellular DDX5 was confirmed to interact with the Nsp9 of PRRSV. • Silencing the DDX5 gene impacted the replication of PRRSV in MARC-145 cells. • The over-expression of DDX5 slightly enhanced the replication of PRRSV. • DDX5 positively regulated the replication of PRRSV via its interaction with Nsp9., The nonstructural protein 9 (Nsp9) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to play important roles in viral replication. The present study first screened that the DEAD-box RNA helicase 5 (DDX5) was a cellular protein interacting with the Nsp9 of PRRSV by a yeast two-hybrid method in a pulmonary alveolar macrophages (PAMs) cDNA library. Next, DDX5 was shown to interact with viral Nsp9 in the co-transfected HEK293 cells with the DDX5- and Nsp9-expressing plasmids, and the interaction between endogenous DDX5 and Nsp9 was also confirmed in MARC-145 cells infected with the Nsp9-expressing lentiviruses. Then, the interacting domains between DDX5 and Nsp9 were determined to be the DEXDc and HELICc domains in DDX5 and the RdRp domain in Nsp9, respectively. Moreover, in the HEK293 cells, MARC-145 cells and PAM cell lines co-transfected with the DDX5- and Nsp9-expressing plasmids, Nsp9 was shown to co-localize with DDX5 in the cytoplasm with a perinuclear pattern, and meanwhile in PRRSV-infected MARC-145 cells and PAMs, endogenous DDX5 was also found to co-localize with Nsp9. Finally, silencing the DDX5 gene in MARC-145 cells significantly impacted the replication of PRRSV, and while the over-expression of DDX5 could slightly enhance viral replication. These findings indicate that DDX5 positively regulates the replication of PRRSV via its interaction with viral Nsp9 in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to modulate the innate immune response of host. However, little is known about the effects of PRRSV infection on porcine intrinsic virus-restriction factors. This study presents the first demonstration that the nonstructural protein 3 (Nsp3) or envelope (E) protein of PRRSV interacted with porcine intrinsic virus-restriction factor IFITM1 or Tetherin. Next, in PRRSV-infected MARC-145 cells and the transfected cells with the IFITM1- or Tetherin-expressing plasmid, IFITM1 was shown to be mainly distributed perinuclear, and Tetherin was proposed to be partially removed away from cell surface. Moreover, the overexpression of IFITM1 and Tetherin were shown to have no obvious effects on the replication of PRRSV in MARC-145 cells. The Nsp3 of PRRSV was demonstrated to induce the proteasome-dependent degradation of IFITM1 upon PRRSV infection. These findings suggest that PRRSV might counteract the antiviral functions of IFITM1 and Tetherin by the interaction of the Nsp3 with IFITM1 and the E protein with Tetherin, providing a novel clue for exploring possible mechanisms associated with the evasion of PRRSV from immune recognition of host.

Non-structural protein1β (Nsp1β) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be involved in suppressing the host innate immune response and mediating viral subgenomic mRNA transcription. In the present study, we have analyzed the interaction of Nsp1β of Chinese highly pathogenic PRRSV (HP-PRRSV) with cellular poly(C)-binding 2 (PCBP2) by means of the yeast two-hybrid screening in a pulmonary alveolar macrophages (PAMs) cDNA library and co-immunoprecipitation (Co-IP) assay. Our results indicated that the Nsp1β of the HP-PRRSV is able to bind and interact with cellular PCBP2 strongly in both the infected cells and plasmid transfected cells. Their minimal binding regions were identified to be the residues 85-203 aa (PCPβ and CTE domains) for the Nsp1β and the residues 96-168 aa (KH2 domain) for PCBP2, respectively. Next, we used confocal immunofluorescence analysis and discovered that, during PRRSV infection in MARC-145 cells and/or plasmid-transfected cells, the Nsp1β and PCBP2 mainly colocalized in the cytoplasm and perinuclear pattern. Moreover, the siRNA-mediated silencing of PCBP2 gene in the MARC-145 cells resulted in significant reduction of the virus titer in supernatants as well as viral proteins, while no significant effects on the expression of the type I interferon α and interferon β, suggesting that the interaction of the Nsp1β with cellular PCBP2 is beneficial to Chinese HP-PRRSV replication in MARC-145 cells.

Apoptosis of host cells plays a critical role in pathogenesis of virus infection. MAPK kinases especially stress-activated protein kinases c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 are often involved in virus-mediated apoptosis. It has been shown that porcine reproductive and respiratory syndrome virus (PRRSV) infection resulted in apoptosis of the host cells both in vitro and in vivo. The current investigation was initiated to determine whether stress-activated protein kinases JNK and p38 play a role in apoptosis induction by PRRSV infection. We examined phosphorylation of JNK and p38, and found that JNK but not p38 was activated in response to PRRSV infection. We then examined effects of this kinase on apoptosis induction and virus replication by using specific inhibitor. We found that JNK inhibition by its inhibitor SP600125 led to the abolishment of PRRSV-mediated apoptosis, but did not suppress virus replication. Further studies demonstrated that ROS generation was involved in JNK activation, and Bcl-2 family anti-apoptotic proteins Mcl-1 and Bcl-xl were downstream targets of JNK to mediate apoptosis. We conclude that activation of JNK signaling pathway is essential for PRRSV-mediated apoptosis but not for virus replication.

Porcine circovirus type 2 (PCV2) has been recognized as an important vial pathogen for global swine industry. The virus is associated with postweaning multisystemic wasting syndrome (PMWS) and other syndrome diseases collectively known as porcine circovirus-associated disease (PCVAD). PCV2 infection and PMWS have caused great impact on the pig production in China during the past 10 years. This review will involve in the history of PCVAD, serological epidemiology of PCV2 infection, and clinical aspect of PCVAD, and the genomic characteristics, variation and genotyping of PCV2, as well as control situation of PCVAD in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is characteristic of genetically extensive variation. In this study, five SPF pigs were co-infected with two strains of PRRSV (JXwn06-81c and HB-1/3.9c), and 352 viruses were cloned by plaque assay from the sera of the infected pigs on days 3, 5, 7, 10, 14, 21 postinfection (pi), and the recombinant events between the two viruses were systematically investigated by sequencing the ORF5, ORF3 and Nsp2 genes of each cloned virus and using SimPlot and Genetic Algorithm for Recombination Detection (GARD) analysis. Totally, 133 recombinant viruses out of the plaque viruses were acquired from four of five infected pigs during days 7–21 pi upon co-infection with JXwn06-81c and HB-1/3.9c. The intragenic recombination and intergenic fragment exchange of the ORF5, ORF3 and Nsp2 genes between the two viruses exhibited different patterns, and the recombination for ORF5 gene and Nsp2 occurred as early as on day 7 pi. The recombination between the ORF5, ORF3 or Nsp2 gene resulted in the generation of chimeric GP5, GP3 or Nsp2. Of the three genes, Nsp2 gene exhibited more complicated recombination situation. Meanwhile, the putative recombination breakpoints and hotspots for the three genes were analyzed. Our findings not only provide valuable evidences for understanding that recombination is an important genetic mechanism contributing to the variation and evolution of PRRSV, but also suggest that extensive use of attenuated vaccine of PRRSV undoubtedly contributes to the increased diversity of PRRSV in field.

The emergence of type 1 porcine reproductive and respiratory syndrome virus (PRRSV) has been noticed recently in China. In the present study, the complete genomic characterization of a strain of type 1 PRRSV (designated GZ11-G1) was described and its pathogenicity for piglets was analyzed. The results showed that the complete genome of GZ11-G1 with a size of 15,094 nt, excluding the poly (A) tails, shared 80.2-96.3% identity with the representative strains of type 1 PRRSV, and in particular, it had highest homology (96.3%) with Amervac PRRS, a live vaccine virus of type 1 PRRSV and SHE, a rescued virus from an infectious clone of Amervac PRRS virus. Compared with the vaccine virus, the nonstructural and structural proteins of GZ11-G1 displayed extensive amino acid variations except for its ORF5a. GZ11-G1 was clustered with the strains of type 1 PRRSV including Cresa3267, Cresa3249, Cresa3256, Olot/91, 9625/2012, ESP-1991-Olot91 and Amervac PRRS vaccine virus by further phylogenetic analysis. Moreover, GZ11-G1 was shown to cause fever, higher viremia and lung and lymph node lesions in piglets. Our findings indicate that GZ11-G1 is genetically related to type 1 PRRSV strains within the cluster formed by Cresa3267, Cresa3249, Cresa3256, Olot/91, 9625/2012, ESP-1991-Olot91 and Amervac PRRS vaccine virus, and it is a pathogenic for piglets. This study aids in understanding the genetic variation and evolution of type 1 PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for swine industry. The non-structural protein 2 (Nsp2) is considered to be one of the immunogenic proteins of PRRSV. In this study, the B-cell epitopes of the Nsp2 protein of a North American type Chinese strain PRRSV BJ-4 were identified on a prokaryotic expressed Nsp2 fragment (73-567aa). A total of six monoclonal antibodies (mAbs) recognizing different epitopes on the expressed protein were prepared. All six mAbs exhibited immunoreactivity with the denatured Nsp2 protein in Western blotting and produced strong perinuclear staining in PRRSV infected MARC-145 cells in an immunofluorescence assay. Pepscan analysis revealed six distinct linear epitopes for the six mAbs, respectively, and of which four were identified to be novel linear Nsp2 B-cell epitopes: T(73)LPERVRPPDDWAT(86), D(385)ELKDQMEED(394), P(452)VPAPRRKVGSDCGS(466), and P(467)VSLGGDVPNS(477). All of the six mAb specific peptides could be recognized by porcine PRRSV antiserum, indicating that the epitopes involving these synthetic peptides were immunogenic and immunodominant during PRRSV infection in pigs. Our results provided valuable information for developing novel PRRSV vaccines using the Nsp2 epitopes as potential serological markers.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be one of the most important pathogens severely affecting global swine industry. An increasingly number of studies have paid much attention to the diverse roles of its nonstructural proteins (Nsps) in regulating the innate immune response of host upon PRRSV infection. In the present study, we first discovered that highly pathogenic PRRSV (HP-PRRSV) and low pathogenic PRRSV (LP-PRRSV) infection exhibited a differential TNF-α expression in pulmonary alveolar macrophages (PAMs), showing that HP-PRRSV infection induces lower TNF-α production at protein level in PAMs, compared with LP-PRRSV. Next, HP-PRRSV was confirmed to strongly suppress TNF-α production by inhibiting ERK signaling pathway. Finally, both Nsp1β and Nsp11 were demonstrated to be responsible for the inhibitory effect on TNF-α production induced by HP-PRRSV and the differential TNF-α production in PAMs. These findings contribute to the understanding of the pathogenesis of the Chinese HP-PRRSV.

Type I interferons (IFNs), predominantly IFN-α and β, play important roles in both innate and adaptive immune responses against viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be able to down-regulate the IFN response during in vivo and in vitro infection. In this study, we first analyzed inhibitory effect of each NSP of low pathogenic PRRSV HB-1/3.9 on IFN-β transcription in MARC-145 cells, and the results showed that the IFN-β promoter activation could be suppressed by NSP1α, NSP2, NSP1β, NSP3, NSP4, NSP5 and NSP11. We next confirmed that the inhibitory effect of NSP4 was mainly mediated through suppressing NF-κB activation, whereas not hindering NF-κB phosphorylation and nuclear translocation, and nuclear-localized NSP4 was responsible for inhibiting IFN-β activation. We further found that the NSP4 of different pathogenic PRRSV strains exhibited differential inhibitory effect on IFN-β, NF-κB, and IRF3 transcription, and the NSP4 of highly pathogenic (HP)-PRRSV could display more strong inhibitory effect. Finally, we determined that the amino acid at residue 155 in NSP4 contributed to its inhibitory effect for IFN-β transcription in vitro by altering its subcellular distribution. Our findings suggest that the nucleus-localized NSP4 of PRRSV participates in the modulation of the host type I IFNs system, and also provide novel insight for understanding the pathogenesis of the Chinese HP-PRRSV.

Recently, duck hepatitis A virus 3 (DHAV-3) with genetically distinct characteristics from DHAV-1 and DHAV-2 was recognized in South Korea and China. In this short communication, we successfully constructed a stable full-length infectious cDNA clone derived from DHAV-3 by solving instability of cloned full-length cDNA in Escherichia coli (E. coli). The cDNA fragments amplified from the genome of DHAV-3 were assembled and inserted into a low-copy-number plasmid. Finally, a full-length cDNA clone containing an engineered SacII site that served as a genetic marker was obtained. The cDNA clone showed stable by serial passages in E. coli when propagated at 25°C under low level of antibiotic selection. BHK-21 cells were transfected with transcribed RNA from the full-length cDNA clone; infectious viral particles were rescued, showing its fatality to 10-day-old duck embryos. The results indicated that the constructed full-length cDNA clone of DHAV-3 is infectious. By various virological assays, our results indicated that the rescued virus exhibited similar biological properties with the parental virus. Animal experiments revealed that the rescued virus retained the high pathogenicity to 1-day-old ducklings and could induce a fatal hepatitis indistinguishable from its parental virus. Our present studies provide a useful tool for future research on genomic functions and molecular pathogenesis of DHAV-3.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important viral disease for the pig industry worldwide. This disease has brought great losses to the Chinese pig production in recent years, particularly following the emergence of the highly pathogenic PRRS virus (PRRSV), and has become an intractable problem for the development of pig industry in China. This paper will review the history of PRRS, the epidemic of atypical PRRS caused by the highly pathogenic virus, and the molecular characteristics of the Chinese highly pathogenic PRRSV, and the development of vaccines against PRRS in China, as well as current control status and perspective of PRRS in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is characteristic of genetically extensive variation. The objective of the present study was to analyze the molecular variation and evolution of porcine reproductive and respiratory syndrome virus in China based on the complete genomic sequences of three highly pathogenic Chinese PRRSV strains isolated in 2006 and the sequences of the amplified Nsp2, ORF5 and ORF7 genes from clinical specimens during 2006-2008. Full-length genome sequencing and phylogenetic analysis showed that the three strains (JXwn06, BJsy06 and NX06) had a unique 30-amino-acid discontinuous deletion in Nsp2, and were classified into the same subgroup that consisted of the most Chinese strains isolated during 2006-2007, the pandemic period of atypical PRRS. The evolution analysis suggested that the emergence of the highly pathogenic PRRSV in China experienced a gradual variation and evolution accumulation progress from Chinese domestic virus. The variation analysis of the amplified 41 Nsp2, 59 ORF5 and 59 ORF7 genes indicated that the diversity of PRRSV strain existed in the field, and the highly pathogenic PRRSV strain with the 30-amino-acid deletion in Nsp2 was the dominating virus in China in recent years. Our data contribute to the understanding of molecular variation and epidemiology surveillance of PRRSV in China.

Forty Chinese PCV2 strains collected between 2004 and 2008 were sequenced and their genetic variations were analyzed together with nine previous PCV2 isolates. Phylogenetic analysis indicated that these Chinese PCV2 strains could be divided into four genotypes (PCV-2a, PCV-2b, PCV-2d and PCV-2e), and the genotype PCV-2c defined in Denmark was not found. PCV-2d and PCV-2e were two genotypes firstly determined in our study. Variation analysis of amino acids of capsid protein revealed that Chinese PCV2 strains clustered within PCV-2d had four amino acid marker positions and the isolates within PCV-2e had seven unique amino acid mutations. Our analysis also showed that PCV-2b became dominating in China in recent years. These data contribute to the understanding of PCV2 molecular epidemiology.