Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, has two distinct and highly diverse genotypes (genotype 1 and genotype 2) in the field. Accurate diagnosis and differentiation of the two genotypes of PRRSV are critical to the effective prevention and control of PRRS. The non-structural protein 10 (Nsp10) plays a vital role in viral replication and is one of the most conserved proteins of PRRSV, thus constituting a good candidate for PRRSV diagnosis.In this study, we generated a monoclonal antibody (mAb) 4D9 against Nsp10 by immunizing BALB/c mice with purified recombinant Nsp10 expressed by an Escherichia coli system. Through fine epitope mapping of mAb 4D9 using a panel of eukaryotic expressed polypeptides with GFP-tags, we identified the motif 286AIQPDYRDKL295 as the minimal unit of the linear B-cell epitope recognized by mAb 4D9. Protein sequence alignment indicated that 286AIQPDYRDKL295 was highly conserved in genotype 2 PRRSV strains, whereas genotype 1 PRRSV strains had variable amino acids in this motif. Furthermore, a mutant of the motif carrying two constant amino acids of genotype 1 PRRSV, Cys290 and Glu293, failed to react with mAb 4D9. More importantly, the mAb 4D9 could differentiate genotype 2 PRRSV strains from genotype 1 PRRSV strains using Western blotting and immunofluorescence analysis.Our findings suggest that Nsp10-specific mAb generated in this study could be a useful tool for basic research and may facilitate the establishment of diagnostic methods to discriminate between genotype 1 and genotype 2 PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) actively induces cell apoptosis both in vitro and in vivo, which can contribute critically to viral pathogenesis. Previous studies have shown that the PRRSV nonstructural protein 4 (nsp4) is an important mediator of this process, but the underlying molecular details remain poorly understood. In this study, we found that the PRRSV nsp4 interacted with the mitochondrial inner membrane protein cytochrome c1 (cyto.c1) and induced its proteolytic cleavage. Interestingly, the cleaved N-terminal fragment of cyto.c1 was found to exert apoptotic activity, which could cause mitochondrial fragmentation, resulting in apoptotic cell death. And RNA interference (RNAi) silencing experiments further confirmed the crucial role which cyto.c1 played in nsp4- and PRRSV-induced cell apoptosis. Thus, our data provide an important piece of mechanistic clues for PRRSV-induced cell apoptosis and also elucidate a novel mechanism for the 3C-like proteases in this finding.

•The genomic characterization of a type 2 PRRSV (TJnh1501) was analyzed.•The isolate TJnh1501 was shown to the recombinant virus between NADC30-like and MLV-like.•The isolate TJnh1501 shared intermediate virulence for piglets.Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen for swine industry worldwide. The recombination occurring among PRRSV strains has been recognized as one of important molecular mechanisms for the evolution of PRRSV. Current prevalence of PRRSV NADC30-like causing clinical disease outbreaks is highly concerned in China. In the present study, the genetic characterization of a recombinant type 2 PRRSV (designated TJnh1501) was analyzed and its pathogenicity for piglets was examined. Our study showed that each region of TJnh1501 genome had 96.67–100% nucleotide and 96.5–100% amino acid identities with a Chinese highly pathogenic PRRSV-derived modified-live virus (MLV)-like except for its nonstructural protein 2 (nsp2)-coding region; while its nsp2-coding region shared higher nucleotide (84.44–85.85%) and amino acid (82.44–84.79%) identities with NADC30 and NADC30-like CHsx1401, and in particular, the highly variable region of nsp2 exhibited characteristic 131-aa deletion identical to NADC30 and NADC30-like CHsx1401. Meanwhile, we identified two recombination breakpoints located in the nt1737 and nt3506 of nsp2-coding region, which had higher nucleotide homology with NADC30 and NADC30-like CHsx1401. Moreover, TJnh1501 infection could cause persistent fever, moderate respiratory clinical signs, higher viremia, and obvious gross and microscopic lung lesions in piglets. The virus was shown to have lower pathogenicity than HP-PRRSV JXwn06, but higher than NADC30-like CHsx1401 for piglets. Our findings reveal that TJnh1501 is a recombinant type 2 PRRSV from the recombinant event between NADC30-like and MLV-like derived from the Chinese highly pathogenic PRRSV, and it exhibits intermediate virulence for pigs. This study adds valuable evidence for understanding the role of genomic recombination in the evolution of PRRSV.

•The PRRSV NADC30-like CHsx1401 was shown to be a moderate virulent virus for piglets.•MLV vaccines could not reduce the clinical signs and lung lesions of the pigs challenged.•MLV vaccines were partially efficacious in the reduction of viral loads.•MLV vaccines are shown to provide extremely limited cross-protection against PRRSV NADC30-like.Porcine reproductive and respiratory syndrome reproductive virus is a devastating pathogen causing tremendous economic losses to swine production worldwide. Emergence of novel and variant PRRSV strains always leads to variable protection efficacy of modified-live virus (MLV) vaccines. Prevalence of PRRSV NADC30-like recently emerging in China has brought about clinical outbreaks of the disease. In the present study, the pathogenicity of a NADC30-like strain CHsx1401 for piglets was analyzed, and the potential cross-protective efficacy of three MLV vaccines including two commercial MLV vaccines and an attenuated low pathogenic PRRSV against this virus was further evaluated in piglets. The NADC30-like CHsx1401 was shown to cause fever, respiratory clinical signs, and lung gross and microscopic lesions of the inoculated piglets, suggesting that this virus is moderate virulent for piglets. Vaccination of piglets with the MLV vaccines could not reduce the clinical signs and lung lesions, and was partially efficacious in the reduction of viral loads in sera upon NADC30-like CHsx1401 challenge, indicating that these three MLV vaccines provide extremely limited cross-protection efficacy against the NADC30-like virus infection. Additionally, Ingelvac PRRS MLV appeared to exert some beneficial efficiency in shortening the period of clinical fever and in improving the growth performance of the challenged pigs. Our findings give valuable guidance for the choice and use of PRRSV MLV vaccines to control NADC30-like virus infection in the field.

•The RNA helicase DDX18 interacts with the nsp2 and nsp10 of PRRSV.•DDX18 enhances the replication of PRRSV in cell culture.•Nsp2 and nsp10 relocates DDX18 from nucleus to cytoplasm.•DDX18 play a pro-viral role via its interaction with nsp2 and nsp10.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.

•Epidemiological and clinical characteristics of HP-PRSV infection were mentioned.•Pathological pathogenesis of HP-PRRSV infection was reviewed.•Immunological aspects of HP-PRRSV infection were stated.•Molecular pathogenesis of HP-PRRSV virulence was reviewed.•Control of HP-PRRSV infection in China was stated.Porcine reproductive and respiratory syndrome virus (PRRSV) has remained a major threat to the worldwide swine industry ever since its first discovery in the early 1990s. Under the selective pressures in the field, this positive-stranded RNA virus undergoes rapid genetic evolution that eventually leads to emergence in 2006 of the devastating Chinese highly pathogenic PRRSV (HP-PRRSV). The atypical nature of HP-PRRSV has caused colossal economic losses to the swine producers in China and the surrounding countries. In this review, we summarize the recent advances in our understanding of the pathogenesis, evolution and ongoing field practices on the control of this troubling virus in China.

The M protein, encoded by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF6 gene, is considered to be one of the most conserved PRRSV proteins. In recent decades, highly specific monoclonal antibodies (Mabs) have been exploited to provide reliable diagnoses for many diseases. In this study, two different Mab clones targeting the linear epitopes on the PRRSV M protein were generated and characterized. Both Mabs showed binding activity against the native PRRSV virion and recombinant M protein when analyzed by immunofluorescence assay (IFA) and Western blot. The targeted epitope of each Mab was mapped by serial truncation of the M protein to generate overlapping fragments. Fine epitope mapping was then performed using a panel of expressed polypeptides. The polypeptide sequences of the two epitopes recognized by Mabs 1C8 and 3F7 were 3SSLD6 and 155VLGGRKAVK163, respectively, with the former being a newly identified epitope on the M protein. In both cases, these two epitopes were finely mapped for the first time. Alignments of Mab epitope sequences revealed that the two epitopes on the M protein were highly conserved between the North American-type strains. These Mabs, along with their mapped epitopes, are useful for the development of diagnostic and research tools, including immunofluorescence, ELISA and Western blot.

Here, we present the complete genomic sequence of the Chinese standard challenge strain (CSC) of duck enteritis virus (DEV), which was isolated in China in 1962. The DEV CSC genome is 162,131 bp long and contains 78 predicted open reading frames (ORFs). Comparison of the genomic sequences of DEV CSC and DEV live vaccine strain K at passage 63 (DEV K p63) revealed that the DEV CSC genome is 4,040 bp longer than the DEV K p63 genome, mainly because of 3,513-bp and 528-bp insertions at the 5′ and 3′ ends of the unique long segment, respectively. At the nucleotide level, 63 of the 76 ORFs in the DEV CSC genome were 100 % identical to the ORFs in the DEV K p63 genome. Two ORFs (UL56 and US10) had frameshift mutations in the C-terminal regions, while LORF5 was unique to the DEV K p63 genome. It is difficult to assign attenuated virulence to changes in specific genes. However, the complete DEV CSC genome will further advance our understanding of the genes involved in virulence and evolution. The DEV CSC genome sequence has been deposited in GenBank under accession number JQ673560.

Trichophyton rubrum (T. rubrum) is a common superficial fungus. Molecular and genetic studies of T. rubrum are still limited. In this paper, we report the global analysis of gene expression profiles at different growth phases using cDNA microarray technology. A total of 2044 differentially expressed genes were obtained and clustered into three expression patterns. Our data confirmed previous results that many mRNAs were pre-stored in the conidia of T. rubrum. Transcriptional profiling and function analysis showed that some glycolytic enzymes share similar expression patterns and may be coregulated during the transition of growth phases. Some genes involved in small GTPase signaling pathways, and in cAMP-dependent and MAPK regulation pathways were induced in response to the growth dynamics of T. rubrum. Although the detailed biological roles of these T. rubrum genes are still unknown, our results suggest that these genes may be involved in regulation mechanisms in the life cycle of the fungus.

Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis, myocarditis and diabetes in its natural host, the mouse. In this study, we generated four cDNA clones with a point mutation at position 100 of VP1. The amino acids isoleucine, alanine, serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis, and viable viruses were rescued. Although all mutants and wild-type viruses display different plaque morphologies, they replicate comparably in BHK-21 cells. The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice. The results showed that the isoleucine- (T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality, lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus. These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection, and its mutation alters the pathogenicity of viral infection in mice.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect and replicate preferentially in pulmonary alveolar macrophages (PAMs). We applied proteomic approaches to investigate the change in cellular proteins of PAMs infected with PRRSV in vitro. A total of 23 cellular proteins with significant alteration in different courses postinfection were identified. These proteins could be classified into the functions associated with morphogenesis, protein synthesis, metabolism, and stress response and ubiquitin-proteasome pathway. Of the altered proteins, two proteins, heat shock 27 kDa protein (HSP27) and superoxide dismutase 2 (SOD2), involved in stress response or ubiquitin-proteasome pathway were observed to be up-regulated. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected PAMs by proteomics and provide valuable information for better understanding the function alterations of PAMs induced by PRRSV infection.

A novel agent (hence termed as P2) was isolated from pig sera in China, which contained covalently bound circular genomic DNAs of 993 nucleotides. Sequence analyses indicated that the agent was closely related to the porcine circovirus (PCV). The molecular clone of P2 was constructed subsequently and used for the following studies. Intracytoplasmic inclusions and intranuclear inclusions were only found in PK-15 cells transfected with the tandem dimer of P2 molecular DNA clone. Intracytoplasmic inclusions were round or irregular in shape and 0.1–0.4 μm in diameter, and intranuclear inclusions were electronically denser than intracytoplasmic inclusions and had two general shapes: round/small (0.1 μm in diameter) and hexagonal/large (0.5–1.4 μm in diameter). The inclusions were not membranously bound. The cells transfected with the tandem dimer of P2 molecular DNA clone were tested positive for P2 DNA at passages 5. The P2 antigen could be detected in both transfected and passaged PK-15 cells. This is the first report regarding the complete nucleotide sequence of a small DNA genome in a circovirus-like infectious agent in vitro.

Non-structural protein 2 (Nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the most variable region and postulated to play an important role in cell and tissue tropism of PRRSV. To investigate the role of Nsp2 in the viability and growth of PRRSV in cells in vitro, two cDNA clones were constructed containing a deletion of 63 consecutive nucleotides (pWSK-DCBAd63) or 117 nucleotides (pWSK-DCBAd117) within the Nsp2-encoding region of PRRSV (BJ-4). The clone pWSK-DCBAd63 was infectious and produced viable recombinant virus, whereas clone pWSK-DCBAd117 could not be rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 cells and was stably propagated during sequential in vitro cell passages, like the virus recovered from the full-length cDNA clone of PRRSV BJ-4. In comparison to the parental virus (BJ-4) and the virus recovered from the full-length cDNA clone of the BJ-4 strain, the rescued virus from pWSK-DCBAd63 exhibited enhanced growth kinetics, reaching the peak progeny virus titer by 48 h postinfection. These observations suggest that the Nsp2-encoding region is necessary for productive virus infection, and partial deletion does not influence the viability and propagation of PRRSV in cell culture, which may provide a way to insert a foreign gene into the viral genome as a marker for differentiation.

•A stable cell line expressing recombinant avian antibody against IBDV was established.•Recombinant avian antibody exhibited neutralization activity to IBDV-B87 strain in DF1 cells.•Recombinant avian antibody exhibited good efficacy in treatment of IBD.A stable cell-line was established that expressed the recombinant avian antibody (rAb) against the infectious bursal disease virus (IBDV). rAb exhibited neutralization activity to IBDV-B87 strain in DF1 cells. The minimum rAb concentration required for inhibition of the cytopathic effect (CPE) was 1.563 μg/mL. To test the efficacy of rAb, a 168-h cohabitation challenge experiment was performed to transmit the disease from the chickens challenged with vvIBDV (HLJ0504 strain) to three test groups of chickens, i.e. (1) chickens treated with rAb, (2) chickens treated with yolk antibody, and (3) non-treatment chickens. The survival rates of chickens treated with rAb, yolk antibody and without treatment were 73%, 67% and 20%, respectively. Another batch of chickens was challenged with IBDV (BC6/85 strain) and then injected with rAb (1.0 mg/kg) 6, 24 and 36 h post-challenge. Non-treatment chickens had 100% morbidity, whereas those administered with rAb exhibited only 20% morbidity. Morbidity was evaluated using clinical indicators and bursal histopathological section. This study provides a new approach to treating IBDV and the rAb represents a promising candidate for this IBDV therapy.

•A stable cell line expressing recombinant avian antibody against IBDV was established.•Recombinant avian antibody exhibited neutralization activity to IBDV-B87 strain in DF1 cells.•Recombinant avian antibody exhibited good efficacy in treatment of IBD.A stable cell-line was established that expressed the recombinant avian antibody (rAb) against the infectious bursal disease virus (IBDV). rAb exhibited neutralization activity to IBDV-B87 strain in DF1 cells. The minimum rAb concentration required for inhibition of the cytopathic effect (CPE) was 1.563 μg/mL. To test the efficacy of rAb, a 168-h cohabitation challenge experiment was performed to transmit the disease from the chickens challenged with vvIBDV (HLJ0504 strain) to three test groups of chickens, i.e. (1) chickens treated with rAb, (2) chickens treated with yolk antibody, and (3) non-treatment chickens. The survival rates of chickens treated with rAb, yolk antibody and without treatment were 73%, 67% and 20%, respectively. Another batch of chickens was challenged with IBDV (BC6/85 strain) and then injected with rAb (1.0 mg/kg) 6, 24 and 36 h post-challenge. Non-treatment chickens had 100% morbidity, whereas those administered with rAb exhibited only 20% morbidity. Morbidity was evaluated using clinical indicators and bursal histopathological section. This study provides a new approach to treating IBDV and the rAb represents a promising candidate for this IBDV therapy.

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 pasmid-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.Highlights► We confirm the interaction of Nsp1β of the Chinese HP-PRRSV with cellular PCBP2. ► We determine the minimal binding regions of Nsp1β with PCBP2 each other. ► The interaction of the Nsp1β with cellular PCBP2 is beneficial to Chinese HP-PRRSV replication in MARC-145 cells.

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.

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 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: T73LPERVRPPDDWAT86, D385ELKDQMEED394, P452VPAPRRKVGSDCGS466, and P467VSLGGDVPNS477. 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.

The glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is a multi-functional protein that plays important roles in virus assembly, entry and viral anti-host responses. In the present study, we investigated the cellular binding partners of GP5 by using lentivirus transduction coupled with immunoprecipitation and mass spectrometry. There were about 40 cellular proteins identified with high Confidence Icons by MS/MS. Ingenuity Pathway Analysis (IPA) indicated that these proteins could be assigned to different functional classes and networks. Furthermore, we validated some of the interactions by co-immunoprecipitation (Co-IP) and confocal microscopy, including those with mitofilin, a mitochondrial inner membrane protein that might be involved in PRRSV or GP5-induced apoptosis, and calnexin, a protein chaperone that might facilitate the folding and maturation of GP5. The interactome data contribute to understand the role and molecular mechanisms of GP5 in PRRSV pathogenesis.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) significantly impact the swine industry worldwide. Co-infections with these viruses are common and several lines of evidence suggest that both PRRSV and PCV2 modify host immune responses that facilitate infection. This study examined cytokine mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) from piglets experimentally co-infected with PRRSV and PCV2 to define the influence of co-infection on host immunity. PBMCs from infected and control piglets were stimulated with concanavalin A and the IL-2, IL-4, IL-6, IL-10, IL-12p40, IFN-γ and TNF-α mRNA levels were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). PBMCs from PRRSV/PCV2 co-infected piglets had significantly reduced IL-2, IL-4, IL-6, IL-12p40 and IFN-γ and significantly increased TNF-α mRNA levels compared to those of the piglets infected with either PRRSV or PCV2 alone. The IL-10 mRNA levels in all virus-infected groups were significantly up-regulated early during infection. These results suggested that co-infection synergistically suppresses T helper 1 (Th1)-type and Th2-type cytokine production by PBMCs, indicating that co-infection likely compromises cell-mediated and humoral immune responses resulting in increased severity of the diseases in piglets.

•Autophagy induced by JEV infection could promote virus replication.•The C, M and NS3 of JEV were involved in autophagy induction.•The NS3 of JEV could target immunity-related GTPases M.Japanese encephalitis virus (JEV) is an important zoonotic pathogen causing viral encephalitis in human and reproductive failure in pigs. In the present study, we first examined the autophagy induced by JEV infection in host cells, and then analyzed the JEV proteins involving in autophagy induction, and further investigated the relationship between viral protein and immunity-related GTPases M (IRGM). Our results showed that JEV infection could induce autophagy in host cells and autophagy promoted the replication of JEV in vitro; the cells transfected with individual plasmid that was expressing C, M and NS3 had a significantly higher conversion of LC3-I/II, and enhanced LC3 signals with the fluorescence punctuates accumulation which was completely co-localized with LC3 and increased number of autophagosomes-like vesicles, suggesting that C, M and NS3 are the major viral proteins involving in autophagy induction upon JEV infection; the virus titer in the cells treated by the siRNA specific for IRGM had a significant decrease, and the NS3 signals in the cells transfected with the plasmid that was expressing NS3 were completely co-localized with the IRGM signals, suggesting that the NS3 of JEV could target IRGM which may play a role in the replication of JEV. Our findings help to understand the role of autophagy in JEV and other flaviviruses infections.

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

•The Chinese HP-PRRSV infection suppressed Th17 cells response in peripheral blood of piglets.•The Chinese HP-PRRSV infection caused a reduced Th17 cells in lungs of piglets.•HP-PRRSV-inoculated piglets showed higher bacterial loads in lungs.•Th17 cells suppression possibly correlated with bacterial secondary infection in piglets.Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to immunomodulate innate and adaptive immunity of pigs. The Chinese highly pathogenic PRRSV (HP-PRRSV) infection causes severe bacterial secondary infection in pigs. However, the mechanism in relation to the bacterial secondary infection induced by HP-PRRSV remains unknown. In the present study, Th17 cells response in peripheral blood, lungs, spleens and lymph nodes of piglets were analyzed, and bacterial loads in lungs of piglets were examined upon HP-PRRSV infection. Meanwhile the changes of CD4+ and CD8+ T cells in peripheral blood of the inoculated piglets were analyzed. The results showed that HP-PRRSV-inoculated piglets exhibited a suppressed Th17 cells response in peripheral blood and a reduced number of Th17 cells in lungs, and higher bacterial loads in lungs, compared with low pathogenic PRRSV. Moreover, HP-PRRSV obviously resulted in severe depletion of porcine T cells in peripheral blood at the early stage of infection. These findings indicate that HP-PRRSV infection suppresses the response of Th17 cells that play an important role in combating bacterial infections, suggesting a possible correlation between the suppression of Th17 cells response in vivo and bacterial secondary infection induced by HP-PRRSV. Our present study adds a novel insight into better understanding of the pathogenesis of the Chinese HP-PRRSV.

•PRRSV NSP4 exerts inhibitory effect for IFN-β transcription in vitro.•Nuclear-localized NSP4 is responsible for inhibiting IFN-β activation.•NSP4 of HP-PRRSV displays more strong inhibitory effect.•The amino acid at residue 155 in NSP4 is responsible for its inhibitory effect.•Our findings provide novel insight for understanding the pathogenesis of 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.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are pathogens, which can significantly affect the swine industry worldwide. Field surveys suggest that simultaneous PRRSV and PCV2 infection is common in pigs. The objective of this study was to measure the changes in peripheral blood leukocyte subpopulations in piglets co-infected experimentally with PRRSV and PCV2, in order to analyze the synergistic influence of co-infection on the immune system. Changes in peripheral blood leukocyte subpopulations were systematically measured by flow cytometry (FCM). The levels of antibodies to PRRSV and PCV2 were detected by indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) and the indirect fluorescent antibody test (IFA), respectively. Serum viral loads were measured using real-time PCR. The results showed that piglets co-infected with PRRSV and PCV2 exhibited slower generation and lower levels of antibodies to PRRSV and PCV2, and increased amounts and a prolonged presence of both PRRSV and PCV2 in serum, in comparison to the piglets infected with either virus alone. The major finding in our study was that the total and differential leukocyte counts, including white blood cells (WBCs), monocytes, granulocytes and lymphocytes (T, B and NK cells, as well as T-cell subpopulations), dramatically decreased early during co-infection with PRRSV and PCV2 for about two weeks, in contrast with animals singly infected with either PRRSV or PCV2. These results suggest that PRRSV and PCV2 co-infection results in a synergistic decrease in immune cells in the peripheral blood of piglets. These data contribute to the understanding of the immunosuppressive effects resulting from PRRSV and PCV2 co-infection in pigs.

•The full-length genome of a strain of type 1 PRRSV (GZ11-G1) was sequenced.•GZ11-G1 shares highest homology (96.3%) with the live vaccine virus Amervac PRRS.•GZ11-G1 is clustered with the strains of type PRRSV by phylogenetic analysis.•The evolution of GZ11-G1 is genetically related to the strains of type PRRSV.•GZ11-G1 is a pathogenic type 1 PRRSV for piglets.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.

•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.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) was firstly characterized in 2006 in China. The virus has caused great economic loss to the Chinese swine production during the past years. Herein, we experimentally infected SPF pigs using two strains of PRRSV with different pathogenicity and observed the lung pathological changes looking for new sights on the possible pathogenesis associated with the virulence of HP-PRRSV. The results indicated that the HP-PRRSV-infected pigs died and exhibited severe pathological changes of lungs featuring increased neutrophils, mast cells and mononuclear macrophages, compared with the pigs inoculated with low pathogenic (LP-) PRRSV. Furthermore, the pigs infected with HP-PRRSV showed the higher levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interleukin (IL)-8 and histamine, leukotriene B4 (LTB4), platelet activation factor (PAF) in sera than those inoculated with LP-PRRSV. Additionally, the fibrosis of lung was observed in the HP-PRRSV-infected pigs. At present, our findings suggest that the aberrant immune responses triggered by HP-PRRSV infection are closely related to acute lung injury (ALI), and especially the pathological changes in lung vascular system are of particular significance. These associated pathological changes of lung are in part responsible for the additional morbidity and mortality observed in HP-PRRSV infection.

•The PRRSV Nsp3 or E protein was demonstrated to interact with IFITM1 or Tetherin.•The Nsp3 was shown to induce the proteasome-dependent degradation of IFITM1.•The E protein was proposed to partially remove Tetherin away from cell surface.•PRRSV might counteract cellular IFITM1 and Tetherin by its Nsp3 and E protein.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.

The genomic sequence of a strain of duck enteritis virus (DEV) was determined and analyzed in this study. The size of its genome is 158,091 bp in length and the genome is predicted to encode 78 putative proteins and resembles the members of the Alphaherpesvirinae in genomic organization and gene composition. The genome of the virus is composed of a unique long (UL) region, a unique short (US) region, a unique short internal repeat (IRS) region and a unique short terminal repeat (TRS) region. Its genomic arrangement pattern (UL-IRS-US-TRS) corresponds to D-type herpesvirus and is consistent with the members of Varicellovirus and Iltovirus genera. Sequence analysis reveals that the genome of the virus contains 67 genes having homologs in most members of the Alphaherpesvirinae. Out of these genes, one gene has a homolog in cercopithecine herpesvirus 8 which is a virus of Betaherpesvirinae, and 5 genes have homologs in avian herpesviruses. Furthermore, the genome possesses three unique genes without homologs in any other herpesviruses. Like most members of the Alphaherpesvirinae, the genes in the UL region of its genome are well conserved, whereas the gene arrangement of IRS-US is similar to that of Marek's disease virus and equine herpesviruses 1. Therefore, our data based on the genomic analysis suggest that DEV represents an osculant taxonomic entity within the Alphaherpesvirinae.

In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/β-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome–lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.