Antigen-specific B-cell responses to porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an acute, viremic infection of 4 to 6 weeks, followed by a persistent infection lasting for several months. We characterized antibody and B-cell responses to viral proteins in acute and persistent infection to better understand the immunological basis of the prolonged infection. The humoral immune response to PRRSV was robust overall and varied among individual viral proteins, with the important exception of a delayed and relatively weak response to envelope glycoprotein 5 (GP5). Memory B cells were in secondary lymphoid organs, not in bone marrow or Peyer's patches, in contrast to the case for many mammalian species. Potent anti-PRRSV memory responses were elicited to recall antigen in vitro, even though a second infection did not increase the B-cell response in vivo, suggesting that productive reinfection does not occur in vivo. Antibody titers to several viral proteins decline over time, even though abundant antigen is known to be present in lymphoid tissues, possibly indicating ineffective antigen presentation. The appearance of antibodies to GP5 is delayed relative to the resolution of viremia, suggesting that anti-GP5 antibodies are not crucial for resolving viremia. Lastly, viral infection had no immunosuppressive effect on the humoral response to a second, unrelated antigen. Taking these data together, the active effector and memory B-cell responses to PRRSV are robust, and over time the humoral immune response to PRRSV is effective. However, the delayed response against GP5 early in infection may contribute to the prolonged acute infection and the establishment of persistence.     

重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞

【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。     

Validation and further characterization of a major quantitative trait locus associated with host response to experimental infection with porcine reproductive and respiratory syndrome virus

Infectious diseases are costly to the swine industry; porcine reproductive and respiratory syndrome (PRRS) is the most devastating. In earlier work, a quantitative trait locus associated with resistance/susceptibility to PRRS virus was identified on Sus scrofa chromosome 4 using approximately 560 experimentally infected animals from a commercial cross. The favorable genotype was associated with decreased virus load and increased weight gain (WG). The objective here was to validate and further characterize the association of the chromosome 4 region with PRRS resistance using data from two unrelated commercial crossbred populations. The validation populations consisted of two trials each of approximately 200 pigs sourced from different breeding companies that were infected with PRRS virus and followed for 42 days post‐infection. Across all five trials, heritability estimates were 0.39 and 0.34 for viral load (VL; area under the curve of log‐transformed viremia from 0 to 21 days post‐infection) and WG to 42 days post‐infection respectively. Effect estimates of SNP WUR10000125 in the chromosome 4 region were in the same directions and of similar magnitudes in the two new trials as had been observed in the first three trials. Across all five trials, the 1‐Mb region on chromosome 4 explained 15 percent of genetic variance for VL and 11 percent for WG. The effect of the favorable minor allele at SNP WUR10000125 was dominant. Ordered genotypes for SNP WUR10000125 showed that the effect was present irrespective of whether the favorable allele was paternally or maternally inherited. These results demonstrate that selection for host response to PRRS virus infection could reduce the economic impact of PRRS.     

Divergence time of porcine reproductive and respiratory syndrome virus subtypes

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged in domestic pigs of Western Europe and North America. Although time of emergence was identical on the two continents, genetic composition was markedly different with a clear geographical subtype structure, indicating that subtypes diverged in separate reservoirs prior to emergence. Genetic analyses have shown that the most recent common ancestor (MRCA) of Western European isolates existed around 1980 and that these originate from Eastern European pigs. These findings are challenged by a study of Hanada et al. who place the MRCA of all PRRSV isolates around 1980 and find that no significant subtype divergence occurred before emergence. Here, I discuss problems of information content, methodology, and biological plausibility associated with this study. Using alternative methodology, I reanalyze the existing data and conclude that the MRCA of all PRRSV isolates existed around 1880, 100 years before the date estimated by Hanada et al.     

Overview: Replication of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. The genome replication of PRRSV is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. Replication of the viral RNA genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic RNA template; this replication complex is embedded within particular virus-induced membrane vesicles. PRRSV RNA replication is directed by at least 14 replicase proteins that have both common enzymatic activities, including viral RNA polymerase, and also unusual and poorly understood RNA-processing functions. In this review, we summarize our current understanding of PRRSV replication, which is important for developing a successful strategy for the prevention and control of this pathogen.     

Receptor-determined susceptibility of preimplantation embryos to pseudorabies virus and porcine reproductive and respiratory syndrome virus

In the present study, the in vitro interaction of embryos with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by viral antigen detection and by evaluating the expression of virus receptors, namely, poliovirus receptor-related 1 (PVRL1; formerly known as nectin 1) for PRV and sialoadhesin for PRRSV. Embryonic cells of zona pellucida intact embryos incubated with PRV remained negative for viral antigens. Also, no antigen-positive cells could be detected after PRV incubation of protease-treated embryos, since the protease disrupted the expression of PRVL1. However, starting from the five-cell-stage onwards, viral antigen-positive cells were detected after subzonal microinjection of PRV. At this stage, the first foci of PVRL1, also a known cell adhesion molecule, were expressed. At the expanded blastocyst stage, a lining pattern of PVRL1 in the apicolateral border of trophectoderm cells was present, whereas the expression in the inner cell mass was low. Furthermore, PVRL1-specific monoclonal antibody CK41 significantly blocked PRV infection of trophectoderm cells of hatched blastocysts, while the infection of the inner cell mass was only partly inhibited. Viral antigen-positive cells were never detected after PRRSV exposure of preimplantation embryos up to the hatched blastocyst stage. Also, expression of sialoadhesin in these embryonic stages was not detected. We conclude that the use of protease to investigate the virus embryo interaction can lead to misinterpretation of results. Results also show that blastomeres of five-cell embryos up to the hatched blastocysts can become infected with PRV, but there is no risk of a PRRSV infection.     

Characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells

The porcine reproductive and respiratory syndrome Virus (PRRSV) is an infectious disease that causes abortions and respiratory disorders in swine. In this study, the interaction between PRRSV and porcine dendritic cells generated from CD14(+) monocytes in the presence of GM-CSF and IL-4 was examined. As a result, it was shown that immature and mature dendritic cells can be productively infected with PRRSV. When the expression of surface MHC molecules on infected dendritic cells was determined, MHC classes I and II were found to be downregulated when compared with uninfected dendritic cells. With the exception of the IL-4 and IFN-gamma cytokines, the induction of the IL-10, IL-12, and TNF-alpha cytokines all increased in dendritic cells infected with PRRSV. A mixed lymphocyte reaction showed that peripheral blood mononuclear cells cocultured with PRRSVinfected dendritic cells were less stimulated than peripheral blood mononuclear cells cocultured with dendritic cells treated with PBS, LPS, or UV-inactivated PRRSV. Therefore, these results suggest that PRRSV would appear to modulate the immune stimulatory function of porcine dendritic cells.     

Phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is a cytoplasmic RNA virus with the unique or unusual feature of having a nucleocapsid (N) protein that is specifically transported to the nucleolus of virus-infected cells. In this communication, we show that the N protein is a phosphoprotein. Phosphoamino acid analysis of authentic and recombinant N proteins demonstrated that serine residues were exclusively phosphorylated. The pattern of phosphorylated N protein cellular distribution in comparison with that of [(35)S]methionine-labeled N protein suggested that phosphorylation does not influence subcellular localization of the protein. Time course studies showed that phosphorylation occurred during, or shortly after, synthesis of the N protein and that the protein remained stably phosphorylated throughout the life cycle of the virus to the extent that phosphorylated N protein was found in the mature virion. Two-dimensional electrophoresis and acid-urea gel electrophoresis showed that one species of the N protein is predominant in virus-infected cells, suggesting that multiple phosphorylated isoforms of N do not exist.     

Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells

Lipid rafts play an important role in the life cycle of many viruses. Cholesterol is a critical structural component of lipid rafts. Although the porcine reproductive and respiratory syndrome virus (PRRSV) has restricted cell tropism for cells of the monocyte/macrophage lineage, a non-macrophage cell MARC-145 was susceptible to PRRSV because of the expression of virus receptor CD163 on the cell surface, therefore MARC-145 cells is used as model cell for PRRSV studies. In order to determine if cholesterol is involved in PRRSV infection in MARC-145 cells, we used three pharmacological agents: methyl-β cyclodextrin (MβCD), mevinolin, and filipin complex to deplete cholesterol in MARC-145. Although these agents act by different mechanisms, they all significantly inhibited PRRSV infection. The inhibition could be prevented by addition of exogenous cholesterol. Cell membrane cholesterol depletion after virus infection had no effect on PRRSV production and cholesterol depletion pre-infection did not reduce the virus attachment, suggesting cholesterol is involved in virus entry. Further results showed that cholesterol depletion did not change expression levels of the PRRSV receptor CD163 in MARC-145, had no effect on clathrin-mediated endocytosis, but disturbed lipid-raft-dependent endocytosis. Collectively, these studies suggest that cholesterol is critical for PRRSV entry, which is likely to be mediated by a lipid-raft-dependent pathway.     

Indirect ELISA with recombinant GP5 for detecting antibodies to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV), which has six structural proteins (GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.     

Structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus of the Arteriviridae family, genomically related to the coronaviruses. PRRSV is the causative agent of both severe and persistent respiratory disease and reproductive failure in pigs worldwide. The PRRSV virion contains a core made of the 123 amino acid nucleocapsid (N) protein, a product of the ORF7 gene. We have determined the crystal structure of the capsid-forming domain of N. The structure was solved to 2.6 A resolution by SAD methods using the anomalous signal from sulfur. The N protein exists in the crystal as a tight dimer forming a four-stranded beta sheet floor superposed by two long alpha helices and flanked by two N- and two C-terminal alpha helices. The structure of N represents a new class of viral capsid-forming domains, distinctly different from those of other known enveloped viruses, but reminiscent of the coat protein of bacteriophage MS2.     

Semen changes in boars after experimental infection with porcine reproductive and respiratory syndrome (PRRS) virus

Eleven boars seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) were trained for semen collection: five boars were inoculated intranasally with 6 x 10(6)TCID(50)/ml of PRRSV (Group A); four boars were inoculated intranasally with 6 x 10(4)TCID(50)/ml (Group B); and two boars were used as uninfected control (Group C). Semen samples were collected at 7-d intervals from 49 d prior to experimental inoculation with PRRSV to 70 d after inoculation, and were examined for sperm volume, sperm concentration, sperm morphology, sperm motility and for the presence of PRRSV. The infection in boars was demonstrated by the reisolation of PRRSV from the serum of all inoculated boars. Rectal temperatures and general health of the boars were clinically normal throughout the trial. Differences were observed in the quality of semen collected from boars after experimental infection with PRRSV. This infection induced a significant decrease in sperm motility and in spermatozoa with normal acrosomes. Of the semen samples tested for virus isolation in swine alveolar macrophages PRRSV was only isolated in 1 boar from Group B. The virus was detected in an additional semen sample in Group A by the production of an antibody titer in a biological assay. All attempts to detect PRRSV by RT-PCR in semen samples were unsuccessful. Nevertheless, from our study it is possible to suggest that the PRRSV can occasionally be transmitted in the semen during the initial phase of the disease.     

Genome-wide association and genomic prediction for yield and component traits of Miscanthus sacchariflorus

Accelerating biomass improvement is a major goal of Miscanthus breeding. The development and implementation of genomic-enabled breeding tools, like marker-assisted selection (MAS) and genomic selection, has the potential to improve the efficiency of Miscanthus breeding. The present study conducted genome-wide association (GWA) and genomic prediction of biomass yield and 14 yield-components traits in Miscanthus sacchariflorus. We evaluated a diversity panel with 590 accessions of M. sacchariflorus grown across 4 years in one subtropical and three temperate locations and genotyped with 268,109 single-nucleotide polymorphisms (SNPs). The GWA study identified a total of 835 significant SNPs and 674 candidate genes across all traits and locations. Of the significant SNPs identified, 280 were localized in mapped quantitative trait loci intervals and proximal to SNPs identified for similar traits in previously reported Miscanthus studies, providing additional support for the importance of these genomic regions for biomass yield. Our study gave insights into the genetic basis for yield-component traits in M. sacchariflorus that may facilitate marker-assisted breeding for biomass yield. Genomic prediction accuracy for the yield-related traits ranged from 0.15 to 0.52 across all locations and genetic groups. Prediction accuracies within the six genetic groupings of M. sacchariflorus were limited due to low sample sizes. Nevertheless, the Korea/NE China/Russia (N = 237) genetic group had the highest prediction accuracy of all genetic groups (ranging 0.26–0.71), suggesting that with adequate sample sizes, there is strong potential for genomic selection within the genetic groupings of M. sacchariflorus. This study indicated that MAS and genomic prediction will likely be beneficial for conducting population-improvement of M. sacchariflorus.     

利用噬菌体随机肽库筛选可结合猪繁殖与呼吸综合症病毒的多肽序列

【目的】采用完整的猪繁殖与呼吸综合症病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)颗粒筛选噬菌体肽库,以获得能高亲和力结合并能抑制该病毒复制的特异性多肽。【方法】用纯化的病毒粒子包被ELISA板,再用M13噬菌体随机12肽库进行筛选。经过3轮淘筛,ELISA鉴定噬菌体单克隆与PRRSV的亲和力,选取与PRRSV具有高亲和力的噬菌体单克隆进行DNA测序,据此推导多肽的氨基酸序列。通过TCID50检测其抗病毒复制能力,同时人工合成FITC标记的展示肽用于PRRSV的检测。【结果】经筛选和鉴定得到17个阳性噬菌体克隆能与PRRSV呈高亲和力结合,DNA测序发现各克隆之间有部分共有基序,其中2个克隆体外能明显抑制PRRSV的复制,使TCID50由10-7.3/0.1mL分别降至10-3.2、10-3.6/0.1mL,而FITC标记该展示肽能够在5mg/L工作浓度检测PRRSV。【结论】通过噬菌体肽库能够筛选到具有抗病毒作用的阳性噬菌体克隆,为进一步开发高效PRRSV的诊断和治疗试剂奠定基础。     

Reconciling individual and population levels of porcine reproductive and respiratory syndrome virus evolution

<正>Dear Editor,Porcine reproductive and respiratory syndrome virus(PRRSV),a member of the family Arteriviridae,represents one of the most challenging pathogens in the swine industry,with serious economic impact.Unfortunately,despite widespread application,vaccination has not been     

Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.     

Whole blood microarray analysis of pigs showing extreme phenotypes after a porcine reproductive and respiratory syndrome virus infection

Background

The presence of variability in the response of pigs to Porcine Reproductive and Respiratory Syndrome virus (PRRSv) infection, and recent demonstration of significant genetic control of such responses, leads us to believe that selection towards more disease resistant pigs could be a valid strategy to reduce its economic impact on the swine industry. To find underlying molecular differences in PRRS susceptible versus more resistant pigs, 100 animals with extremely different growth rates and viremia levels after PRRSv infection were selected from a total of 600 infected pigs. A microarray experiment was conducted on whole blood RNA samples taken at 0, 4 and 7 days post infection (dpi) from these pigs. From these data, we examined associations of gene expression with weight gain and viral load phenotypes. The single nucleotide polymorphism (SNP) marker WUR10000125 (WUR) on the porcine 60 K SNP chip was shown to be associated with viral load and weight gain after PRRSv infection, and so the effect of the WUR10000125 (WUR) genotype on expression in whole blood was also examined.

Results

Limited information was obtained through linear modeling of blood gene differential expression (DE) that contrasted pigs with extreme phenotypes, for growth or viral load or between animals with different WUR genotype. However, using network-based approaches, molecular pathway differences between extreme phenotypic classes could be identified. Several gene clusters of interest were found when Weighted Gene Co-expression Network Analysis (WGCNA) was applied to 4dpi contrasted with 0dpi data. The expression pattern of one such cluster of genes correlated with weight gain and WUR genotype, contained numerous immune response genes such as cytokines, chemokines, interferon type I stimulated genes, apoptotic genes and genes regulating complement activation. In addition, Partial Correlation and Information Theory (PCIT) identified differentially hubbed (DH) genes between the phenotypically divergent groups. GO enrichment revealed that the target genes of these DH genes are enriched in adaptive immune pathways.

Conclusion

There are molecular differences in blood RNA patterns between pigs with extreme phenotypes or with a different WUR genotype in early responses to PRRSv infection, though they can be quite subtle and more difficult to discover with conventional DE expression analyses. Co-expression analyses such as WGCNA and PCIT can be used to reveal network differences between such extreme response groups.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1741-8) contains supplementary material, which is available to authorized users.     

Identifying putative candidate genes and pathways involved in immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection

Differences in gene expression were compared between RNAs from lungs of high (HR) and low (LR) porcine reproductive and respiratory syndrome virus (PRRSV) burden pigs using the swine protein-annotated long oligonucleotide microarray, the Pigoligoarray. Pathway analyses were carried out to determine biological processes, pathways and networks that differ between the LR and HR responses. Differences existed between HR and LR pigs for 16 signalling pathways [P < 0.01/-log (P-value) >1.96]. Top canonical pathways included acute phase response signalling, crosstalk between dendritic cells and natural killer cells and tight junction signalling, with numerous immune response genes that were upregulated (SOCS1, SOD2, RBP4, HLA-B, HLA-G, PPP2R1A and TAP1) or downregulated (IL18, TF, C4BPA, C1QA, C1QB and TYROBP). One mechanism, regulation of complement activation, may have been blocked in HR (PRRSV-susceptible) pigs and could account for the poor clearance of PRRSV by infected macrophages. Multiple inhibiting signals may have prevented effective immune responses in susceptible HR pigs, although some protective genes were upregulated in these pigs. It is likely that in HR pigs, expression of genes associated with protection was delayed, so that the immune response was not stimulated early; thus, PRRSV infection prevented protective immune responses.