Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a serious threat to the swine industry worldwide. Exostosin glycosyltransferase 1 (EXT1), an enzyme involved in the biosynthesis of heparin sulfate, has also been reported to be a host factor essential for a wide variety of pathogens. However, the role of EXT1 in PRRSV infection remains uncharted. Here, we identified that PRRSV infection caused an increase of EXT1 expression. EXT1 knockdown promoted virus infection, whereas its overexpression inhibited virus infection, suggesting an inhibitory function of EXT1 to PRRSV infection. We found that EXT1 had no effects on the attachment, internalization, or release of PRRSV but did restrict viral RNA replication. EXT1 was determined to interact with viral nonstructural protein 3 (nsp3) and nsp5 via its N-terminal cytoplasmic tail and to enhance K48-linked polyubiquitination of these two nsps to promote their degradation. Furthermore, the C-terminal glycosyltransferase activity domain of EXT1 was necessary for nsp3 and nsp5 degradation. We also found that EXT2, a EXT1 homolog, interacted with EXT1 and inhibited PRRSV infection. Similarly, EXT1 effectively restricted porcine epidemic diarrhea virus and porcine enteric alphacoronavirus infection in Vero cells. Taken together, this study reveals that EXT1 may serve as a broad-spectrum host restriction factor and suggests a molecular basis for the potential development of therapeutics against PRRSV infection.     

Genetic analysis of ORF5 porcine reproductive and respiratory syndrome virus isolated in Vietnam

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens because it is highly infectious and causes economic losses due to decreased pig productivity. In this study, the 603 bp complete major envelope protein encoding gene (ORF5) of 32 field PRRSV isolates from Vietnam collected during 2008–2012 were sequenced and analyzed. Multiple nucleotide (nt) and deduced amino acid (aa) alignments of ORF5 were performed on the 32 isolates: the representative strains (European and North American genotypes), Chinese strains available in GenBank and vaccine strains licensed for use in Vietnam. The results showed 94.8–100.0% nt identity and 94.0–100% aa similarity among the 32 isolates. These isolates shared similarities with the prototype of the North American PRRSV strain (VR‐2332; nt 87.8–89.3%, aa 87.5–90.0%), and Lelystat virus, the prototype of the European PRRSV strain (LV; nt 61.1–61.9%, aa 55.1‐57.0%). There was greater similarity with QN07 (nt 96.5‐98.5%, aa 96.0‐99.0%) from the 2007 PRRS outbreak in QuangNam Province, CH‐1a (nt 93.2–95.1%, 91.5–93.5%) isolated in China in 1995 and JXA1 (nt 96.5–98.6%, aa 95.0–98.0%), the highly pathogenic strain from China isolated in 2006. The Vietnamese isolates were more similar to JXA1‐R (nt 96.5–98.6%, aa 95.0–98.0%), the strain used in Chinese vaccines, than to Ingelvac MLV/BSL‐PS (nt 87.2–89.0%, aa 86.0–89.0%). Phylogenetic analysis showed that the 32 isolates were of the North American genotype and classified into sub‐lineage 8.7. This sub‐lineage contains highly pathogenic Chinese PRRSV strains. This study documents genetic variation in circulating PRRSV strains and could assist more effective use of PRRS vaccines in Vietnam.     

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.     

Genetic analysis of ORF5 in porcine reproductive and respiratory syndrome virus in Japan

In recent years, no reports regarding genetic information on porcine reproductive and respiratory syndrome virus (PRRSV) with a focus on Japan have been published. To clarify the effect of time on PRRSV genomic evolution, we sequenced the open reading frame 5 (600 or 603 bases) obtained from Japanese PRRSV isolates for three periods (1992-1993, 2000-2001, and 2007-2008) and compared their phylogenetic relationships. Assessment of mean pairwise homology of nucleotide sequences of PRRSV isolates indicated a trend towards increasing heterogeneity over time. In addition, we newly detected a virus classified in cluster IV, indicative of the increasing genetic variation of PRRSV in Japan.     

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.     

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.

The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.     

猪繁殖与呼吸障碍综合征病毒(PRRSV)3′末端非翻译区中调控序列的研究

以北美株PRRSV感染性克隆pCBC2为平台进行反向遗传操作,将3′UTR中的一级结构进行了系列缺失或插入突变,并改变二级结构中的一个保守的茎环结构,构建全长PRRSV突变体克隆,解析3′UTR突变对病毒感染性的影响,旨在界定调控PRRSV3′UTR的启动子序列及二级结构,即复制过程中的最小调控元件。以空斑和Northern blot来研究拯救后重组病毒的复制、转录和生长特性,发现重组病毒感染动力学与亲本病毒无可见差别。结果表明PRRSV3′UTR的5′端可耐受一定数目的核苷酸的缺失(41nt)与插入(23nt)突变,但进一步9nt缺失造成保守的环结构突变后就使病毒失去了感染性。证明了这是3′UTR中控制PRRSV复制过程的的必需序列及二级结构,为进一步解析PRRSV复制过程的调控元件奠定了基础。     

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.     

体外共表达的猪繁殖与呼吸综合征病毒GP5和M蛋白可形成异源二聚体

为了探讨猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因编码的GP5蛋白和ORF6编码的M蛋白体外共表达特性,分别构建了PRRSV ORF5、ORF6单基因或双基因共表达的真核表达质粒pCI-ORF5、pCI-ORF6和pCI-ORF5/ORF6,转染BHK-21细胞,Western blot检测证实共表达的GP5和M蛋白能够形成异源二聚体。同时,以绿色荧光蛋白(EGFP)和红色荧光蛋白(RFP)为示踪,发现当ORF5-EGFP和ORF6-RFP共表达时,能促进GP5蛋白从内质网向高尔基体转运,提示GP5-M异源二聚体的形成可能与GP5蛋白的翻译后修饰、转运、定位有关。     

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.     

Application of GP5 protein to develop monoclonal antibody against porcine reproductive and respiratory syndrome virus

In this study,a panel of monoclonal antibodies (mAbs) against Porcine reproductive and respiratory syndrome virus(PRRSV),named as 8C9 and4B4,were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV (TCID50=5.5),screened by the indirect ELISA and subjected to several limiting dilutions.mAbs were then identified by biological characterization.Among the two fusion cell strains,8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass.The titers in cell culture supernatant and abdomen liquor reached to 1:104and 1:105,respectively.The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively,and no reaction with Classical swine fever virus (CSFV) or Swine vesicular disease virus (SVDV).The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa,respectively.In neutralization activity tests,the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1:512,but mAB 8C9 has no neutralization activities to PRRSV.     

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.     

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.     

Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain

After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

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