Porcine arterivirus attachment to the macrophage-specific receptor sialoadhesin is dependent on the sialic acid-binding activity of the N-terminal immunoglobulin domain of sialoadhesin

The sialic acid-binding lectin sialoadhesin (Sn) is a macrophage-restricted receptor for porcine reproductive and respiratory syndrome virus (PRRSV). To investigate the importance of pSn sialic acid-binding activity for PRRSV infection, an R₁₁₆-to-E mutation was introduced in the predicted sialic acid-binding domain of pSn, resulting in a mutant, pSn^RE, that could not bind sialic acids. PSn, but not pSn^RE, allowed PRRSV binding and internalization. These data show that the sialic acid-binding activity of pSn is essential for PRRSV attachment to pSn and thus identifies the variable, N-terminal domain of Sn as a PRRSV binding domain.     

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.     

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP₃, GP₄ and GP₅ envelope glycoproteins, only the M/GP₅ glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP₅. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.     

Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages

The porcine reproductive and respiratory syndrome virus (PRRSV) has a very restricted tropism for well-differentiated cells of the monocyte-macrophage lineage, which is probably determined by specific receptors on these cells. In this study, the importance of heparinlike molecules on porcine alveolar macrophages (PAM) for PRRSV infection was determined. Heparin interacted with the virus and reduced infection of PAM up to 92 or 88% for the American and European types of PRRSV, respectively. Other glycosaminoglycans, similar to heparin, had no significant effect on infection while heparinase treatment of PAM resulted in a significant reduction of the infection. Analysis of infection kinetics showed that PRRSV attachment to heparan sulfate occurs early in infection. A heparin-sensitive binding step was observed which converted completely into a heparin-resistant binding after 120 min at 4 degrees C. Using heparin-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was observed that the structural matrix (M) and nucleocapsid (N) proteins attached to heparin. Nonreducing SDS-PAGE revealed that M bound to heparin mainly as a complex with glycoprotein GP(5) and that the N protein bound to heparin as a homodimer. GP(3), which was identified as a minor structural protein of European types of PRRSV, did not bind to heparin. Since the N protein is not exposed on the virion surface, it was concluded that the structural M protein and the M-GP(5) complex contribute to PRRSV attachment on a heparinlike receptor on PAM. This is the first report that identifies a PRRSV ligand for a cell surface heparinlike receptor on PAM.     

An Intact Sialoadhesin (Sn/SIGLEC1/CD169) Is Not Required for Attachment/Internalization of the Porcine Reproductive and Respiratory Syndrome Virus

Surface expression of SIGLEC1, also known as sialoadhesin or CD169, is considered a primary determinant of the permissiveness of porcine alveolar macrophages for infection by porcine reproductive and respiratory syndrome virus (PRRSV). In vitro, the attachment and internalization of PRRSV are dependent on the interaction between sialic acid on the virion surface and the sialic acid binding domain of the SIGLEC1 gene. To test the role of SIGLEC1 in PRRSV infection, a SIGLEC1 gene knockout pig was created by removing part of exon 1 and all of exons 2 and 3 of the SIGLEC1 gene. The resulting knockout ablated SIGLEC1 expression on the surface of alveolar macrophages but had no effect on the expression of CD163, a coreceptor for PRRSV. After infection, PRRSV viremia in SIGLEC1^−/− pigs followed the same course as in SIGLEC1^−/+ and SIGLEC1^+/+ littermates. The absence of SIGLEC1 had no measurable effect on other aspects of PRRSV infection, including clinical disease course and histopathology. The results demonstrate that the expression of the SIGLEC1 gene is not required for infection of pigs with PRRSV and that the absence of SIGLEC1 does not contribute to the pathogenesis of acute disease.     

Identification of a Putative Receptor for Porcine Reproductive and Respiratory Syndrome Virus on Porcine Alveolar Macrophages

To identify the receptor which may determine the macrophage tropism of porcine reproductive and respiratory syndrome virus (PRRSV), monoclonal antibodies (MAbs) against porcine alveolar macrophages (PAM) were produced. Two MAbs (41D3 and 41D5) which completely blocked PRRSV infection of PAM were further characterized. It was found that they reduce the attachment of PRRSV to PAM and immunoprecipitate a 210-kDa membrane protein from PAM. This protein was detected on the cell membranes of PAM but not of PRRSV-nonpermissive cells. A colocalization was found between the reactive sites of MAb 41D3 and PRRSV on PAM membranes. All PRRSV-infected cells in tissues of experimentally infected pigs reacted with MAb 41D3. Taken together, all these data suggest that the identified 210-kDa membrane protein is a putative receptor for PRRSV on porcine macrophages.     

Binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.     

Cutting edge: CD43 functions as a T cell counterreceptor for the macrophage adhesion receptor sialoadhesin (Siglec-1)

Sialoadhesin (Siglec-1) is a macrophage-restricted sialic acid-binding receptor that mediates interactions with hemopoietic cells, including lymphocytes. In this study, we identify sialoadhesin counterreceptors on T lymphocytes. Several major glycoproteins (85, 130, 240 kDa) were precipitated by sialoadhesin-Fc fusion proteins from a murine T cell line (TK-1). Binding of sialoadhesin to these glycoproteins was sialic acid dependent and was abolished by mutation of a critical residue (R97A) of the sialic acid binding site in the membrane distal Ig-like domain of sialoadhesin. The 130- and 240-kDa sialoadhesin-binding glycoproteins were identified as the sialomucins CD43 and P-selectin glycoprotein ligand 1 (CD162), respectively. CD43 expressed in COS cells supported increased binding to immobilized sialoadhesin. Finally, sialoadhesin bound different glycoforms of CD43 expressed in Chinese hamster ovary cells, including unbranched (core 1) and branched (core 2) O:-linked glycans, that are normally found on CD43 in resting and activated T cells, respectively. These results identify CD43 as a T cell counterreceptor for sialoadhesin and suggest that in addition to its anti-adhesive role CD43 may promote cell-cell interactions.     

Alpha2,3 and alpha2,6 N-linked sialic acids facilitate efficient binding and transduction by adeno-associated virus types 1 and 6

Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either alpha2,3-linked or alpha2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcalpha2-3GalNAcbeta1-4GlcNAc, as well as two glycoproteins with alpha2,3 and alpha2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support alpha2,3 and alpha2,6 sialic acids that are present on N-linked glycoproteins as primary receptors for efficient AAV1 and AAV6 viral infection.     

The polymorphism analysis of CD169 and CD163 related with the risk of porcine reproductive and respiratory syndrome virus (PRRSV) infection

Porcine reproductive and respiratory syndrome virus (PRRSV) could infect porcine alveolar macrophages (PAM), and the CD169 and CD163 are identified as critical receptors on the surface of PAM, but whether the single nucleotide polymorphisms (SNPs) of these genes could influence the infection is remain unclear. In this study, we identified totally 6 SNPs for CD169 (G1640T, C1654A, C4175T) and CD163 (G2277A, A2552G and C2700A), and evaluated their associations with PRRSV infection using two classified methods in a 524 pig population to investigate the effects of mutations on the PRRSV receptors. The pigs with genotypes of AA of CD169-C1654A, CT of CD169-C4175T and AA of CD163-A2552G appeared to resistant to the PRRSV infection by the combination of two classified results. The results provided fundamental molecular investigation to promote pig breeding with disease resistance. However, the identification of functional changes induced by SNPs and molecular mechanism were need further research.     

Sialic acid functions in enterovirus 70 binding and infection

The interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decay-accelerating factor (DAF/CD55) is an attachment receptor for enterovirus 70 (EV70), a member of the Picornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.     

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。     

N-Linked Glycosylation of GP5 of Porcine Reproductive and Respiratory Syndrome Virus Is Critically Important for Virus Replication In Vivo

It has been proposed that the N-linked glycan addition at certain sites in GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) is important for production of infectious viruses and viral infectivity. However, such specific N-linked glycosylation sites do not exist in some field PRRSV isolates. This implies that the existence of GP5-associated glycan per se is not vital to the virus life cycle. In this study, we found that mutation of individual glycosylation sites at N30, N35, N44, and N51 in GP5 did not affect virus infectivity in cultured cells. However, the mutants carrying multiple mutations at N-linked glycosylation sites in GP5 had significantly reduced virus yields compared with the wild-type (wt) virus. As a result, no viremia and antibody response were detected in piglets that were injected with a mutant without all N-linked glycans in GP5. These results suggest that the N-linked glycosylation of GP5 is critically important for virus replication in vivo. The study also showed that removal of N44-linked glycan from GP5 increased the sensitivity of mutant virus to convalescent-phase serum samples but did not elicit a high-level neutralizing antibody response to wt PRRSV. The results obtained from the present study have made significant contributions to better understanding the importance of glycosylation of GP5 in the biology of PRRSV.     

携带CD163报告载体的猪诱导多能干细胞株的建立

猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)是包括猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)在内的多种高致病病毒的受体细胞,是研究病毒与宿主互作机制的重要模型。然而PAM来源有限,难以满足当前需求。利用猪诱导性多能干细胞(induced pluripotent stem cells, iPSCs)向巨噬细胞定向诱导是解决PAM细胞数量不足的有效方法。CD163是PAM细胞的重要标记,也是PRRSV等病毒的主要受体。建立实时报告CD163激活程度的报告系统对于建立并优化猪iPSCs向PAM的诱导分化体系具有指导意义。本研究利用CRISPR/Cas9介导的基因编辑系统,设计靶向CD163终止密码子的sgRNA并构建相应的打靶载体,将其导入到猪PAM中的检测报告系统。进一步将该报告系统导入猪iPSCs中,通过碱性磷酸酶染色、免疫荧光染色和EDU染色等手段来检测其安全性。将猪内源CD163的报告载体系统转染至原代PAM中,检测到了红色荧光的表达,证明了该载体系...     

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.     

A comparative study of the asparagine-linked oligosaccharides on siglec-5, siglec-7 and siglec-8, expressed in a CHO cell line, and their contribution to ligand recognition.

The siglecs (sialic acid-binding immunoglobulin-like lectins) mediate sialic acid-dependent cellular interactions and may in some cases signal through SH2-binding domains. In addition to the previously characterized siglecs, sialoadhesin, CD22, CD33 and myelin-associated glycoprotein, several new ones, siglec-5, siglec-7 and siglec-8, have recently been cloned. Although these novel receptors have generated considerable interest as therapeutic targets because of their expression pattern on immune cells, very little is known about how their lectin activity is regulated. Previous studies with sialoadhesin, CD22 and CD33 have shown that siglec glycosylation has significant effects on binding. To determine any differences in the glycan composition of siglec-5, siglec-7 and siglec-8 that may modify their function, we released and characterized the N-linked oligosaccharide distribution in these three glycoproteins. The glycan pools from siglec-5 and siglec-7 contained a larger proportion of sialylated and core-fucosylated biantennary, triantennary and tetra-antennary oligosaccharides, whereas the carbohydrate mixture released from siglec-8 is noticeably less sialylated and is more abundant in 'high-mannose'-type glycans. In addition, we show that, in contrast with CD22 and CD33, mutating the conserved potentially N-linked glycosylation site in the first domain has no effect on binding mediated by siglec-5 or siglec-7.     

Direct correlation between sialic acid binding and infection of cells by two human polyomaviruses (JC virus and BK virus)

For the human polyomaviruses JC virus (JCV) and BK virus (BKV), the first step to a successful infection involves binding to sialic acid moieties located on the surfaces of host cells. By stripping and then reconstituting specific sialic acid linkages on host cells, we show that JCV uses both α(2,3)-linked and α(2,6)-linked sialic acids on N-linked glycoproteins to infect cells. For both JCV and BKV, the sialic acid linkages required for cell surface binding directly correlate with the linkages required for infection. In addition to sialic acid linkage data, these data suggest that the third sugar from the carbohydrate chain terminus is important for virus binding and infection.     

Oligosaccharides as receptors for JC virus

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy. Its hemagglutination activity and entry into host cells have been reported to depend on an N-linked glycoprotein containing sialic acid. In order to identify the receptors of JCV, we generated virus-like particles (VLP) consisting of major viral capsid protein VP1. We then developed an indirect VLP overlay assay to detect VLP binding to glycoproteins and a panel of glycolipids. We found that VLP bound to sialoglycoproteins, including alpha1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on alpha2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal alpha2-3- or alpha2-6-linked sialic acid or the branched alpha2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal alpha2-6-linked sialic acid had the highest affinity for VLP, inhibited the hemagglutination activity of VLP and JCV, and inhibited the attachment of VLP to cells. We also demonstrate that VLP bound to specific glycolipids, such as lactosylceramide, and gangliosides, including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and that VLP bound weakly to GD1a but did not bind to GM1a, GM2, or galactocerebroside. Furthermore, the neoglycoprotein containing the terminal alpha2-6-linked sialic acid and the ganglioside GT1b inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycoproteins and glycolipids work as JCV receptors and may be feasible as anti-JCV agents.     

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.