Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with GP5 in PRRSV-Infected PAMs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus-host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development.

The Molecular and Function Characterization of Porcine MID2.

Midline2 (MID2/TRIM1) is a member of the tripartite motif-containing (TRIM) family, which is involved in a wide range of cellular processes. However, fundamental studies on porcine MID2 (pMID2) are still lacking. In this study, we identified and characterized the full length MID2 gene of pig (Sus scrofa). The sequence alignment analysis results showed that pMID2 had an N-terminal RING zinc-finger domain, BBC domain, and C-terminal COS box, FN3 motif, and PRY-SPRY domain that were conserved and similar to those of other vertebrates. Furthermore, pMID2 had the highest expression levels in porcine lung and spleen. Serial deletion and site-directed mutagenesis showed that the putative nuclear factor-κB (NF-κB) binding site may be an essential transcription factor for regulating the transcription expression of pMID2. Furthermore, the immunofluorescence assay indicated that pMID2 presented in the cell membrane and cytoplasm. To further study the functions of pMID2, we identified and determined its potential ability to perceive poly (I:C) and IFN-α stimulation. Stimulation experiments showed pMID2 enhanced poly (I:C)-/IFN-α-induced JAK-STAT signaling pathway, indicating that pMID2 might participate in the immune responses. In conclusion, we systematically and comprehensively analyzed the characterizations and functions of pMID2, which provide valuable information to explore the pMID2 functions in innate immunity. Our findings not only enrich the current knowledge of MID2 in IFN signaling regulation but also offer the basis for future research of pig MID2 gene.

The Host E3-Ubiquitin Ligase TRIM28 Impedes Viral Protein GP4 Ubiquitination and Promotes PRRSV Replication.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a highly pathogenic porcine virus that brings tremendous economic losses to the global swine industry. PRRSVs have evolved multiple elegant strategies to manipulate the host proteins and circumvent against the antiviral responses to establish infection. Therefore, the identification of virus-host interactions is critical for understanding the pathogenesis of PRRSVs. Tripartite motif protein 28 (TRIM28) is a transcriptional co-repressor involved in the regulation of viral and cellular transcriptional programs; however, its precise role in regulating PRRSV infection remains unknown. In this study, we found that the mRNA and protein levels of TRIM28 were up-regulated in PRRSV-infected porcine alveolar macrophages (PAMs) and MARC-145 cells. Ectopic TRIM28 expression dramatically increased viral yields, whereas the siRNA-mediated knockdown of TRIM28 significantly inhibited PRRSV replication. Furthermore, we used a co-immunoprecipitation (co-IP) assay to demonstrate that TRIM28 interacted with envelope glycoprotein 4 (GP4) among PRRSV viral proteins. Intriguingly, TRIM28 inhibited the degradation of PRRSV GP4 by impeding its ubiquitination. Taken together, our work provides evidence that the host E3-ubiquitin ligase TRIM28 suppresses GP4 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM28, as a potential target in the development of anti-viral drugs against PRRSV.

Immune Molecules' mRNA Expression in Porcine Alveolar Macrophages Co-Infected with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) are economically important pathogens in swine, and pigs with dual infections of PCV2 and PRRSV consistently have more severe clinical symptoms and interstitial pneumonia. However, the synergistic pathogenesis mechanism induced by PRRSV and PCV2 co-infection has not yet been illuminated. Therefore, the aim of this study was to characterize the kinetic changes of immune regulatory molecules, inflammatory factors and immune checkpoint molecules in porcine alveolar macrophages (PAMs) in individuals infected or co-infected with PRRSV and/or PCV2. The experiment was divided into six groups: a negative control group (mock, no infected virus), a group infected with PCV2 alone (PCV2), a group infected with PRRSV alone (PRRSV), a PCV2-PRRSV co-infected group (PCV2-PRRSV inoculated with PCV2, followed by PRRSV 12 h later), a PRRSV-PCV2 co-infected group (PRRSV-PCV2 inoculated with PRRSV, followed by PCV2 12 h later) and a PCV2 + PRRSV co-infected group (PCV2 + PRRSV, inoculated with PCV2 and PRRSV at the same time). Then, PAM samples from the different infection groups and the mock group were collected at 6, 12, 24, 36 and 48 h post-infection (hpi) to detect the viral loads of PCV2 and PRRSV and the relative quantification of immune regulatory molecules, inflammatory factors and immune checkpoint molecules. The results indicated that PCV2 and PRRSV co-infection, regardless of the order of infection, had no effect on promoting PCV2 replication, while PRRSV and PCV2 co-infection was able to promote PRRSV replication. The immune regulatory molecules (IFN-α and IFN-γ) were significantly down-regulated, while inflammatory factors (TNF-α, IL-1ß, IL-10 and TGF-ß) and immune checkpoint molecules (PD-1, LAG-3, CTLA-4 and TIM-3) were significantly up-regulated in the PRRSV and PCV2 co-infection groups, especially in PAMs with PCV2 inoculation first followed by PRRSV. The dynamic changes in the aforementioned immune molecules were associated with a high viral load, immunosuppression and cell exhaustion, which may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions by dual infection with PCV2 and PRRSV in PAMs.

Activating Fc Gamma Receptors and Viral Receptors Are Required for Antibody-Dependent Enhancement of Porcine Reproductive and Respiratory Syndrome Virus Infection.

Antibody-dependent enhancement (ADE) is an event in preexisting sub-, or non-neutralizing antibodies increasing the viral replication in its target cells. ADE is one crucial factor that intensifies porcine reproductive and respiratory syndrome virus (PRRSV) infection and results in PRRSV-persistent infection. Nevertheless, the exact mechanisms of PRRSV-ADE infection are poorly understood. In the current research, the results of the ADE assay showed that porcine immunoglobulin G (IgG) specific for the PRRSV significantly enhanced PRRSV proliferation in porcine alveolar macrophages (PAMs), suggesting that the ADE activity of PRRSV infection existed in pig anti-PRRSV IgG. The results of the RNA interference assay showed that knockdown of the Fc gamma receptor I (FcγRI) or FcγRIII gene significantly suppressed the ADE activity of PRRSV infection in PAMs, suggesting that FcγRI and FcγRIII were responsible for mediating PRRSV-ADE infection. In addition, the results of the antibody blocking assay showed that specific blocking of the Sn1, 2, 3, 4, 5, or 6 extracellular domain of the sialoadhesin (Sn) protein or selective blockade of the scavenger receptor cysteine-rich (SRCR) 5 domain of the CD163 molecule significantly repressed the ADE activity of PRRSV infection in PAMs, suggesting that Sn and CD163 were involved in FcγR-mediated PRRSV-ADE infection. The Sn1-6 domains of porcine Sn protein and the SRCR 5 domain of porcine CD163 molecule might play central roles in the ADE of PRRSV infection. In summary, our studies indicated that activating FcγRs (FcγRI and FcγRIII) and viral receptors (Sn and CD163) were required for ADE of PRRSV infection. Our findings provided a new insight into PRRSV infection that could be enhanced by FcγRs and PRRSV receptors-mediated PRRSV-antibody immune complexes (ICs), which would deepen our understanding of the mechanisms of PRRSV-persistent infection via the ADE pathway.

Differences in Humoral Immune Response against the Type 2 Porcine Reproductive and Respiratory Syndrome Virus via Different Immune Pathways.

The intramuscular vaccine is the principal strategy to protect pigs from porcine reproductive and respiratory syndrome virus (PRRSV), However, it is still difficult to control PRRSV effectively. This study infected piglets with PRRSV through intramuscular and intranasal inoculation. Subsequently, viral loads, anti-PRRSV antibody levels, and neutralizing antibodies (NAs) titers in both serum and saliva were monitored for 43 days. Meanwhile, tissues were obtained through necropsy at 43 days post-inoculation (dpi) to detect viral loads. The results indicated that viremia lasted from 3 to 31 dpi in both the inoculation groups, but the viruses survived in the lungs and lymph nodes after viremia clearance. The antibody response was detected from 11 dpi, but the response of NAs was delayed until 3-4 weeks. Furthermore, intranasal inoculation induced lower viral load levels than injection inoculation. In addition, positive SIgA and NAs levels were produced early, with higher levels through intranasal inoculation. Therefore, our data indicated that a more robust antibody response and lower virus loads could be induced by intranasal inoculation, and mucosal inoculation could be a suitable pathway for PRRSV vaccines.

MicroRNA-376b-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication by Targeting Viral Restriction Factor TRIM22.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically significant pathogen and has evolved several strategies to evade host antiviral response and provide favorable conditions for survival. In the present study, we demonstrated that a host microRNA, miR-376b-3p, was upregulated by PRRSV infection through the viral components, nsp4 and nsp11, and that miR-376b-3p can directly target tripartite motif-containing 22 (TRIM22) to impair its anti-PRRSV activity, thus facilitating the replication of PRRSV. Meanwhile, we found that TRIM22 induced degradation of the nucleocapsid protein (N) of PRRSV by interacting with N protein to inhibit PRRSV replication, and further study indicated that TRIM22 could enhance the activation of the lysosomal pathway by interacting with LC3 to induce lysosomal degradation of N protein. In conclusion, PRRSV increased miR-376b-3p expression and hijacked the host miR-376b-3p to promote PRRSV replication by impairing the antiviral effect of TRIM22. Therefore, our finding outlines a novel strategy of immune evasion exerted by PRRSV, which is helpful for better understanding the pathogenesis of PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes enormous economic losses each year in the swine industry worldwide. MicroRNAs (miRNAs) play important roles during viral infections via modulating the expression of viral or host genes at the posttranscriptional level. TRIM22 has recently been identified as a key restriction factor that inhibited the replication of a number of human viruses, such as HIV, encephalomyocarditis virus (ECMV), hepatitis C virus (HCV), HBV, influenza A virus (IAV), and respiratory syncytial virus (RSV). In this study, we showed that host miR-376b-3p could be upregulated by PRRSV and functioned to impair the anti-PRRSV role of TRIM22 to facilitate PRRSV replication. Meanwhile, we found that TRIM22 inhibited the replication of PRRSV by interacting with viral N protein and accelerating its degradation through the lysosomal pathway. Collectively, the findings reveal a novel mechanism that PRRSV used to exploit the host miR-376b-3p to evade antiviral responses and provide new insight into the study of virus-host interactions.

Proteomic Characterization of PAMs with PRRSV-ADE Infection.

The antibody-dependent enhancement (ADE) effect of a PRRSV infection is that the preexisting sub- or non-neutralizing antibodies specific against PRRSV can facilitate the virus entry and replication, and it is likely to be a great obstacle for the selection of immune strategies and the development of high-efficiency PRRSV vaccines. However, the proteomic characterization of primary alveolar macrophages (PAMs) with a PRRSV-ADE infection has not yet been investigated so far. Therefore, we performed a tandem mass tag (TMT)-based quantitative proteomic analysis of PAMs with a PRRSV-ADE infection in this study. The results showed that a total of 3935 differentially expressed proteins (DEPs) were identified in the PAMs infected with PRRSV-ADE, including 2004 up-regulated proteins and 1931 down-regulated proteins. Further, the bioinformatics analysis for these DEPs revealed that a PRRSV-ADE infection might disturb the functions of ribosome, proteasome and mitochondria. Interestingly, we also found that the expression of the key molecules in the innate immune pathways and antiviral proteins were significantly down-regulated during a PRRSV-ADE infection. This study was the first attempt to analyze the proteomic characterization of PAMs with a PRRSV-ADE infection in vitro. Additionally, the findings will provide valuable information for a better understanding of the mechanism of virus-antibody-host interactions during a PRRSV-ADE infection.

Activation of Fc gamma receptor IIb up-regulates the production of interferon-alpha and interferon-gamma in porcine alveolar macrophages during PRRSV infection.

Porcine Fc gamma receptor IIb (FcγRIIb) has been cloned and characterized for many years. However, the role of FcγRIIb in innate antiviral response to porcine reproductive and respiratory syndrome virus (PRRSV) infection has not yet been well investigated. In current study, our results showed that specific activation of FcγRIIb in porcine alveolar macrophages (PAMs) significantly enhanced the production of interferon-alpha (IFN-α) and interferon-gamma (IFN-γ), and significantly repressed the production of transforming growth factor beta 1 (TGF-ß1). In addition, our results showed that specific activation of FcγRIIb in PAMs cells in PRRSV infection not only significantly increased the production of IFN-α and IFN-γ, but also significantly decreased the production of TGF-ß1, and significantly inhibited PRRSV replication level. In summary, our studies indicated that FcγRIIb signaling up-regulated the production of IFN-α and IFN-γ in PAMs cells in vitro, in response to PRRSV infection.

Antibody-Mediated Porcine Reproductive and Respiratory Syndrome Virus Infection Downregulates the Production of Interferon-α and Tumor Necrosis Factor-α in Porcine Alveolar Macrophages via Fc Gamma Receptor I and III.

Antibody-dependent enhancement (ADE) contributes to the pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV)-persistent infection. However, the mechanisms of PRRSV-ADE infection are still confusing. A clear understanding of the event upon virus infection by the ADE pathway has become crucial for developing efficient intervention of the PRRSV infection. In this study, an ADE assay showed that PRRSV-ADE infection in porcine alveolar macrophages (AMs) significantly decreased the production of interferon-α (IFN-α) and tumor necrosis factor-α (TNF-α), and significantly increased the production of interleukine-10 (IL-10). A gene knockdown assay based on small interfering RNA (siRNA) showed that both Fc gamma receptor I (FcγRI) and FcγRIII in porcine AMs were involved in PRRSV-ADE infection. An activation assay showed that specific activation of FcγRI or FcγRIII in porcine AMs during PRRSV infection not only significantly decreased the production of IFN-α and TNF-α, but also significantly increased the production of IL-10 and significantly facilitated PRRSV replication. In conclusion, our studies suggested that ADE downregulated the production of IFN-α and TNF-α in porcine AMs maybe via FcγRI and FcγRIII, thereby leading to enhanced PRRSV infection.

Activation of activating Fc gamma receptors down-regulates the levels of interferon ß, interferon γ and interferon λ1 in porcine alveolar macrophages during PRRSV infection.

Porcine activating Fc gamma receptors (FcγRI and FcγRIII) have been cloned and characterized for many years. However, their roles in interferon (IFN) antiviral immune response to porcine reproductive and respiratory syndrome virus (PRRSV) infection have not yet been investigated extensively. In this study, PRRSV infection assay showed that PRRSV increased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1 in porcine alveolar macrophages (PAMs) in early infection and decreased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1 in PAMs in late infection. Activation assay showed that specific activation of FcγRI or FcγRIII in PAMs decreased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1 and increased significantly the transcription of transforming growth factor ß1 (TGF-ß1). PRRSV infection assay mediated by FcγRI and FcγRIII showed that specific activation of FcγRI or FcγRIII in PAMs during PRRSV infection decreased significantly the transcription of IFN-ß, IFN-γ and IFN-λ1, but increased significantly the transcription of TGF-ß1 and enhanced significantly viral replication. In conclusion, our studies suggested that activating FcγR signaling inhibited the transcriptional levels of IFN-ß, IFN-γ and IFN-λ1 in PAMs in response to PRRSV infection.

Activation of sialoadhesin down-regulates the levels of innate antiviral cytokines in porcine alveolar macrophages in vitro.

Porcine sialoadhesin (pSn) is a crucial porcine reproductive and respiratory syndrome virus (PRRSV) receptor mediating the attachment and internalization of virus into its major target cells, porcine alveolar macrophages (PAMs). However, the role of pSn in innate antiviral immune response has not yet been investigated. In this study, our results showed that PRRSV down-regulated significantly the mRNA levels of IFN-α, IFN-ß, IFN-γ, IFN-λ1, IFN-λ3 and IFN-λ4 and up-regulated significantly the mRNA levels of IL-10 and pSn in infected PAMs in vitro, suggesting that PRRSV infection inhibited the transcription of innate antiviral cytokines in host cells. Our results also showed that selective activation of pSn down-regulated significantly the mRNA levels of IFN-α, IFN-ß, IFN-γ, IFN-λ1, IFN-λ3, IFN-λ4 and TNF-α and up-regulated significantly the mRNA level of IL-10 in PAMs in vitro, suggesting that pSn signaling inhibited the transcription of innate antiviral cytokines. Further results showed that pSn1, pSn2, pSn3, pSn4 and pSn5 domains of pSn were responsible for the inhibition of levels of innate antiviral cytokines. In conclusion, our results suggested that pSn suppressed innate antiviral immune response by down-regulating the levels of innate antiviral cytokines in PAMs. It was possible that PRRSV-pSn interaction may suppress innate antiviral immune response to PRRSV infection by repressing the production of innate antiviral cytokines.

Genetic diversity and phylogenetic analysis of the ORF5 gene of PRRSV from central China.

To more fully understand the genetic diversity and molecular epidemiology of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan province of China, 112 full-length ORF5 gene sequences, originating from Henan province between 2006 and 2015, were subjected to sequence variation and phylogenetic analysis. Phylogenetic analysis revealed that all Henan isolates belonged to the Type 2 genotype and could be further divided into three subgroups. Subgroup 1 and 2 viruses predominated in Henan and subgroup 2 overtook subgroup 1 as the most prevalent PRRSV between 2006 and 2015. Highly pathogenic PRRSV (HP-PRRSV) isolates predominated in Henan and eight RespPRRSV MLV vaccine-like isolates were observed in subgroup 3. Sequence variation analysis revealed that the ORF5 genes of all Henan isolates shared >83.3% nucleotide and >80.1% amino acid sequence identity with each other. Primary neutralizing epitope (PNE) analysis revealed that, relative to the attenuated RespPRRSV MLV vaccine isolate, all but one of the subgroup 1 Henan isolates had mutations at amino acid 39 within the key PNE of GP5. Analysis of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in GP5 revealed that all but two of the Henan isolates had a highly conserved sequence between amino acids 77 and 82 positions of GP5. N-linked glycosylation site (NGS) analysis revealed a novel potential NGS at GP5 amino acid position 59 in two of the subgroup 2 Henan isolates. Another novel GP5 amino acid mutation (44N→44D) was found in a single subgroup 1 Henan isolate (HeNan-A9) in a glycosylation site that is known to be crucial for PRRSV infectivity.

Ligation of porcine Fc gamma receptor III inhibits levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto macrophages through Fc receptor-mediated endocytosis. FcγR III is the activating receptor with a tyrosine-based activating motif (ITAM) in its cytoplasmic tail, where up-regulates phagocytosis. However, porcine FcγR III's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγR III in PAM cells down-regulated significantly mRNA levels of IFN-α and TNF-α post-pretreatment, and up-regulated significantly mRNA level of IL-10 post-pretreatment, suggesting that porcine FcγR III signal can inhibit the transcriptional levels of innate antiviral cytokine in host cells. Simultaneously, PRRSV infection assay mediated by FcγR III indicated that selective activation of porcine FcγR III in PAM cells inhibited significantly mRNA levels of IFN-α and TNF-α, and enhanced significantly mRNA level of IL-10, and increased significantly viral mRNA levels, in response to PRRSV infection, suggesting that FcγR III ligation can inhibit the antiviral immune response to PRRSV infection. These results elucidated that the one mechanism of PRRSV-ADE regulated via porcine FcγRIII may be by decreasing antiviral cytokine levels, facilitating viral replication.

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China.

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003-2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003-2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.

Ligation of porcine Fc gamma receptor I inhibits levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto macrophages through Fc receptor-mediated endocytosis. FcγRI is the activating receptor with a tyrosine-based activating motif (ITAM) in its cytoplasmic tail, where up-regulates phagocytosis. However, porcine FcγRI's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγRI in PAM cells down-regulated significantly mRNA levels of IFN-α and TNF-α post-pretreatment, suggesting that porcine FcγRI signal can inhibit the innate antiviral response of host cells. PRRSV infection assay mediated by FcγRI indicated that selective activation of porcine FcγRI in PAM cells inhibited significantly mRNA levels of antiviral cytokine (IFN-α and TNF-α) in response to PRRSV infection, suggesting that FcγRI ligation can inhibit the antiviral immune response to PRRSV infection.

Genetic variation and phylogenetic analysis of porcine circovirus type 2 infections in central China.

Porcine circovirus type 2 (PCV2) infection causes postweaning multisystemic wasting syndrome and porcine circovirus-associated diseases in many regions. A total of 77 sequences, including 31 sampled from Henan province of China, were retrieved from GenBank and subjected to amino acid variation and phylogenetic analyses. The two PCV genotypes prevailing in Henan were PCV-2a and PCV-2b with PCV-2b accounting for 93.5 % (29/31) of the Henan isolates. The 31 Henan isolates all shared between 92.7 and 100 % sequence similarity. Amino acid variation analysis of the capsid protein revealed that Henan PCV2 strains tended to accumulate more substitutions within epitopic regions-a substitution pattern consistent with host immune system-mediated selection of virus immune escape variants. The analysed PCV sequences carry evidence of at least six unique recombination events. Selective pressure analysis of the relative recombination-free ORF2 sequences of these viruses revealed evidence of sites that are likely evolving in response to host-driven immune pressures-a finding that coupled with information on the prevalent diversity in Henan PCV2 isolates of known immunoreactive genomic loci will aid in future studies aiming to assess the evolutionary responses of PCV2 in China to the widespread deployment of anti-PCV vaccines in the country.

Ligation of Fc gamma receptor IIB enhances levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto its host cells, such as monocytes and macrophages, through Fc receptor-mediated endocytosis. FcγRIIB is the only inhibitory receptor with a tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, where counters the "ITAM triggered" activation signals and down-regulates phagocytosis. However, porcine FcγRIIB's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγRIIB in PAM cells up-regulated significantly mRNA levels of IFN-α and TNF-α at any time point post-pretreatment, suggesting that porcine FcγRIIB signal can enhance the innate antiviral response of host cells. PRRSV infection assay mediated by FcγRIIB indicated that selective activation of porcine FcγRIIB in PAM cells enhanced mRNA levels of antiviral cytokine (IFN-α and TNF-α) and repressed mRNA levels of IL-10 in response to PRRSV infection, suggesting that FcγRIIB ligation can enhance the antiviral immune response to PRRSV infection. In addition, FcγRIIB ligation to infection indicated that PRRSV replication in PAM was not positive correlation with increasing of IFN-α mRNA levels and decreasing of IL-10 mRNA levels, suggesting that there is complex viral replication mechanism in immune cells such as PAM for PRRSV.

Porcine Fc gamma RIIb sub-isoforms are generated by alternative splicing.

Receptors for the Fc portion of IgG (FcγRs) are expressed on various leukocytes and they modulate both humoral and cell-mediated immune responses with different capacities for IgG binding and phagocytosis. Four different types of FcγRs, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIV, have been identified. There are three FcγRII isoforms (activating FcγRIIa and FcγRIIc, and inhibitory FcγRIIb) in humans, one isoform (inhibitory FcγRIIb) in mice, and two isoforms (inhibitory FcγRIIb and activating FcγRIIc) in cattle. Two alternativly spliced isoforms of FcγRIIb, b1 and b2, have been identified in humans, mice and cattle, however, only two porcine FcγRIIb transcripts have been reported. In this study, we report the identification of three new porcine FcγRIIb transcript and analyze the sequences of five porcine FcγRIIb transcript generated by alternative splicing. The porcine transcript 1 and porcine transcript 2 have a high homology and structural similarity with human b1 and b2, respectively, while there is only one alanine residue difference at the signal peptide region between porcine transcript 1 and transcript 4, as well as porcine transcript 2 and transcript 3. This is the first time that an alternativly spliced isoform of porcine transcript 5 is described in pigs rather than humans or other animals. All the five transcripts have the consensus sequence of an ITIM (ITYSLL) in their cytoplasmic tails. Analysis results indicate that the five transcripts serve as inhibitory receptors and are these sub-isoforms or alternativly spliced isoforms. Immunoglobulin-binding assays show that transcript 1, transcript 2, transcript 3 and transcript 4 have binding activity for IgG immune complexes, whereas transcripts 5 without domain 2 can not bind IgG-complexes. It is now clear that porcine FcγRIIb exists as five sub-isoforms at least. These sub-isoforms may individually modulate FcγRIIb-mediated immune responses in the porcine immune system.

Molecular cloning and characterization of a porcine Fc gamma RIIb sub-isoform(FcγRIIb1).

Receptors for the Fc fragment of IgG (FcγRs) constitute one of the main effector mechanisms through which IgG immune complexes exert their action. Four FcγRs, FcγRI (CD64) with high affinity, FcγRI with intermediate affinity, FcγRII (CD32) and FcγRIII (CD16) with low affinity, have been identified. There are three FcγRII isoforms (activating FcγRIIa and FcγRIIc, and inhibiting FcγRIIb) existing in humans, one isoform in mice (inhibiting FcγRIIb), and two isoforms in cattle (inhibiting FcγRIIb, activating FcγRIIc). Two splice sub-isoforms of FcγRIIb, FcγRIIb1(b1) and FcγRIIb2(b2), have been identified in humans, mice and cattle, however, few of FcγRIIb sub-isoforms have been investigated in pig. In this study, we describe the molecular cloning, sequencing and characterization of a porcine FcγRIIb sub-isoform, FcγRIIb1. The cDNA encoding porcine FcγRIIb1 was isolated from peripheral blood leucocytes RNA with RT-PCR. The porcine FcγRIIb1 cDNA contains a 951bp open-reading frame, encoding a 316 amino acid transmembrane glycoprotein composed of two immunoglobulin (Ig)-like extracellular domains, a transmembrane region and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibiting motif (ITIM). The porcine FcγRIIb1 shares 98.3% homology and has a 19 amino acid in-frame insertion in cytoplasmic tail when compared with amino acid sequence of DQ026064. Immunofluorescence analysis showed that the glycoprotein encoded by the porcine FcγRIIb1 cDNA was expressed in the stable transfected COS-7 cells, and an immunoglobulin-binding assay showed that it had binding activity for IgG immune complexes. Identification of the porcine FcγRIIb1 will help our understanding of the molecular basis of IgG-FcγR interaction in the porcine immune response.