Porcine Reproductive And Respiratory Syndrome Virus Receptor Research Articles

Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant to PRRS virus assault

Porcine reproductive and respiratory syndrome virus (PRRSV) infects placental and lung macrophages, causing a global epidemic with economic loss. Attempts to develop an effective vaccine to control the disease have not been effective. Currently, developing PRRSV disease-resistant pigs via a gene editing (GE) strategy to mutate the PRRSV receptor or to delete the binding domain on the macrophage appears promising. In this study, we used the strategy of Edinburg University to construct two guide RNAs (gRNAs) located on the proximal front and post sites of exon 7. Directive microinjection of two gRNAs and Cas9 mRNA into the cytoplasm of pronuclear zygotes efficiently generated four piglets confirmed as CD163 knockout (KO) and/or CD163 exon 7 deleted (CD163ΔE7). In four GE piglets, three pigs carried two chromosome CD163 KO or ΔE7. Peripheral blood mononuclear cells (PBMCs) from three GE and wild-type (WT) pigs were activated into macrophages for in vitro transfection. The results showed that the activated macrophages from all GE pigs were significantly more viable than those from WT pig. Current results suggest that we have successfully generated PRRSV-resistant pigs, although in vivo challenge is needed to validate that the pigs are PRRSV resistant.

Genome-wide CRISPR/Cas9 screen identifies host factors important for porcine reproductive and respiratory syndrome virus replication

Porcine reproductive and respiratory syndrome (PRRS), a viral infection caused by PRRS virus (PRRSV) can result in severe reproductive failure, and respiratory disease in the pigs thus causing enormous economic losses to the global swine industry. Although the cellular receptors for PRRSV have been identified, but mechanisms underlying PPRSV replication remain obscure. Here, we have performed a genome-scale CRISPR/Cas9 knockout screen in the pig kidney cells with PRRSV. Several genes were found to be highly enriched post-PRRSV selection, just like KxDL Motif Containing 1(KXD1), Proteasome 26S Subunit, Non-ATPase 3 (PSMD3) and Galectin 2 (LGALS2) and soon on. Importantly, we have identified that loss of KXD1 resulted in the restricted autophagy and inhibited replication of PRRSV. Therefore, our study demonstrates that CRISPR/Cas9 system can be effectively used for the screening of pig factors responsible for PRRSV replication.

Broad antiviral peptides against PRRSV based on novel linear epitopes on porcine CD163

PRRSV causes major economic losses to swine industry world-wide, which requires innovative antiviral agents. Porcine scavenger receptor CD163 has been identified as an essential fusion receptor for Porcine reproductive and respiratory Syndrome Virus (PRRSV) infection. In this study, novel antiviral peptides from pCD163 against PRRSV were developed based on broad neutralizing monoclonal antibodies. SRCR-5-9 of pCD163 from baculovirus efficiently binds to PRRSVs of lineage 8 and lineage 1, blocking infection in PAMs. A batch of monoclonal antibodies targeting SRCR-5-9 were generated and characterized. 8H2 and 4H7 block PRRSV infection by the disruption in viral attachment to PAMs. Virus titer reduced 100–1000 folds in average and the virus copy number decreased about 104 folds with these antibodies. Linear epitopes of 8H2 and 4H7 were individually localized in SRCR6 (1-30 aa) and PSTI(1-15aa) of pCD163. Mutations of SRCR6 NI1718KT and PST SS1314AA abolished the recognition of 8H2 and 4H7 to the corresponding region individually. Peptides derived from the linear epitopes displayed a broad inhibitory effect on PRRSVs of different lineages in a dose-dependent manner and further modulated PRRSV-related NF-κB pathway. In conclusion, these findings deepen the understanding in the interaction between PRRSV and pCD163 receptor and provide alternative universal antiviral strategies against PRRSV.

Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response

The polarization status of porcine alveolar macrophages (PAMs) determines the infectivity of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection skews macrophage polarization toward an M2 phenotype, followed by T-cells inactivation. CD163, one of the scavenger receptors of M2 macrophages, has been described as a putative receptor for PRRSV. In this study, we examined two types of PRRSV-2-derived recombinant antigens, A1 (g6Ld10T) and A2 (lipo-M5Nt), for their ability to mediate PAM polarization and T helper (Th1) response. A1 and A2 were composed of different combination of ORF5, ORF6, and ORF7 in full or partial length. To enhance the adaptive immunity, they were conjugated with T cells epitopes or lipidated elements, respectively. Our results showed that CD163+ expression on PAMs significantly decreased after being challenged with A1 but not A2, followed by a significant increase in pro-inflammatory genes (TNF-α, IL-6, and IL-12). In addition, next generation sequencing (NGS) data show an increase in T-cell receptor signaling in PAMs challenged with A1. Using a co-culture system, PAMs challenged with A1 can induce Th1 activation by boosting IFN-γ and IL-12 secretion and TNF-α expression. In terms of innate and T-cell-mediated immunity, we conclude that A1 is regarded as a potential vaccine for immunization against PRRSV infection due to its ability to reverse the polarization status of PAMs toward pro-inflammatory phenotypes, which in turn reduces CD163 expression for viral entry and increases immunomodulation for Th1-type response.

Goat AKAP12: Indel Mutation Detection, Association Analysis With Litter Size and Alternative Splicing Variant Expression.

A-kinase anchoring protein 12 (AKAP12) plays key roles in male germ cells and female ovarian granulosa cells, whereas its influence on livestock litter size remains unclear. Herein we detected the genetic variants of AKAP12 gene and their effects on litter size as well as alternative splicing variants expression in Shaanbei white cashmere (SBWC) goats, aiming at exploring theoretical basis for goat molecular breeding. We identified two Insertion/deletions (Indels) (7- and 13-bp) within the AKAP12 gene. Statistical analyses demonstrated that the 13-bp indel mutation in the 3′ UTR was significantly associated with litter size (n = 1,019), and the carriers with DD genotypes presented lower litter sizes compared with other carriers (P < 0.01). Bioinformatics analysis predicted that this 13-bp deletion sequence could bind to the seed region of miR-181, which has been documented to suppress porcine reproductive and respiratory syndrome virus (PRRSV) infection by targeting PRRSV receptor CD163 and affect the pig litter size. Therefore, luciferase assay for this 13-bp indel binding with miRNA-181 was performed, and the luciferase activity of pcDNA-miR-181-13bp-Deletion-allele vector was significantly lower than that of the pcDNA-miR-181-13bp-Insertion-allele vector (P < 0.05), suggesting the reduced binding capability with miR-181 in DD genotype. Given that alternative spliced variants and their expression considerably account for the Indel genetic effects on phenotypic traits, we therefore detected the expression of the alternative spliced variants in different tissues and identified that AKAP12-AS2 exhibited the highest expression levels in testis tissues. Interestingly, the AKAP12-AS2 expression levels of homozygote DD carriers were significantly lower than that of individuals with heterozygote ID, in both testis and ovarian tissues (P < 0.05), which is consistent with the effect of the 13-bp deletion on the reduced litter size. Taken together, our results here suggest that this 13-bp indel mutation within goat AKAP12 might be utilized as a novel molecular marker for improving litter size in goat breeding.

Recent Advances in PRRS Virus Receptors and the Targeting of Receptor-Ligand for Control.

Cellular receptors play a critical role in viral infection. At least seven cellular molecules have been identified as putative viral entry mediators for porcine reproductive and respiratory syndrome virus (PRRSV). Accumulating data indicate that among these candidates, CD163, a cysteine-rich scavenger receptor on macrophages, is the major receptor for PRRSV. This review discusses the recent advances and understanding of the entry of PRRSV into cells, viral pathogenesis in CD163 gene-edited swine, and CD163 as a potential target of receptor–ligand for the control of PRRS.

Intradermal co-inoculation of codon pair deoptimization (CPD)-attenuated chimeric porcine reproductive and respiratory syndrome virus (PRRSV) with Toll like receptor (TLR) agonists enhanced the protective effects in pigs against heterologous challenge

The objective of this study was to assess protective efficacy of vaccination using CPD-attenuated chimeric PRRSV and Toll like receptor (TLR) agonists (HSP70 c-terminal domain and HSPX) as adjuvants through different inoculation routes. In this study, a chimeric PRRSV composed of two field isolates was synthesized and attenuated by CPD in NSP1 as described in the previous study. The infection of the CPD-attenuated chimeric PRRSV to pigs of 3 weeks-old showed no clinical signs without pathological lesions in necropsy, while it induced improved cross immunity between its parent strains. The TLR agonists were expressed in E. coli and purified to be used. In challenge experiment, pigs of 3 weeks-old were vaccinated using the CPD-attenuated chimeric virus with the prepared TLR agonists through intramuscular or intradermal route, following heterologous challenge after 4 weeks of vaccination. In results, intramuscular or intradermal inoculation of the CPD-attenuated chimeric virus demonstrated excellent protective efficacy against heterologous challenges. Importantly, intradermal inoculation with the TLR agonists enhanced protective effects as shown in the significantly increased level of PRRSV-specific IFN-γ-SCs and cytokines in sera, and the significant reduction of pathological lesion and viral load in lung. This study suggested that the intradermal inoculation of CPD-attenuated chimeric PRRSV plus TLR agonists should be more effective for protection of pigs against diverse PRRS field viruses.

Development of a Monoclonal Antibody Against Porcine CD163 SRCR5 Domain Which Partially Blocks Infection of PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV), which seriously endangers the world pig industry, invades host cells through receptor-mediated endocytosis involving clathrin. CD163 is an essential receptor for PRRSV during its infection of cells. The scavenger receptor cysteine-rich 5 (SRCR5) domain of the CD163 molecule is necessary for PRRSV infection, and interacts with glycoproteins GP2a and GP4 of PRRSV, allowing the virus to infect the host cells. In this study, a monoclonal antibody (mAb) against the SRCR5-6 region of porcine CD163 was developed, and the target epitope of the mAb was determined as 497TWGTVCDSDF506, which is directly adjacent to the ligand-binding pocket (LBP) domain (487-495aa) of CD163. Further study indicated that the mAb could partially block PRRSV infection of its target cells, pulmonary alveolar macrophages. The mAb developed in the study may provide a foundation of antiviral therapy for PRRSV.

Susceptibility of porcine pulmonary microvascular endothelial cells to porcine reproductive and respiratory syndrome virus.

Microvascular endothelial cells possess versatile functions and their roles in a variety of viral infections have been documented. Porcine reproductive and respiratory syndrome virus (PRRSV) infection induces severe lung inflammatory lesions in piglets, which is manifested as pulmonary endothelial dysfunction. However, the underlying mechanism of PRRSV affecting porcine pulmonary microvascular endothelial cells (PMECs) remains unknown. This study aimed to evaluate the susceptibility of PMECs to PRRSV. Primary PMECs were isolated and purified from piglet lungs, and the expression of three PRRSV receptors was characterized using immunofluorescence. Overt cytopathic effects of the PRRSV strain HN in PMECs were observed at day five post-infection, and PRRSV antigens in PMECs were determined at both RNA and protein levels using immunofluorescence and quantitative RT-PCR assays. The viral antigen significantly increased at 96 hr post-infection, and infectious virus was recovered from the supernatant of the infected PMECs. The results show that PMECs can be infected with the PRRSV strain HN, and that their receptor expression pattern is different from that of alveolar macrophages. The results of this study shed light on the potential roles of PMECs in PRRSV infection and provide a comprehensive understanding of the pathogenesis underlying its severe manifestation.

Cellular protein profiles altered by PRRSV infection of porcine monocytes-derived dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play an important role in inducing primary antigen-specific immune responses. Some viruses have evolved to specifically target DCs to circumvent the host immune responses for their persistence in the host. One example is porcine reproductive and respiratory syndrome virus (PRRSV) that causes a persistent infection in pigs through modulating DC-mediated antiviral response. To study the cellular protein responses in PRRSV-infected monocyte-derived dendritic cells (MoDCs), two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins in PRRSV-infected MoDCs and the control cells. A total of 252 cellular proteins in MoDCs that were significantly altered at different time periods post-infection were identified. Differentially expressed proteins that are involved in the endocytosis pathway, actin cytoskeleton network, antigen processing and presentation, JAK-STAT signaling pathway and PRRSV receptors were identified and further analyzed. Among them, the expression changes of STAT1, Mx1, PICALM and SLA-DR were further verified by Western blotting. The protein profiles associated with PRRSV infection of MoDCs should offer novel insights to further investigation of PRRSV-mediated antiviral evasion mechanism and its pathobiology in swine.

Identification of Differentially Expressed Non-coding RNA in Porcine Alveolar Macrophages from Tongcheng and Large White Pigs Responded to PRRSV

Porcine reproductive and respiratory syndrome (PRRS) is one of the most ruinous diseases in pig production. Our previous work showed that Tongcheng pigs (TC) were less susceptible to PRRS virus (PRRSV) than Large White (LW) pigs. To elucidate the difference in PRRSV resistance between the two breeds, small RNA-seq and ribo-zero RNA-seq were used to identify differentially expressed non-coding RNAs (including miRNAs and lincRNAs) responded to PRRSV in porcine alveolar macrophages (PAMs) from TC and LW pigs. Totally, 250 known mature miRNAs were detected. For LW pigs, there were 44 down-regulated and 67 up-regulated miRNAs in infection group; while for TC pigs, 12 down-regulated and 23 up-regulated miRNAs in TC infection group were identified. The target genes of the common differentially expressed miRNAs (DEmiRNAs) in these two breeds were enriched in immune-related processes, including apoptosis process, inflammatory response, T cell receptor signaling pathway and so on. In addition, 5 shared DEmiRNAs (miR-181, miR-1343, miR-296-3p, miR-199a-3p and miR-34c) were predicted to target PRRSV receptors, of which miR-199a-3p was validated to inhibit the expression of CD151. Interestingly, miR-378 and miR-10a-5p, which could inhibit PRRSV replication, displayed higher expression level in TC control group than that in LW control group. Contrarily, miR-145-5p and miR-328, which were specifically down-regulated in LW pigs, could target inhibitory immunoreceptors and may involve in immunosuppression caused by PRRSV. This indicates that DEmiRNAs are involved in the regulation of the immunosuppression and immune escape of the two breeds. Furthermore, we identified 616 lincRNA transcripts, of which 48 and 30 lincRNAs were differentially expressed in LW and TC pigs, respectively. LincRNA TCONS_00125566 may play an important role in the entire regulatory network, and was predicted to regulate the expression of immune-related genes through binding with miR-1343 competitively. In conclusion, this study provides an important resource for further revealing the interaction between host and virus, which will specify a new direction for anti-PRRSV research.

Characterization of the attachment and infection by Porcine reproductive and respiratory syndrome virus 1 isolates in bone marrow-derived dendritic cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect porcine dendritic cells (DC). Previous studies indicated that different PRRSV1 isolates regulated differently the cytokine profiles and expression of surface molecules of DC. However, the characterisation of the infection is lacking. The current study aimed to characterise the replication and attachment of different PRRSV1 isolates in bone marrow-derived DC (BMDC). For this purpose, immature (i) and mature (m) BMDC were infected with three PRRSV1 isolates. The replication kinetics showed that titres in iBMDC were significantly (p < 0.05) higher than in mBMDC by 24 hpi, and for two isolates titres peaked earlier in iBMDC, suggesting that iBMDC were more efficient in supporting PRRSV1 replication than mBMDC. The attachment was revealed by a three-colour confocal microscopy staining. All three isolates were seen attached to iBMDC even in cells lacking CD163 -the essential receptor for PRRSV- or porcine sialoadhesin (PoSn). The attachment was not fully avoided after removal of heparan sulphate by heparinase I. Furthermore, the infection was examined with regards to CD163 expression. By flow cytometry and confocal microscopy, positive signals of PRRSV1 nucleocapsid could be observed in CD163− iBMDC. Additional sorting experiment demonstrated that CD163− iBMDC were infected only when CD163lo/hi cells were present. This can be interpreted in different ways: susceptible CD163− cells arose as result of milieu created by CD163+ infected BMDC; CD163− cells were infected by receptor-independent mechanisms (i.e. exosomes) or, some cells expressed CD163 at levels beyond the technical sensitivity.

CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses to current swine production worldwide. Highly pathogenic PRRSV (HP-PRRSV), originated from a genotype 2 PRRSV, is more virulent than classical PRRSV and further exacerbates the economic impact. HP-PRRSV has become the predominant circulating field strain in China since 2006. CD163 is a cellular receptor for PRRSV. The depletion of CD163 whole protein or SRCR5 region (interaction site for the virus) confers resistance to infection of several PRRSV isolates in pigs or cultured host cells. In this study, we described the generation of a CD163 knockout (KO) pig in which the CD163 protein was ablated by using CRISPR/Cas9 gene targeting and somatic cell nuclear transfer (SCNT) technologies. Challenge with HP-PRRSV TP strain showed that CD163 KO pigs are completely resistant to viral infection manifested by the absence of viremia, antibody response, high fever or any other PRRS-associated clinical signs. By comparison, wild-type (WT) controls displayed typical signs of PRRSV infection and died within 2 weeks after infection. Deletion of CD163 showed no adverse effects to the macrophages on immunophenotyping and biological activity as hemoglobin–haptoglobin scavenger. The results demonstrated that CD163 knockout confers full resistance to HP-PRRSV infection to pigs without impairing the biological function associated with the gene.

Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.

Developing a Triple Transgenic Cell Line for High-Efficiency Porcine Reproductive and Respiratory Syndrome Virus Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens in the swine industry worldwide. Due to the lack of robust cell lines and small animal models, the pathogenesis of PRRSV infection and mechanism for protective vaccination are still not yet well understood. To obtain useful cell lines, several groups have attempted to construct different transgenic cell lines with three PRRSV receptors: CD163, CD169, and CD151. The results showed that CD163 is essential for PRRSV entry into target cells and replication, and both CD169 and CD151 play key roles during PRRSV infection. However, their interplay and combined effect remains unclear. In this study, we generated transgenic BHK-21 derived cell lines co-expressing different combinations of the three receptors, which were transfected with CD163 alone, or the combination of CD163 and CD169, or the combination of CD163 and CD151, or the combination of CD163, CD169, and CD151 using the PiggyBac transposon system. Our results showed that the synergistic interaction among the three receptors was important to improve the susceptibility of cells during PRRSV infection. Through a series of comparable analyses, we confirmed that the cell line co-expressing triple receptors sustained viral infection and replication, and was superior to the current cell platform used for the PRRSV study, MARC-145 cells. Moreover, we found that PRRSV infection of the transgenic cell lines could trigger IFN-stimulated gene responses similar to those of porcine alveolar macrophages and MARC-145 cells. In summary, we developed a stable transgenic cell line susceptible to PRRSV, which may not only provide a useful tool for virus propagation, vaccine development, and pathogenesis studies, but also establish the foundation for small animal model development.

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has heavily impacted the global swine industry. The PRRSV nonstructural protein 2 (nsp2) plays crucial roles in viral replication and host immune regulation, most likely by interacting with viral or cellular proteins that have not yet been identified. In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify nsp2-interacting proteins in PRRSV-infected cells with an nsp2-specific monoclonal antibody. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2. Nsp2-interacting cellular proteins were classified into different functional groups and an interactome network of nsp2 was generated. Interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, the nsp2 interactome under the condition of virus infection clarifies a role of nsp2 in PRRSV replication and immune evasion. Biological significanceViral proteins must interact with other virus-encoded proteins and/or host cellular proteins to function, and interactome analysis is an ideal approach for identifying such interacting proteins. In this study, we used the quantitative interactome methodology to identify the viral and cellular proteins that potentially interact with the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) under virus infection conditions, thus providing a rich source of potential viral and cellular interaction partners for PRRSV nsp2. Based on the interactome data, we further demonstrated that PRRSV nsp2 and nucleocapsid protein together with cellular vimentin, form a complex that may be essential for viral attachment and replication, which partly explains the role of nsp2 in PRRSV replication and immune evasion.

Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread.

Intercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two distant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain structural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV-infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor proteins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1β, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response. Extracellular virus particles transmit infection between organisms, but within infected hosts intercellular infection can be spread by additional mechanisms. In this study, we describe an alternative pathway for intercellular transmission of PRRSV in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases, and certain structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection.

PRRSV receptors and their roles in virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted cell tropism and prefers to invade well-differentiated cells of the monocyte/macrophage lineage, such as pulmonary alveolar macrophages and African green monkey kidney cell line MA-104 and its derivatives, such as Marc-145, Vero and CL-2621. PRRSV infection of the host cells actually is a receptor-mediated endocytosis and replication process. The presence and absence of the cellular receptors decide whether the cell lines are permissive or non-permissive to PRRSV infection. Several PRRSV non-permissive cell lines, such as BHK-21, PK-15 and CHO-K1, have been shown to become sensitive to the virus infection upon expression of the recombinant receptor proteins. Up to now, heparin sulfate, sialoadhesin, CD163, CD151 and vimentin have been identified as the important PRRSV receptors via their involvement in virus attachment, internalization or uncoating. Each receptor is characterized by the distribution in different cells, the function in virus different infection stages and the interaction model with the viral proteins or genes. Joint forces of the receptors recently attract attentions due to the specific function. PRRSV receptors have become the targets for designing the new anti-viral reagents or the recombinant cell lines used for isolating the viruses or developing more effective vaccines due to their more conserved sequences compared with the genetic variation of the virus. In this paper, the role of PRRSV receptors and the molecular mechanism of the interaction between the virus and the receptors are reviewed.

The Influence of Porcine Reproductive and Respiratory Syndrome Virus Infection on the Expression of Cellular Prion Protein in Marc-145 Cells

Cellular prion protein (PrPC) represents a potential regulator of cellular immunity and has been confirmed to be an invasive receptor exploited by bacteria such as Brucella abortus to facilitate the infection in host cells. Up to now, there are few informations about the role of PrPC in virus replication in host cells. Porcine reproductive and respiratory syndrome virus (PRRSV), a single positive-stranded RNA virus, prefers to infect African green monkey kidney cell line MA-104 and its derivatives, such as Marc-145. We found that PrPC is expressed on the surfaces of Marc-145 cells and its expression profiles greatly changed after PRRSV incubation with these cells. PrPC expression reached the peak at 12 hrs after PRRSV inoculation and then decreased sharply to recover to the original status while CD163, a PRRSV receptor in Marc-145 cells, achieved the climax at 48 hrs after virus infection and then slowly lowered its expression level. Here, we firstly suggest that PRRSV infection has an influence on the expression of PrPC on the surfaces of Marc-145 cells and PrPC may be involved in PRRSV invasion of host cells.

Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine Reproductive and Respiratory Syndrome Virus Entry

As a consequence of their effects on ectodomain shedding, members of the A disintegrin and metalloprotease (ADAM) family have been implicated in the control of various cellular processes. Although ADAM family members are also involved in cancer, inflammation, and other pathologies, it is unclear whether they affect porcine reproductive and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine alveolar macrophages (PAMs). We also demonstrate that the inhibition of ADAM17 upregulates membrane CD163 expression, a putative PRRSV receptor that is exogenously expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore, overexpression of ADAM17 induced downregulation of CD163 expression and a reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific small interfering RNA resulted in upregulation of CD163 expression with a corresponding increase in PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV nonpermissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate that ADAM17 downregulates CD163 expression and hinders PRRSV entry. Hence, downregulation of ADAM17 particular substrates may be an additional component of the anti-infection defenses. ADAM17 is one of the important membrane-associated metalloproteases that mediate various cellular events, as well as inflammation, cancer, and other pathologies. Here, we investigate for the first time the role of the metalloprotease ADAM17 in PRRSV infection. By using inhibitor and genetic modification methods, we demonstrate that ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More specifically, ADAM 17 mediates the downregulation of the PRRSV cellular receptor CD163. The reduction in CD163 expression represents another component of the anti-infection response initiated by ADAM17.