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Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen affecting global swine industry. Our recent study has shown that the first four Ig-like domains of sialoadhesin (Sn4D) and the scavenger receptor cysteine-rich domains 5-9 (SRCR59) of CD163 can act as the soluble viral receptors (SVRs) of PRRSV. Co-injection with the two SVR-expressing recombinant adenovirus (rAd) vectors can protect pigs from the lethal challenge with three PRRSV strains. However, the in vivo expression of the two SVRs persists for only two weeks and thus their long-term anti-PRRSV effects remain to be improved. In this study, we fused the two SVRs with a flexible linker or self-cleaving peptide and expressed them with a single recombinant adeno-associated virus (rAAV) vector. The two rAAVs, namely rAAV-Sn4D-SRCR59-Fc and rAAV-SRCR59-Fc/Sn4D-Fc, were generated by using baculovirus-insect cell system. Western blotting analysis showed that the two SVR fusions were efficiently expressed in and secreted from the rAAV-transduced cells. Viral infection blocking assay showed that PRRSV titers in porcine alveolar macrophage (PAM) cells were reduced by 1.6-2.7 log10 after co-cultivation with rAAV-Sn4D-SRCR59-Fc-transduced cells or by 1.9-3.2 log10 after co-cultivation with rAAV-SRCR59-Fc/Sn4D-Fc-transduced cells. After single-dose injection of mice with the rAAV vectors, the expression of two SVR fusions persisted for at least 35 days, which was significantly longer than SRCR59-Fc expression in rAd-SRCR59-Fc-injected mice. Among the two SVR fusions expressed, both expression level and anti-PRRSV activity of SRCR59-Fc/Sn4D-Fc were higher than that of Sn4D-SRCR59-Fc. Therefore, rAAV-SRCR59-Fc/Sn4D-Fc generated can be developed as a novel anti-PRRSV reagent., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases affecting pig industry worldwide. Sialoadehesin (Sn) and CD163 are the two specific receptors for PRRSV infection of porcine alveolar macrophages. Our previous study showed that the soluble Sn receptor Sn4D-Fc and soluble CD163 receptor SRCR59-Fc expressed by the two recombinant adenoviral (rAd) vectors have an additive anti-PRRSV effect in vitro. In the present study, rAd-Sn4D-Fc and rAd-SRCR59-Fc were inoculated into pigs, and the efficient expression of Sn4D-Fc and SRCR59-Fc proteins was detected by ELISA. Then, PRRSV-naïve pigs were inoculated with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc before contagious infection with different PRRSV strains. Among the three rAd inoculation groups, simultaneous inoculation with the two rAd vectors provided the best protection against highly pathogenic JXA1 strain PRRSV, followed by rAd-SRCR59-Fc inoculation and rAd-Sn4D-Fc inoculation. Clinical observation and quantitative RT-PCR analyses showed that all of the double rAd-inoculated pigs (n = 9) survived from the contagious infection with highly pathogenic JXA1, JS07 or SH1705 strain PRRSV with significantly alleviated clinical scores, viremia, fecal viral emission and tissue virus loads. These data suggest that rAd-Sn4D-Fc and rAd-SRCR59-Fc can be developed further as the universal therapeutic vaccine to facilitate PRRSV eradication., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Environmental surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) represents a key issue in control of the disease. CD151 has recently been recognized as one of several receptors for PRRSV. We describe here a novel method for concentration of PRRSV from the environmental samples by CD151-binding capture. After fusion to self-aggregating peptide ELK16, the large extracellular loop (LEL) of porcine CD151 and its two segments (namely N63 and C63) were expressed in E. coli as protein aggregates. The three fusion proteins were purified to high purities by regular centrifugation and washing with Triton X-100. Viral binding assay showed that the C63-ELK16 protein, but not ELK16-N63 protein, had the specific binding affinity for PRRSV. The C63-ELK16 protein could bind to, and eluted from, PRRSV in pH-, temperature-, and time-dependent manners with a final virus recovery of 44.7%. By using PRRSV-spiked and experimentally infected pig fecal slurry samples, the C63-ELK16 binding capture-combined quantitative RT-PCR was shown to have higher detection sensitivity than the conventional RT-PCR. Although the viral RNA could be detected in the experimentally infected pig samples with or without C63-ELK16 binding capture, infectious PRRSV was not isolated without C63-ELK16 binding capture. Therefore, the CD151-binding capture method established offers sufficient recovery and quickness and will facilitate environmental PRRSV surveillance.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease. As part of the development of RNA interference (RNAi) strategy against the disease, in this study a recombinant adenovirus (rAd) expressing the artificial microRNA (amiRNA) targeting the 3' untranslated region (UTR) was used to investigate the exosome-mediated amiRNA transfer from different pig cell types to porcine alveolar macrophages (PAMs). Quantitative RT-PCR showed that the sequence-specific amiRNA was expressed in and secreted via exosomes from the rAd-transduced pig kidney cell line PK-15, PAM cell line 3D4/163, kidney fibroblast cells (PFCs) and endometrial endothelial cells (PEECs) with different secretion efficiencies. Fluorescent microscopy revealed that the dye-labeled amiRNA-containing exosomes of different cell origins were efficiently taken up by all of the five types of pig cells tested, including primary PAMs. Quantitative RT-PCR showed that the amiRNA-containing exosomes of different cell origins were taken up by primary PAMs in both time- and dose-dependent manners. Both quantitative RT-PCR and viral titration assays showed that the exosome-delivered amiRNA had potent anti-viral effects against three different PRRSV strains. These data suggest that the exosomes derived from pig cells could serve as an efficient miRNA transfer vehicle, and that the exosome-delivered amiRNA had potent anti-viral effects against different PRRSV strains., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described., Methods: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration., Results: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains., Conclusion: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease to the swine industry worldwide. Current PRRS vaccines are only partially effective and new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two essential receptors for PRRSV infection of porcine alveolar macrophage (PAM). To investigate the feasibility of the soluble viral receptors for PRRS control, in the present study we generated recombinant adenovirus (rAd) expressing the four N-terminal Ig-like domains of porcine Sn (Sn4D), the fifth SRCR domain (SRCR5) or domains 5-9 (SRCR59) of porcine CD163 as porcine Fc (pFc) fusion proteins. Efficient expression of the soluble viral receptors in the rAd-transduced cells was confirmed by RT-PCR and Western blotting. To detect their antiviral activities, the soluble viral receptors were purified from the media of rAd-transduced cells and identified by Western blotting. The viral binding assay showed that the soluble receptors Sn4D-Fc and SRCR59-Fc, but not SRCR5-Fc and the control pFc, were able to bind to PRRSV particles. The viral infection blocking assays showed that co-treatment of PRRSV with different concentrations of Sn4D-Fc and SRCR59-Fc proteins resulted in a much higher (72.1%-77.6%) reduction in PRRSV-positive cell number than the single protein treatment (45.1%-60.0% or 44.0%-56.2%). To investigate the feasibility of delivering the soluble viral receptors to PAM, two pig cell lines were transduced with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc using a transwell culture system. PAM cells were infected with PRRSV and then co-cultured with the rAd-transduced cells. Viral titration assay showed that co-cultivation of the infected PAM with rAd-Sn4D-Fc- and rAd-SRCR59-Fc-transduced cells resulted in much higher (by ∼3.5 log) reduction in the viral titers (TCID50) than that of co-cultivation with the single vector-transduced cells (by ∼1.0 log). Further studies showed that the rAd co-delivered soluble receptors Sn4D-Fc and SRCR59-Fc had dose-dependent and temporal antiviral effect against three different PRRSV strains. Since the data presented indicate an additive anti-PRRSV activity between the soluble receptors Sn4D-Fc and SRCR59-Fc, we conclude that the two rAd vectors generated will be useful for development a novel reagent for PRRS control., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: In order to study the role of porcine CD151 in infection of porcine cells by porcine reproductive and respiratory syndrome virus (PRRSV), we established a porcine CD151 transgenic PK-15 cell line., Methods: The full-length complementary DNA (cDNA) for porcine CD151 was amplified from porcine alveolar macrophages by reverse transcription-polymerase chain reaction (RT-PCR), and subcloned into eukaryotic expression vector pcDNA3. The recombinant vector pcDNA-CD151 was transfected into PK-15 cells and the transgenic cell line was generated after G418 selection. Transcription of the CD151 cDNA in transgenic cell line was detected by RT-PCR and immunofluorescence. The cell line, together with control cell lines PK-15, 3D4-CD163 and MARC-145, was infected with PRRSV, and the viral RNA genome or antigens in the infected cells was detected by RT-PCR or immunofluorescence. At different time points post-infection, the virus was harvested and titrated on MARC-145 cells., Results: The expected size of porcine CD151 cDNA was amplified with a sequence identity of 99.7% to the published sequence. From the pcDNA-CD151-transfected cell culture, a transgenic cell line PK15-CD151 was generated and correct expression of porcine CD151 was confirmed. After PRRSV infection, the viral RNA genome and antigens were detected in the cell-line. Although apparent cytopathic effect was not observed in the virally infected cell line, the infectious virus with a high viral title was detected. The cell line had been passed for more than 30 generations and no significant difference in viral title was observed among generations 10, 20 and 30 after PRRSV infection., Conclusion: Transfection of non-permissive PK-15 cells with porcine CD151 cDNA conferred the susceptibility to PRRSV infection, indicating an important role of the porcine CD151 in PRRSV infection of porcine cells.

A robust artificial microRNA (amiRNA) strategy against porcine reproductive and respiratory syndrome virus (PRRSV) was developed by targeting the untranslated regions (UTRs). Six candidate amiRNAs targeting the 5' or 3' UTR were used for vector construction, and four effective amiRNAs were selected for further study using a vector transfection/virus infection assay. In cell cultures stably transfected with the four amiRNA vectors, expression of the sequence-specific amiRNAs was confirmed using poly(A)-tailed RT-PCR. After infection with three different PRRSV strains, the viral RNA genome and/or transcript were inhibited by ~90 % (semi-quantitative RT-PCR), and the viral titers were decreased by more than six log CCID(50) (viral titration assay) before day 3 postinfection. The potent anti-PRRSV effects lasted for at least 5 days. Sequence analysis showed that the amiRNA antiviral activities were not compromised by the presence of one or two mismatches in their binding targets. This work constitutes a step towards developing a more effective RNAi strategy against PRRSV.

Previous studies on the underlying mechanism for porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive failure have been focused on the viral replication in the endothelial macrophages, and the susceptibility of porcine endometrial endothelial (PEE) cells to PRRSV has not yet been investigated. Therefore, in the present study we generated a PEE cell line by transfection of the primary cells with a SV40 large T antigen expression vector. The PEE cell line maintained the endothelial morphology with a significantly faster growth rate, shorter population doubling time and higher plating efficiency than the primary cells. The endothelial origination of the cell line was confirmed by detection of the endothelial cell-specific markers. The PEE cell line had been passed successively for 60 generations with an unlimited growth potential. To further characterize the PEE cell line, cells of different passages were infected with different PRRSV strains and analyzed for the viral antigen and replication. Overt cytopathic effect was observed from 36h postinfection (HPI) and the viral antigen detected as early as 12 HPI. The infectious virus was recovered from the infected PEE cells with a titer higher than that in MARC-145 cells. Since the data presented indicate a high susceptibility of PEE cells to PRRSV, we conclude that the PEE cell line generated will be useful for growth of PRRSV and further studies on the underlying mechanism for PRRSV infection of PEE cells. The finding of the susceptibility of PEE cells to PRRSV may provide an alternative explanation for PRRSV-induced reproductive failure., (Copyright © 2012 Elsevier B.V. All rights reserved.)