PDCD4 restricts PRRSV replication in an eIF4A-dependent manner and is antagonized by the viral nonstructural protein 9.

As obligate parasites, viruses have evolved multiple strategies to evade the host immune defense. Manipulation of the host proteasome system to degrade specific detrimental factors is a common viral countermeasure. To identify host proteins targeted for proteasomal degradation by porcine reproductive and respiratory syndrome virus (PRRSV), we conducted a quantitative proteomics screen of PRRSV-infected Marc-145 cells under the treatment with proteasome inhibitor MG132. The data revealed that the expression levels of programmed cell death 4 (PDCD4) were strongly downregulated by PRRSV and significantly rescued by MG132. Further investigation confirmed that PRRSV infection induced the translocation of PDCD4 from the nucleus to the cytoplasm, and the viral nonstructural protein 9 (Nsp9) promoted PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway. The C-terminal domain of Nsp9 was responsible for PDCD4 degradation. As for the role of PDCD4 during PRRSV infection, we demonstrated that PDCD4 knockdown favored viral replication, while its overexpression significantly attenuated replication, suggesting that PDCD4 acts as a restriction factor for PRRSV. Mechanistically, we discovered eukaryotic translation initiation factor 4A (eIF4A) was required for PRRSV. PDCD4 interacted with eIF4A through four sites (E249, D253, D414, and D418) within its two MA3 domains, disrupting eIF4A-mediated translation initiation in the 5'-untranslated region of PRRSV, thereby inhibiting PRRSV infection. Together, our study reveals the antiviral function of PDCD4 and the viral strategy to antagonize PDCD4. These results will contribute to our understanding of the immune evasion strategies employed by PRRSV and offer valuable insights for developing new antiviral targets.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in major economic losses in the global swine industry and is difficult to control effectively. Here, using a quantitative proteomics screen, we identified programmed cell death 4 (PDCD4) as a host protein targeted for proteasomal degradation by PRRSV. We demonstrated that PDCD4 restricts PRRSV replication by interacting with eukaryotic translation initiation factor 4A, which is required for translation initiation in the viral 5'-untranslated region. Additionally, four sites within two MA3 domains of PDCD4 are identified to be responsible for its antiviral function. Conversely, PRRSV nonstructural protein 9 promotes PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway, thus weakening the anti-PRRSV function. Our work unveils PDCD4 as a previously unrecognized host restriction factor for PRRSV and reveals that PRRSV develops countermeasures to overcome PDCD4. This will provide new insights into virus-host interactions and the development of new antiviral targets.

Unveiling Shared Immune Responses in Porcine Alveolar Macrophages during ASFV and PRRSV Infection Using Single-Cell RNA-seq.

African swine fever virus (ASFV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections lead to severe respiratory diseases in pigs, resulting in significant economic losses for the global swine industry. While numerous studies have focused on specific gene functions or pathway activities during infection, an investigation of shared immune responses in porcine alveolar macrophages (PAMs) after ASFV and PRRSV infections was lacking. In this study, we conducted a comparison using two single-cell transcriptomic datasets generated from PAMs under ASFV and PRRSV infection. Pattern recognition receptors (PRRs) RIG-I (DDX58), MDA5 (IFIH1), and LGP2 (DHX58) were identified as particularly recognizing ASFV and PRRSV, triggering cellular defense responses, including the upregulation of four cytokine families (CCL, CXCL, IL, and TNF) and the induction of pyroptosis. Through weighted gene co-expression network analysis and protein-protein interaction analysis, we identified thirteen gene and protein interactions shared by both scRNA-seq analyses, suggesting the ability to inhibit both ASFV and PRRSV viral replication. We discovered six proteins (PARP12, PARP14, HERC5, DDX60, RSAD2, and MNDA) in PAMs as inhibitors of ASFV and PRRSV replication. Collectively, our findings showed detailed characterizations of the immune responses in PAMs during ASFV and PRRSV infections, which may facilitate the treatments of these viral diseases.

MARCO Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection through Intensifying Viral GP5-Induced Apoptosis.

Studying viral glycoprotein-host membrane protein interactions contributes to the discovery of novel cell receptors or entry facilitators for viruses. Glycoprotein 5 (GP5), which is a major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) virions, is a key target for the control of the virus. Here, the macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified as one of the host interactors of GP5 through a DUALmembrane yeast two-hybrid screening. MARCO was specifically expressed on porcine alveolar macrophages (PAMs), and PRRSV infection downregulated MARCO expression both in vitro and in vivo. MARCO was not involved in viral adsorption and internalization processes, indicating that MARCO may not be a PRRSV-entry facilitator. Contrarily, MARCO served as a host restriction factor for PRRSV. The knockdown of MARCO in PAMs enhanced PRRSV proliferation, whereas overexpression suppressed viral proliferation. The N-terminal cytoplasmic region of MARCO was responsible for its inhibitory effect on PRRSV. Further, we found that MARCO was a proapoptotic factor in PRRSV-infected PAMs. MARCO knockdown weakened virus-induced apoptosis, whereas overexpression aggravated apoptosis. MARCO aggravated GP5-induced apoptosis, which may result in its proapoptotic function in PAMs. The interaction between MARCO and GP5 may contribute to the intensified apoptosis induced by GP5. Additionally, the inhibition of apoptosis during PRRSV infection weakened the antiviral function of MARCO, suggesting that MARCO inhibits PRRSV through the regulation of apoptosis. Taken together, the results of this study reveal a novel antiviral mechanism of MARCO and suggest a molecular basis for the potential development of therapeutics against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the most serious threats to the global swine industry. Glycoprotein 5 (GP5) exposed on the surface of PRRSV virions is a major glycoprotein, and it is involved in viral entry into host cells. A macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified to interact with PRRSV GP5 in a DUALmembrane yeast two-hybrid screening. Further investigation demonstrated that MARCO may not serve as a potential receptor to mediate PRRSV entry. Instead, MARCO was a host restriction factor for the virus, and the N-terminal cytoplasmic region of MARCO was responsible for its anti-PRRSV effect. Mechanistically, MARCO inhibited PRRSV infection through intensifying virus-induced apoptosis in PAMs. The interaction between MARCO and GP5 may contribute to GP5-induced apoptosis. Our work reveals a novel antiviral mechanism of MARCO and advances the development of control strategies for the virus.

Recent advances in inhibition of porcine reproductive and respiratory syndrome virus through targeting CD163.

Porcine reproductive and respiratory syndrome virus (PRRSV) has plagued the pig industry for more than 30 years and causes great economic losses. At present different commercial vaccines are available but limited tools. Until now at least six potential host factors are identified as the key receptors for PRRSV infection. Among them, CD163 molecule is the most important and critical in PRRSV life cycle responsible for mediating virus uncoating and genome release. It determines the susceptibility of target cells to the virus. Several PRRSV non-permissive cells (such as PK-15, 3D4/21, and BHK-21) are demonstrated to become completely susceptible to PRRSV infection in the presence of expression of porcine CD163 protein. Therefore, CD163 has become the target for the design of novel antiviral molecules disrupting the interaction between CD163 and viral glycoproteins, or the breeding of gene-modified animals against PRRSV infection. In this review, we comprehensively summarize the recent progress in inhibition of PRRSV replication via targeting CD163 receptor. In addition, whether there are other potential molecules interacting with CD163 in the process of uncoating of virus life cycle is also discussed.

Corrigendum: Chlorine Dioxide Inhibits African Swine Fever Virus by Blocking Viral Attachment and Destroying Viral Nucleic Acids and Proteins.

[This corrects the article DOI: 10.3389/fvets.2022.844058.].

Chlorine Dioxide Inhibits African Swine Fever Virus by Blocking Viral Attachment and Destroying Viral Nucleic Acids and Proteins.

African swine fever (ASF) is a highly contagious disease and provokes severe economic losses and health threats. At present no effective vaccine or treatment is available to prevent or cure ASF. Consequently, there is an urgent need to develop effective drugs against ASF virus (ASFV). Chlorine dioxide (ClO2), an ideal biocide, has broad-spectrum antibacterial activity and no drug resistance. Here, we found that ClO2 strongly inhibited ASFV replication in porcine alveolar macrophages (PAMs). The inhibitory effect of ClO2 occurred during viral attachment rather than entry, indicating that ClO2 suppressed the early stage of virus life cycle. ClO2 showed a potent anti-ASFV effect when added either before, simultaneously with, or after virus infection. Furthermore, ClO2 could destroy viral nucleic acids and proteins, which may contribute to its capacity of inactivating ASFV virions. The minimum concentration of degradation of ASFV nucleic acids by ClO2 is 1.2 µg/mL, and the degradation is a temperature-dependent manner. These have guiding significance for ClO2 prevention and control of ASFV infection in pig farms. In addition, ClO2 decreased the expression of ASFV-induced inflammatory cytokines. Overall, our findings suggest that ClO2 may be an ideal candidate for the development of novel anti-ASFV prophylactic and therapeutic drugs in swine industry.

Genetic Characterization and Variation of African Swine Fever Virus China/GD/2019 Strain in Domestic Pigs.

African swine fever (ASF) was first introduced into Northern China in 2018 and has spread through China since then. Here, we extracted the viral DNA from the blood samples from an ASF outbreak farm in Guangdong province, China and sequenced the whole genome. We assembled the full length genomic sequence of this strain, named China/GD/2019. The whole genome was 188,642 bp long (terminal inverted repeats and loops were not sequenced), encoding 175 open reading frames (ORF). The China/GD/2019 strain belonged to p72 genotype II and p54 genotype IIa. Phylogenetic analysis relationships based on single nucleotide polymorphisms (SNPs) also demonstrated that it grouped into genotype II. A certain number of ORFs mainly belonging to multigene families (MGFs) were absent in the China/GD/2019 strain in comparison to the China/ASFV/SY-18 strain. A deletion of approximately 1 kb was found in the China/GD/2019 genome which was located at the EP153R and EP402R genes in comparison to the China/2018/AnhuiXCGQ strain. We revealed a synonymous mutation site at gene F317L and a non-synonymous mutation site at gene MGF_360-6L in China/GD/2019 comparing to three known Chinese strains. Pair-wise comparison revealed 165 SNP sites in MGF_360-1L between Estonia 2014 and the China/GD/2019 strain. Comparing to China/GD/2019, we revealed a base deletion located at gene D1133L in China/Pig/HLJ/2018 and China/DB/LN/2018, which results in a frameshift mutation to alter the encoding protein. Our findings indicate that China/GD/2019 is a new variant with certain deletions and mutations. This study deepens our understanding of the genomic diversity and genetic variation of ASFV.

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

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

Antiviral Mechanism of Tea Polyphenols against Porcine Reproductive and Respiratory Syndrome Virus.

Neither inactivated nor attenuated vaccines can effectively prevent and control the infection and spread of porcine reproductive and respiratory syndrome virus (PRRSV). Therefore, it is necessary to broaden new horizons and to conceive effective preventive strategies. The main components of Tea polyphenol (TPP) are catechins and their derivatives. TPP has many physiological activities and has certain antiviral and antifungal effects. However, whether TPP shows anti-PRRSV activity remains unclear. We found that TPP effectively inhibited PRRSV infection in Marc-145 cells by suppressing the stages of viral attachment, internalization, replication, and release. TPP exhibited a potent anti-PRRSV effect regardless of pre-treatment or post-treatment. In addition, we demonstrated that TPP restrained PRRSV-induced p65 entry into the nucleus to suppress the activation of the NF-κB signaling pathway, which ultimately leads to the inhibition of the expression of inflammatory cytokines. Furthermore, TPP limited the synthesis of viral non-structural protein 2 (nsp2), the core component of viral replication transcription complexes, which may contribute to the inhibition of viral RNA replication. TPP has the potential to develop into an effective antiviral agent for PRRSV prevention and control in the future.

Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus.

BACKGROUND: As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. RESULTS: Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. CONCLUSIONS: In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

TREM2 suppresses the proinflammatory response to facilitate PRRSV infection via PI3K/NF-κB signaling.

Triggering receptor expressed on myeloid cells 2 (TREM2) serves as an anti-inflammatory receptor, negatively regulating the innate immune response. TREM2 is mainly expressed on dendritic cells and macrophages, the target cells of porcine reproductive and respiratory syndrome virus (PRRSV). Thus, we investigated the potential role of TREM2 in PRRSV infection in porcine alveolar macrophages (PAMs). We found that there was an increased expression of TREM2 upon PRRSV infection in vitro. TREM2 silencing restrained the replication of PRRSV, whereas TREM2 overexpression facilitated viral replication. The cytoplasmic tail domain of TREM2 interacted with PRRSV Nsp2 to promote infection. TREM2 downregulation led to early activation of PI3K/NF-κB signaling, thus reinforcing the expression of proinflammatory cytokines and type I interferons. Due to the enhanced cytokine expression, a disintegrin and metalloproteinase 17 was activated to promote the cleavage of membrane CD163, which resulted in suppression of infection. Furthermore, exogenous soluble TREM2 (sTREM2)-mediated inhibition of PRRSV attachment might be attributed to its competitive binding to viral envelope proteins. In pigs, following PRRSV challenge in vivo, the expression of TREM2 in lungs and lymph nodes as well as the production of sTREM2 were significantly increased. These novel findings indicate that TREM2 plays a role in regulating PRRSV replication via the inflammatory response. Therefore, our work describes a novel antiviral mechanism against PRRSV infection and suggests that targeting TREM2 could be a new approach in the control of the PRRSV infection.

Lipopolysaccharide Downregulates CD163 Expression to Inhibit PRRSV Infection via TLR4-NF-κB Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to induce proinflammatory cytokine production and modulate the host interferon (IFN) system. Proinflammatory cytokines and type I IFNs contribute to the prevention of viral infection. Lipopolysaccharide (LPS), a specific agonist to Toll-like receptor 4 (TLR4), provokes signal transduction and activates immune response in vivo and in vitro. Here we identified LPS inhibited PRRSV infection in porcine alveolar macrophages (PAMs) and in Marc-145 cells. To investigate the possible mechanism, we found TLR4-NF-κB pathway was obviously activated in LPS-treated PAMs at the early stage of PRRSV infection. As a result, the expression of proinflammatory cytokines was strongly induced following LPS and PRRSV co-treatment. Due to the enhanced proinflammatory response, CD163 expression was significantly reduced and a disintegrin and metalloproteinase 17 was activated, which promotes the cleavage of membrane CD163. Ultimately, CD163 down-regulation led to the suppression of PRRSV replication. Our data demonstrate that LPS has an impact on PRRSV infection via inflammation response, which provides a new insight of inflammation-mediated antiviral immunity and a new strategy to control PRRSV infection.

Highly Efficient Generation of Pigs Harboring a Partial Deletion of the CD163 SRCR5 Domain, Which Are Fully Resistant to Porcine Reproductive and Respiratory Syndrome Virus 2 Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2 differ in their recognition of CD163. Substitution of porcine CD163 SRCR5 domain with a human CD163-like SRCR8 confers resistance to PRRSV 1 but not PRRSV 2. The deletion of CD163 SRCR5 has been shown to confer resistance to PRRSV 1 in vivo and both PRRSV 1 and 2 in vitro. However, the anti-PRRSV 2 activity of modifying the CD163 SRCR5 domain has not yet been reported. Here, we describe the highly efficient generation of two pig breeds (Liang Guang Small Spotted and Large White pigs) lacking a short region of CD163 SRCR5, including the ligand-binding pocket. We generated a large number of gene-edited Large White pigs of the F0 generation for use in viral challenge studies. The results of this study show that these pigs are completely resistant to infection by species 2 PRRSV, JXA1, and MY strains. There were no clinical symptoms, pathological abnormalities, viremia, or anti-PRRSV antibodies in the CD163 SRCR5-edited pigs compared to wild-type controls after viral challenge. Porcine alveolar macrophages (PAMs) isolated from CD163 SRCR5-edited Large White pigs also displayed resistance to PRRSV in vitro. In addition, CD163 SRCR5-edited PAMs still exhibited a cytokine response to PRRSV infection, and no significant difference was observed in cytokine expression compared to wild-type PAMs. Taken together, these data suggest that CD163 SRCR5-edited pigs are resistant to PRRSV 2, providing a basis for the establishment of PRRSV-resistant pig lines for commercial application and further investigation of the essential region of SRCR5 involved in virus infection.

Inhibitory effect of iota-carrageenan on porcine reproductive and respiratory syndrome virus in vitro.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen and causes significant economic losses to the swine industry worldwide each year. Current vaccination strategies do not effectively prevent and control the virus. Consequently, it is necessary to develop novel antiviral strategies. Carrageenan, extracted from marine red algae, exhibits anti-coagulant, anti-tumour, anti-virus and immunomodulatory activities. METHODS: We investigate the inhibitory effect of iota-carrageenan (CG) on PRRSV strain CH-1a via antiviral assay and viral binding, entry and release assays. RESULTS: We found that CG effectively inhibited CH-1a replication at mRNA and protein levels in both Marc-145 cells and porcine alveolar macrophages (PAMs). The antiviral activity of CG occurred during viral attachment and entry in virus life cycle. In addition, CG suppressed viral release in Marc-145 cells, as well as blocked CH-1a-induced apoptosis during the late period of infection. Furthermore, CG inhibited CH-1a-induced NF-κB activation, thus interfering with cytokine production in Marc-145 cells and PAMs, which contributes to its anti-PRRSV activity. CONCLUSIONS: Taken together, our data imply that CG might be an ideal candidate that is worthwhile developing into a new anti-PRRSV prophylactic and therapeutic drug.

CD163ΔSRCR5 MARC-145 Cells Resist PRRSV-2 Infection via Inhibiting Virus Uncoating, Which Requires the Interaction of CD163 With Calpain 1.

Porcine alveolar macrophages without the CD163 SRCR5 domain are resistant to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, whether the deletion of CD163 SRCR5 in MARC-145 cells confers resistance to PRRSV and interaction of which of the host proteins with CD163 is involved in virus uncoating remain unclear. Here we deleted the SRCR5 domain of CD163 in MARC-145 cells using CRISPR/Cas9 to generate a CD163ΔSRCR5 MARC-145 cell line. The modification of CD163 had no impact on CD163 expression. CD163ΔSRCR5 cells were completely resistant to infection by PRRSV-2 strains Li11, CHR6, TJM, and VR2332. The modified cells showed no cytokine response to PRRSV-2 infection and maintained normal cell vitality comparable with the WT cells. The resistant phenotype of the cells was stably maintained through cell passages. There were no replication transcription complexes in the CD163ΔSRCR5 cells. SRCR5 deletion did not disturb the colocalization of CD163 and PRRSV-N in early endosomes (EE). However, the interaction of the viral proteins GP2a, GP3, or GP5 with CD163, which is involved in virus uncoating was affected. Furthermore, 77 CD163-binding cellular proteins affected by the SRCR5 deletion were identified by LC-MS/MS. Inhibition of calpain 1 trapped the virions in EE and forced then into late endosomes but did not block viral attachment and internalization, suggesting that calpain 1 is involved in the uncoating. Overall, CD163ΔSRCR5 MARC-145 cells are fully resistant to PRRSV-2 infection and calpain 1 is identified as a novel host protein that interacts with CD163 to facilitate PRRSV uncoating.

Chlorine dioxide inhibits the replication of porcine reproductive and respiratory syndrome virus by blocking viral attachment.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a great economic loss to the swine industry globally. Current prevention and treatment measures are not effective to control the outbreak and spread of porcine reproductive and respiratory syndrome (PRRS). In other words, new antiviral strategies are urgently needed. Chlorine dioxide (ClO2) is regarded as a broad-spectrum disinfectant with strong inhibitory effects on microbes and parasites. The purpose of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of ClO2 against PRRSV infection in vitro. Here, we identified ClO2 (the purity is 99%) could inhibit the infection and replication of PRRSV in both Marc-145 cells and porcine alveolar macrophages (PAMs). ClO2 could block PRRSV binding to cells rather than internalization and release, suggesting that ClO2 blocks the first stage of the virus life cycle. We also demonstrated that the inhibition exerted by ClO2 was attributed to the degradation of PRRSV genome and proteins. Moreover, we confirmed that ClO2 could decrease the expression of inflammatory cytokines induced by PRRSV. In summary, ClO2 is an efficient agent and potently suppressed PRRSV infection in vitro.

An integrated analysis of membrane remodeling during porcine reproductive and respiratory syndrome virus replication and assembly.

BACKGROUND: Recently, three-dimensional (3D) imaging techniques have been used to detect viral invasion and the appearance of specialized structures established in virus-infected cells. These methods have had a positive impact in the field of virology and helped to further our knowledge of how viruses invade cells. Nearly all positive-strand RNA viruses propagate their viral genomes in part through intracellular membranes. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus, accumulates viral RNA that forms replication complexes (RCs) in infected cells. In this study, using immunofluorescence and electron microscopy (EM), we dissected PRRSV-induced membrane structures in infected cells and determined the correlations between PRRSV particles and vesicles stimulated by PRRSV to understand the structural and dynamic aspects of PRRSV infection. METHODS: We identified the appropriate time point by determining the 50% tissue culture infectious dose (TCID50) and using qRT-PCR and Western blotting. The co-localization of viruses and organelles was determined by immunofluorescence and immune-electron microscopy (IEM). The ultrastructure of cells infected by PRRSV was observed using EM and electron tomography (ET). RESULTS: In our study, we found that PRRSV dsRNA was located at the endoplasmic reticulum (ER) and autophagosomes; in addition, the N protein was located at the mitochondria, ER and autophagosomes. Vesicles induced by PRRSV appeared at 16 hours post-infection (h.p.i.) and increased in size with time during the infection period. In addition, our findings demonstrated that the virus vesicles originated from the ER, and these two organelle structures connected with each other to form a reticulovesicular network (RVN) that provided a site for virus replication and assembly. CONCLUSION: Our results revealed that membrane vesicles induced by PRRSV were derived from the ER. The vesicles may provide a location for PRRSV replication and assembly.