Porcine cis-acting lnc-CAST positively regulates CXCL8 expression through histone H3K27ac.

The chemokine CXCL8, also known as the neutrophil chemotactic factor, plays a crucial role in mediating inflammatory responses and managing cellular immune reactions during viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) primarily infects pulmonary alveolar macrophages (PAMs), leading to acute pulmonary infections. In this study, we explored a novel long non-coding RNA (lncRNA), termed lnc-CAST, situated within the Cxcl8 gene locus. This lncRNA was found to be highly expressed in porcine macrophages. We observed that both lnc-CAST and CXCL8 were significantly upregulated in PAMs following PRRSV infection, and after treatments with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Furthermore, we noticed a concurrent upregulation of lnc-CAST and CXCL8 expression in lungs of PRRSV-infected pigs. We then determined that lnc-CAST positively influenced CXCL8 expression in PAMs. Overexpression of lnc-CAST led to an increase in CXCL8 production, which in turn enhanced the migration of epithelial cells and the recruitment of neutrophils. Conversely, inhibiting lnc-CAST expression resulted in reduced CXCL8 production in PAMs, leading to decreased migration levels of epithelial cells and neutrophils. From a mechanistic perspective, we found that lnc-CAST, localized in the nucleus, facilitated the enrichment of histone H3K27ac in CXCL8 promoter region, thereby stimulating CXCL8 transcription in a cis-regulatory manner. In conclusion, our study underscores the pivotal critical role of lnc-CAST in regulating CXCL8 production, offering valuable insights into chemokine regulation and lung damage during PRRSV infection.

Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.

MiR-320 inhibits PRRSV replication by targeting PRRSV ORF6 and porcine CEBPB.

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), results in huge economic losses to the world pig industry. MiRNAs have been reported to be involved in regulation of viral infection. In our study, miR-320 was one of 21 common differentially expressed miRNAs of Meishan, Pietrain, and Landrace pig breeds at 9-h post-infection (hpi). Bioinformatics and experiments found that PRRSV replication was inhibited by miR-320 through directly targeting PRRSV ORF6. In addition, the expression of CCAAT enhancer binding protein beta (CEBPB) was also inhibited by miR-320 by targeting the 3' UTR of CEBPB, which significantly promotes PRRSV replication. Intramuscular injection of pEGFP-N1-miR-320 verified that miR-320 significantly inhibited the replication of PRRSV and alleviated the symptoms caused by PRRSV in piglets. Taken together, miR-320 have significant roles in the infection and may be promising therapeutic target for PRRS.

Fuzhengjiedu San inhibits porcine reproductive and respiratory syndrome virus by activating the PI3K/AKT pathway.

BACKGROUND: Traditional Chinese medicine (TCM) has been garnering ever-increasing worldwide attention as the herbal extracts and formulas prove to have potency against disease. Fuzhengjiedu San (FZJDS), has been extensively used to treat viral diseases in pigs, but its bioactive components and therapeutic mechanisms remain unclear. METHODS: In this study, we conducted an integrative approach of network pharmacology and experimental study to elucidate the mechanisms underlying FZJDS's action in treating porcine reproductive and respiratory syndrome virus (PRRSV). We constructed PPI network and screened the core targets according to their degree of value. GO and KEGG enrichment analyses were also carried out to identify relevant pathways. Lastly, qRT-PCR, flow cytometry and western blotting were used to determine the effects of FZJDS on core gene expression in PRRSV-infected monkey kidney (MARC-145) cells to further expand the results of network pharmacological analysis. RESULTS: Network pharmacology data revealed that quercetin, kaempferol, and luteolin were the main active compounds of FZJDS. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway was deemed the cellular target as it has been shown to participate most in PRRSV replication and other PRRSV-related functions. Analysis by qRT-PCR and western blotting demonstrated that FZJDS significantly reduced the expression of P65, JNK, TLR4, N protein, Bax and IĸBa in MARC-145 cells, and increased the expression of Bcl-2, consistent with network pharmacology results. This study provides that FZJDS has significant antiviral activity through its effects on the PI3K/AKT signaling pathway. CONCLUSION: We conclude that FZJDS is a promising candidate herbal formulation for treating PRRSV and deserves further investigation.

Porcine reproductive and respiratory syndrome virus nsp4-mediated ß2M downregulation contributes to SLA-I decrease and virus infection in vivo and in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection inhibits swine leukocyte antigen class I (SLA-I) expression in pigs, resulting in inefficient antigen presentation and subsequent low levels of cellular PRRSV-specific immunity as well as persistent viremia. We previously observed that the non-structural protein 4 (nsp4) of PRRSV contributed to inhibition of the ß2-microglobulin (ß2M) and SLA-I expression in cells. Here, we constructed a series of nsp4 mutants with different combination of amino acid mutations to attenuate the inhibitory effect of nsp4 on ß2M and SLA-I expression. Almost all nsp4 mutants exogenously expressed in cells showed an attenuated effect on inhibition of ß2M and SLA-I expression, but the recombinant PRRSV harboring these nsp4 mutants failed to be rescued with exception of the rPRRSV-nsp4-mut10 harboring three amino acid mutations. However, infection of rPRRSV-nsp4-mut10 not only enhanced ß2M and SLA-I expression in both cells and pigs but also promoted the DCs to active the CD3+CD8+T lymphocytes more efficiently, as compared with its parental PRRSV (rPRRVS-nsp4-wt). These data suggested that the inhibition of nsp4-mediated ß2M downregulation improved ß2M/SLA-I expression in pigs.

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus.

Minor envelope proteins from GP2a to GP4 contribute to the spread pattern and yield of type 2 PRRSV in MARC-145 cells.

In China, porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are widely used. These vaccines, which contain inactivated and live attenuated vaccines (LAVs), are produced by MARC-145 cells derived from the monkey kidney cell line. However, some PRRSV strains in MARC-145 cells have a low yield. Here, we used two type 2 PRRSV strains (CH-1R and HuN4) to identify the genes responsible for virus yield in MARC-145 cells. Our findings indicate that the two viruses have different spread patterns, which ultimately determine their yield. By replacing the viral envelope genes with a reverse genetics system, we discovered that the minor envelope proteins, from GP2a to GP4, play a crucial role in determining the spread pattern and yield of type 2 PRRSV in MARC-145 cells. The cell-free transmission pattern of type 2 PRRSV appears to be more efficient than the cell-to-cell transmission pattern. Overall, these findings suggest that GP2a to GP4 contributes to the spread pattern and yield of type 2 PRRSV.

Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18.

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.

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.

The mRNA Vaccine Expressing Single and Fused Structural Proteins of Porcine Reproductive and Respiratory Syndrome Induces Strong Cellular and Humoral Immune Responses in BalB/C Mice.

PRRS is a viral disease that profoundly impacts the global swine industry, causing significant economic losses. The development of a novel and effective vaccine is crucial to halt the rapid transmission of this virus. There have been several vaccination attempts against PRRSV using both traditional and alternative vaccine design development approaches. Unfortunately, there is no currently available vaccine that can completely control this disease. Thus, our study aimed to develop an mRNA vaccine using the antigens expressed by single or fused PRRSV structural proteins. In this study, the nucleotide sequence of the immunogenic mRNA was determined by considering the antigenicity of structural proteins and the stability of spatial structure. Purified GP5 protein served as the detection antigen in the immunological evaluation. Furthermore, cellular mRNA expression was detected by immunofluorescence and western blotting. In a mice experiment, the Ab titer in serum and the activation of spleen lymphocytes triggered by the antigen were detected by ELISA and ICS, respectively. Our findings demonstrated that both mRNA vaccines can significantly stimulate cellular and humoral immune responses. More specifically, the GP5-mRNA exhibited an immunological response that was similar to that of the commercially available vaccine when administered in high doses. To conclude, our vaccine may show promising results against the wild-type virus in a natural host.

Heat shock protein 71 restricts mutation of porcine reproductive and respiratory syndrome virus nsp2 in vitro.

porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection, is an important swine infectious disease that causes substantial losses worldwide each year. PRRSV is a positive-sense single-stranded RNA virus that is highly susceptible to mutation and recombination, making vaccine and drug research for the disease extremely difficult. In this study, the binding of PRRSV nsp2 to HSP71 protein was detected by using the IP/MS technique. And the inhibitory effect of HSP71 on nsp2 antagonistic activity was validated by measuring NF-kB luciferase reporter. According to stress from inhibitory effects, the amino acid variation profile of PRRSV nsp2 under HSP71 stress was further analyzed using second-generation sequencing. Surprisingly, the results indicated that HSP71 pressure limits the random mutations of PRRSV nsp2 and maintains the dominant PRRSV strain within the population. Mutant strain showed weaker antagonistic activity and replication capability in cell. These results imply the binding of HSP71 with PRRSV nsp2 may lead to maintain the stability of highly virulent strains of PRRSV.

Role of microRNAs in host defense against porcine reproductive and respiratory syndrome virus infection: a hidden front line.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most globally devastating viruses threatening the swine industry worldwide. Substantial advancements have been achieved in recent years towards comprehending the pathogenesis of PRRSV infection and the host response, involving both innate and adaptive immune responses. Not only a multitude of host proteins actively participate in intricate interactions with viral proteins, but microRNAs (miRNAs) also play a pivotal role in the host response to PRRSV infection. If a PRRSV-host interaction at the protein level is conceptualized as the front line of the battle between pathogens and host cells, then their fight at the RNA level resembles the hidden front line. miRNAs are endogenous small non-coding RNAs of approximately 20-25 nucleotides (nt) that primarily regulate the degradation or translation inhibition of target genes by binding to the 3'-untranslated regions (UTRs). Insights into the roles played by viral proteins and miRNAs in the host response can enhance our comprehensive understanding of the pathogenesis of PRRSV infection. The intricate interplay between viral proteins and cellular targets during PRRSV infection has been extensively explored. This review predominantly centers on the contemporary understanding of the host response to PRRSV infection at the RNA level, in particular, focusing on the twenty-six miRNAs that affect viral replication and the innate immune response.

Computational antigenic insights into the novel NADC-34-like Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) isolate YC-2020.

In this computational study, we advanced the understanding of the antigenic properties of the NADC-34-like isolate of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), named YC-2020, relevant in veterinary pathology. We utilized sequence comparison analyses of the M and N proteins, comparing them with those of NADC34, identifying substantial amino acid homology that allowed us to highlight conserved epitopes and crucial variants. Through the application of Clustal Omega for multiple sequence alignment and platforms like Vaxijen and AllerTOP for predicting antigenic and allergenic potential, our analyses revealed important insights into the conservation and variation of epitopes essential for the development of effective diagnostic tools and vaccines. Our findings, aligned with initial experimental studies, underscore the importance of these epitopes in the development of targeted immunodiagnostic platforms and significantly contribute to the management and control of PRRSV. However, further studies are required to validate the computational predictions of antigenicity for this new viral isolate. This approach underscores the potential of computational models to enable ongoing monitoring and control of PRRSV evolution in swine. While this study provides valuable insights into the antigenic properties of the novel PRRSV isolate YC-2020 through computational analysis, it is important to acknowledge the limitations inherent to in silico predictions, specifically, the absence of laboratory validation.

Immune response enhancement by dietary supplementation with Caesalpinia sappan extract in weaned pigs challenged with porcine reproductive and respiratory syndrome virus.

BACKGROUND: At present, porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe epidemics impacting pig farming globally. Despite the fact that a number of studies have been conducted on potential solutions to this problem, none have proven effective. The focus of problem solving is the use of natural ingredients such as plant extracts. Popular throughout Asia, Caesalpinia sappan (CS) is a therapeutic plant that inhibits PRRSV in vitro. Therefore, this study was performed to determine the efficacy of CS extract dietary supplementation on the productive performance, antibody levels, immunological indicators, and lung pathology of PRRSV-challenged weaned pigs. A total of 32 weaned piglets (28 days old) were randomized into 4 groups and kept separately for 14 days. The treatments were organized in a 2 × 2 factorial design involving two factors: PRRSV challenge and supplementation with 1 mg/kg CS extract. The pigs in the PRRSV-challenged groups were intranasally inoculated with 2 mL of PRRSV (VR2332) containing 104 TCID50/mL, while those in the groups not challenged with PRRSV were inoculated with 2 mL of normal saline. RESULTS: In the PRRSV-challenged group (CS + PRRSV), supplementation with CS extract led to an increase in white blood cells (WBCs) on Day 7 post infection (p < 0.05) and particularly in lymphocytes on Days 7 and 14. The antibody titer was significantly greater in the CS + PRRSV group than in the PRRSV-challenged group not administered CS (PRRSV group) on Day 14 postinfection (S/P = 1.19 vs. 0.78). In addition, CS extract administration decreased the prevalence of pulmonary lesions, which were more prevalent in the PRRSV-challenged pigs that did not receive the CS extract. CONCLUSION: The findings of this study suggest that supplementation with CS extract is beneficial for increasing WBC counts, especially lymphocytes, increasing the levels of antibodies and reducing the prevalence of lung lesions in PRRSV-infected pigs.

Caffeic acid phenethyl ester: an effective antiviral agent against porcine reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome (PRRS) presents a formidable viral challenge in swine husbandry. Confronting the constraints of existing veterinary pharmaceuticals and vaccines, this investigation centers on Caffeic Acid Phenethyl Ester (CAPE) as a prospective clinical suppressant for the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The study adopts an integrated methodology to evaluate CAPE's antiviral attributes. This encompasses a dual-phase analysis of CAPE's interaction with PRRSV, both in vitro and in vivo, and an examination of its influence on viral replication. Varied dosages of CAPE were subjected to empirical testing in animal models to quantify its efficacy in combating PRRSV infections. The findings reveal a pronounced antiviral potency, notably in prophylactic scenarios. As a predominant component of propolis, CAPE stands out as a promising candidate for clinical suppression, showing exceptional effectiveness in pre-exposure prophylaxis regimes. This highlights the potential of CAPE in spearheading cutting-edge strategies for the management of future PRRSV outbreaks.

Contrasting PRRSV temporal lineage patterns at the individual farm, production system, and regional levels in Ohio and neighboring states from 2017 to 2021.

Porcine reproductive and respiratory virus (PRRSV), one of the most significant viruses in the swine industry, has been challenging to control due to its high mutation and recombination rates and complexity. This retrospective study aimed to describe and compare the distribution of PRRSV lineages obtained at the individual farm, production system, and regional levels. PRRSV-2 (type 2) sequences (n = 482) identified between 2017 - 2021 were provided by a regional state laboratory (Ohio Department of Agriculture, Animal Disease Diagnostic Center (ODA-ADDL)) collected from swine farms in Ohio and neighboring states, including Indiana, Michigan, Pennsylvania, and West Virginia. Additional sequences (n = 138) were provided by one collaborating swine production system. The MUSCLE algorithm on Geneious Prime® was used to align the ORF5 region of PRRSV-2 sequences along with PRRSV live attenuated vaccine strains (n = 6) and lineage anchors (n = 169). Sequenced PRRSV-2 were assigned to the most identical lineage anchors/vaccine strains. Among all sequences (n = 620), 29.8% (185/620) were ≥ 98.0% identity with the vaccine strains, where 93.5% (173/185) and 6.5% (12/185) were identical with the L5 Ingelvac PRRS® MLV and L8 Fostera® PRRS vaccine strains, respectively, and excluded from the analysis. At the regional level across five years, the top five most identified lineages included L1A, L5, L1H, L1C, and L8. Among non-vaccine sequences with production system known, L1A sequences were mostly identified (64.3% - 100.0%) in five systems, followed by L1H (0.0% - 28.6%), L1C (0.0% - 10.5%), L5 (0.0% - 14.4%), L8 (0.0% - 1.3%), and L1F (0.0% - 0.5%). Furthermore, among non-vaccine sequences with the premise identification available (n = 262), the majority of sequences from five individual farms were either classified into L1A or L5. L1A and L5 sequences coexisted in three farms, while samples submitted by one farm contained L1A, L1H, and L5 sequences. Additionally, the lineage classification results of non-vaccine sequences were associated with their restriction fragment length polymorphism (RFLP) patterns (Fisher's exact test, p < 0.05). Overall, our results show that individual farm and production system-level PRRSV-2 lineage patterns do not necessarily correspond to regional-level patterns, highlighting the influence of individual farms and systems in shaping PRRSV occurrence within those levels, and highlighting the crucial goal of within-farm and system monitoring and early detection for accurate knowledge on PRRSV-2 lineage occurrence and emergence.

Porcine HERC6 acts as major E3 ligase for ISGylation and is auto-ISGylated for effective ISGylation in porcine.

Interferon-stimulated gene product 15 (ISG15) can be conjugated to substrates through ISGylation. Currently, the E3 ligase for porcine ISGylation remains unclear. Here, we identified porcine HERC5 and HERC6 (pHERC5/6) as ISGylation E3 ligases with pHERC6 acting as a major one by reconstitution of porcine ISGylation system in HEK-293 T cell via co-transfecting E1, E2 and porcine ISG15(pISG15) genes. Meanwhile, our data demonstrated that co-transfection of pISG15 and pHERC5/6 was sufficient to confer ISGylation, suggesting E1 and E2 of ISGylation are interchangeable between human and porcine. Using an immunoprecipitation based ISGylation analysis, our data revealed pHERC6 was a substrate for ISGylation and confirmed that K707 and K993 of pHERC6 were auto-ISGylation sites. Mutation of these sites reduced pHERC6 half-life and inhibited ISGylation, suggesting that auto-ISGylation of pHERC6 was required for effective ISGylation. Conversely, sustained ISGylation induced by overexpression of pISG15 and pHERC6 could be inhibited by a well-defined porcine ISGylation antagonist, the ovarian tumor (OTU) protease domain of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-nsp2 and PRRSV-nsp1ß, further indicating such method could be used for identification of virus-encoded ISG15 antagonist. In conclusion, our study contributes new insights towards porcine ISGylation system and provides a novel tool for screening viral-encoded ISG15 antagonist.

Isolation and pathogenicity comparison of two novel natural recombinant porcine reproductive and respiratory syndrome viruses with different recombination patterns in Southwest China.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the swine industry. Frequent mutations and recombinations account for PRRSV immune evasion and the emergence of novel strains. In this study, we isolated and characterized two novel PRRSV-2 strains from Southwest China exhibiting distinct recombination patterns. They were designated SCABTC-202305 and SCABTC-202309. Phylogenetic results indicated that SCABTC-202305 was classified as lineage 8, and SCABTC-202309 was classified as lineage 1.8. Amino acid mutation analysis identified unique amino acid substitutions and deletions in ORF5 and Nsp2 genes. The results of the recombination analysis revealed that SCABTC-202305 is a recombinant with JXA1 as the major parental strain and NADC30 as the minor parental strain. At the same time, SCABTC-202309 is identified as a recombinant with NADC30 as the major parental strain and JXA1 as the minor parental strain. In this study, we infected piglets with SCABTC-202305, SCABTC-202309, or mock inoculum (control) to study the pathogenicity of these isolates. Although both isolated strains were pathogenic, SCABTC-202305-infected piglets exhibited more severe clinical signs and higher mortality, viral load, and antibody response than SCABTC-202309-infected piglets. SCABTC-202305 also caused more extensive lung lesions based on histopathology. Our findings suggest that the divergent pathogenicity observed between the two novel PRRSV isolates may be attributed to variations in the genetic information encoded by specific genomic regions. Elucidating the genetic determinants governing PRRSV virulence and transmissibility will inform efforts to control this devastating swine pathogen.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most critical pathogens impacting the global swine industry. Frequent mutations and recombinations have made the control of PRRSV increasingly difficult. Following the NADC30-like PRRSV pandemic, recombination events involving PRRSV strains have further increased. We isolated two novel field PRRSV recombinant strains, SCABTC-202305 and SCABTC-202309, exhibiting different recombination patterns and compared their pathogenicity in animal experiments. The isolates caused higher viral loads, persistent fever, marked weight loss, moderate respiratory clinical signs, and severe histopathologic lung lesions in piglets. Elucidating correlations between recombinant regions and pathogenicity in these isolates can inform epidemiologic tracking of emerging strains and investigations into viral adaptive mechanisms underlying PRRSV immunity evasion. Our findings underscore the importance of continued genomic surveillance to curb this economically damaging pathogen.

GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Antigenicity, epitope mapping, and intracellular distribution of the NSP7α protein of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge economic losses to the global pig industry. Nonstructural protein 7α (NSP7α) of PRRSV is highly conserved among different lineages of PRRSV and could be a potential target for the development of detection methods. In this study, NSP7α was expressed in prokaryote (Escherichia coli) and purified. An NSP7α-ab-ELISA detection method was established, the NSP7α-ab-ELISA has 93.1 % coincidence rate with IDEXX PRRS X3 ab test kit. NSP7α antibody was detected in pig serum by ELISA 14 days following PRRSV infection. Three monoclonal antibodies (4H9, 3F2, and C10) against NSP7α prepared by a hybridoma technique were used for epitope mapping by indirect immunofluorescence. The 4H9, 3F2, and C10 antibodies all recognized the C-terminal 72-149 amino acid region of NSP7α. 4H9 reacted with amino acids 135-143, but 3F2 and C10 did not react with any truncated polypeptide. In addition, by using the monoclonal antibodies, NSP7α was localized solely in the cytoplasm, while the N protein was distributed in the cytoplasm and nucleus. The collective findings of the antigenicity and epitope of NSP7α will be helpful for understanding the antigenicity of NSP7α and developing PRRSV diagnostic methods.