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.

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.

Establishment and Application of a Quadruplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, Porcine Circovirus Type 3, and Streptococcus suis.

Respiratory illnesses present a significant threat to porcine health, with co-infections involving Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Streptococcus suis (SS), Porcine Circovirus Type 2 (PCV2), and Porcine Circovirus Type 3 (PCV3) acting as the primary causative agents. As a result, the precise diagnosis of PRRSV, PCV2, PCV3 and SS is of paramount importance in the prevention and control of respiratory diseases in swine. Therefore, we conducted a molecular bioinformatical analysis to concurrently detect and differentiate PRRSV, PCV2, PCV3 and SS. We selected the ORF6 gene of PRRSV, the ORF2 gene of PCV2 and PCV3, and the glutamate dehydrogenase (GDH) gene of SS as targets. Specific primers and probes were designed for each pathogen, and following meticulous optimization of reaction conditions, we established a multiple TaqMan fluorescence quantitative PCR detection method. Subsequently, we subjected this method to a comprehensive assessment, evaluating its specificity, sensitivity, and repeatability. The research results demonstrated that the established multiple TaqMan fluorescence quantitative PCR detection method displays displayed exemplary specificity, with no instances of cross-reactivity with other pathogens. The method's minimum detection concentrations for PRRSV, PCV2, PCV3, and SS were 2.80 × 101 copies/µL, 1.96 × 102 copies/µL, 2.30 × 102 copies/µL, and 1.75 × 103 copies/µL, respectively. When applied to the analysis of 30 clinical samples, the results closely mirrored those obtained through Chinese standard uniplex real-time qPCR detection method for PRRSV, as well as the general PCR methods for SS, PCV2, and PCV3. This study underscores the robust specificity, high sensitivity, and consistent stability of the multiple TaqMan fluorescence quantitative PCR detection method that we have developed. It is ideally suited to the clinical monitoring of PRRSV, PCV2, PCV3, and SS, and it carries significant importance in ongoing efforts to prevent and manage respiratory diseases in porcine populations.

Corrigendum: Isolation, identification, recombination analysis and pathogenicity experiment of a PRRSV recombinant strain in Sichuan Province, China.

[This corrects the article DOI: 10.3389/fmicb.2024.1362471.].

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.

Type I and II interferons, transcription factors and major histocompatibility complexes were enhanced by knocking down the PRRSV-induced transforming growth factor beta in monocytes co-cultured with peripheral blood lymphocytes.

The innate and adaptive immune responses elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection are known to be poor. This study investigates the impact of PRRSV-induced transforming growth factor beta 1 (TGFß1) on the expressions of type I and II interferons (IFNs), transcription factors, major histocompatibility complexes (MHC), anti-inflammatory and pro-inflammatory cytokines in PRRSV-infected co-cultures of monocytes and peripheral blood lymphocytes (PBL). Phosphorothioate-modified antisense oligodeoxynucleotide (AS ODN) specific to the AUG region of porcine TGFß1 mRNA was synthesized and successfully knocked down TGFß1 mRNA expression and protein translation. Monocytes transfected with TGFßAS1 ODN, then simultaneously co-cultured with PBL and inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) showed a significant reduction in TGFß1 mRNA expression and a significant increase in the mRNA expressions of IFNα, IFNγ, MHC-I, MHC-II, signal transducer and activator of transcription 1 (STAT1), and STAT2. Additionally, transfection of TGFßAS1 ODN in the monocyte and PBL co-culture inoculated with cPRRSV-2 significantly increased the mRNA expression of interleukin-12p40 (IL-12p40). PRRSV-2 RNA copy numbers were significantly reduced in monocytes and PBL co-culture transfected with TGFßAS1 ODN compared to the untransfected control. The yields of PRRSV-2 RNA copy numbers in PRRSV-2-inoculated monocytes and PBL co-culture were sustained and reduced by porcine TGFß1 (rTGFß1) and recombinant porcine IFNα (rIFNα), respectively. These findings highlight the strategy employed by PRRSV to suppress the innate immune response through the induction of TGFß expression. The inclusion of TGFß as a parameter for future PRRSV vaccine and vaccine adjuvant candidates is recommended.

Highly pathogenic PRRSV upregulates IL-13 production through nonstructural protein 9-mediated inhibition of N6-methyladenosine demethylase FTO.

Porcine reproductive and respiratory syndrome virus (PRRSV), a highly infectious virus, causes severe losses in the swine industry by regulating the inflammatory response, inducing tissue damage, suppressing the innate immune response, and promoting persistent infection in hosts. Interleukin-13 (IL-13) is a cytokine that plays a critical role in regulating immune responses and inflammation, particularly in immune-related disorders, certain types of cancer, and numerous bacterial and viral infections; however, the underlying mechanisms of IL-13 regulation during PRRSV infection are not well understood. In this study, we demonstrated that PRRSV infection elevates IL-13 levels in porcine alveolar macrophages. PRRSV enhances m6A-methylated RNA levels while reducing the expression of fat mass and obesity associated protein (FTO, an m6A demethylase), thereby augmenting IL-13 production. PRRSV nonstructural protein 9 (nsp9) was a key factor for this modulation. Furthermore, we found that the residues Asp567, Tyr586, Leu593, and Asp595 were essential for nsp9 to induce IL-13 production via attenuation of FTO expression. These insights delineate PRRSV nsp9's role in FTO-mediated IL-13 release, advancing our understanding of PRRSV's impact on host immune and inflammatory responses.

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.

Isolation, identification, recombination analysis and pathogenicity experiment of a PRRSV recombinant strain in Sichuan Province, China.

Since 2013, the porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2), lineage 1.8 (NADC30-like PRRSV) has emerged and become widely prevalent in China. The NADC30-like PRRSV poses significant challenges for disease control, primarily because of its propensity for frequent mutations and recombinations. We successfully isolated and identified a NADC30-like strain, designated SCCD22, in Chengdu, Sichuan Province, China. We meticulously examined the genetic recombination properties and evaluated its pathogenicity in 28-day-old piglets. SCCD22 showed 93.02% nucleotide homology with the NADC30 PRRSV strain, and its non-structural protein 2 coding region showed the same 131 amino acid deletion pattern as that seen in NADC30. Furthermore, we identified two recombination events in SCCD22: one in the NSP2 region (1,028-3,290 nt), where it was highly similar to the JXA1-like strain GZ106; and another in the NSP10 ~ 12 region (9,985-12,279 nt), closely resembling the NADC30-like strain CY2-1604. Piglets infected with SCCD22 exhibited clinical symptoms such as elevated body temperature, prolonged fever, reduced appetite, and roughened fur. Postmortem examinations underscored the typical lung pathology associated with PRRSV, indicating that the lungs were the primary affected organs. Furthermore, extended viral shedding accompanied by progressive viremia was observed in the serum and nasal excretions of infected piglets. In summary, this study reports a domestic PRRSV recombination strain in the Sichuan Province that can provide critical insights into preventing and controlling PRRSV in this region.

A chimeric porcine reproductive and respiratory syndrome virus 1 strain containing synthetic ORF2-6 genes can trigger T follicular helper cell and heterologous neutralizing antibody responses and confer enhanced cross-protection.

The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.

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.

Genomic surveillance and evolutionary dynamics of type 2 porcine reproductive and respiratory syndrome virus in China spanning the African swine fever outbreak.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a serious threat to the pig industry in China. Our previous study demonstrated that PRRSV persists with local circulations and overseas imports in China and has formed a relatively stable epidemic pattern. However, the sudden African swine fever (ASF) outbreak in 2018 caused serious damage to China's pig industry structure, which resulted in about 40 per cent of pigs being slaughtered. The pig yields recovered by the end of 2019. Thus, whether the ASF outbreak reframed PRRSV evolution with changes in pig populations and further posed new threats to the pig industry becomes a matter of concern. For this purpose, we conducted genomic surveillance and recombination, NSP2 polymorphism, population dynamics, and geographical spread analysis of PRRSV-2, which is dominant in China. The results showed that the prevalence of ASF had no significant effects on genetic diversities like lineage composition, recombination patterns, and NSP2 insertion and deletion patterns but was likely to lead to changes in PRRSV-2 recombination frequency. As for circulation of the two major sub-lineages of Lineage 1, there was no apparent transmission of NADC30-like among provinces, while NADC34-like had obvious signs of inter-provincial transmission and foreign importation during the ASF epidemic. In addition, two suspected vaccine recombinant epidemic strains suggest a slight safety issue of vaccine use. Herein, the interference of ASF to the PRRSV-2 evolutionary pattern was evaluated and vaccine safety was analyzed, in order to monitor the potential threat of PRRSV-2 to China's pig industry in the post-epidemic era of ASF.

MiR-339-5p inhibits replication of porcine reproductive and respiratory syndrome virus by targeting viral gene regions.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a variable virus, whose spread cannot be totally stopped by vaccination. PRRSV infection results in abortion and respiratory symptoms in pregnant pigs. One crucial component of the anti-viral infection strategy is microRNA (miRNA), a class of multifunctional small molecules. It is unknown whether miR-339-5p can specifically target the PRRSV gene and prevent the virus from replicating, despite the fact that miR-339-5p is markedly up-regulated during the PRRSV infection. In this pursuit, the present study revealed that the two PRRSV areas targeted by miR-339-5p were PRRSV nsp2-3378 to 3403 and PRRSV nsp2-3112 to 3133 using the miRanda program. Dual luciferase reporter assays showed that the miR-339-5p target region of the PRRSV gene sequence exhibited 100% homology and was highly conserved. Furthermore, the ability of miR-339-5p to target PRRSV gene areas was verified. It was found that the overexpression of miR-339-5p markedly reduced the PRRSV replication through PRRSV infection trials. The precursor sequence of ssc-miR-339-5p was amplified using the DNA of pig lung tissue as a template in order to create a fragment of 402 bp of porcine-derived miR-339-5p precursor sequence, which was then used to produce the eukaryotic expression plasmid of miR-339-5p. In conclusion, miR-339-5p can target the specific PRRSV gene areas and prevent PRRSV replication, offering fresh perspectives for the creation of medications that combat the PRRSV infection.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths.

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at - 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Characterization of a novel recombinant NADC30­like porcine reproductive and respiratory syndrome virus in Shanxi Province, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens affecting the swine industry. In this report, a novel PRRSV strain SXht2012 was isolated from Shanxi province in China. To identify genetic characteristics of SXht2012, we conducted phylogenetic and homology analyses after sequencing its complete genome. The results revealed that SXht2012 belonged to NADC30-like strain and shared 91.3% nucleotide (nt) identity with strain NADC30. Notably, sequence alignment showed that a distinctive feature in the NSP2 region, where a 131-amino acid (aa) deletion was found in the hypervariable region (HVR). Additionally, variations were also detected in the GP5 protein, specifically in the decoy peptide, T cell peptide, and a potential glycosylation site (aa 32). Furthermore, we also found that SXht2012 was likely a recombination virus originating from NADC30-like and JXA1-like strains, and three recombination breakpoints were identified in the genome at nt positions 1516, 5280 and 6851, which correspond to the NSP2, NSP3, and NSP7 regions. Overall, these findings have significant implications for understanding the genetic variation and evolutionary dynamics of PRRSV strains.

A descriptive study on spatial and temporal distributions of genetic clusters of porcine reproductive and respiratory syndrome virus infecting pig sites in Quebec, Canada, between 2010 and 2019.

BACKGROUND: The wide diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains combined with incomplete heterologous cross-protection complicates the management of the disease at both the herd and the regional levels. The objectives of this study were to describe the spatial and temporal distribution of various PRRSV genetic clusters infecting pig sites in Quebec, Canada, and to compare PRRSV regional diversity of wild-type sequences over the years. MATERIALS AND METHODS: A retrospective surveillance-based study was conducted on all pig sites which had PRRSV ORF5 sequences from field submissions transferred into the Laboratoire d'épidémiologie et de médecine porcine database from January 1, 2010 to December 31, 2019. A maximum likelihood phylogenetic tree inferred from multiple sequence alignment was used to identify genetic clusters. For each wild-type cluster gathering ≥ 15 sequences, the number of pig sites in which the cluster was detected per administrative region and per year were displayed on bubble charts and the spatiotemporal distribution of pig sites was illustrated using pie chart maps. A molecular analysis of variance was performed to compare PRRSV wild-type sequence diversity according to the administrative region for each year. RESULTS: A total of 32 wild-type clusters gathering 1653 PRRSV2 sequences from 693 pig sites were described. Each cluster was detected on up to 132 pig sites and 7 administrative regions over the 10-year period. Annually, the mean (min-max) number of wild-type clusters detected in at least one pig site reached 24 (17-29). Some clusters remained localized on a few sites over time whereas others were widespread over the territory during a few or many years. For each year, regional differences were also observed in PRRSV diversity of wild-type sequences. CONCLUSIONS: The differences observed in both the spatiotemporal distributions of PRRSV clusters and in the regional diversity of wild-type sequences highlight the importance of ongoing provincial surveillance to improve collective PRRS management strategies.

PRRSV degrades MDA5 via dual autophagy receptors P62 and CCT2 to evade antiviral innate immunity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically devastating pathogen that has evolved various strategies to evade innate immunity. Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5 (MDA5), a receptor that senses viral RNA. In this study, the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed, and the detailed mechanisms were explored. We found that the interaction between P62 and MDA5 is enhanced due to two factors: the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2α and the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells. As a result of these modifications, the classic P62-mediated autophagy is triggered. Additionally, porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2 (CCT2), which is enhanced by PRRSV nsp3. This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination. In summary, enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways: the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2, leading to intense innate immune suppression. The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies.

Evaluation of Truck Cab Decontamination Procedures following Inoculation with Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus.

This experiment aimed to evaluate commercially available disinfectants and their application methods against porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) on truck cab surfaces. Plastic, fabric, and rubber surfaces inoculated with PEDV or PRRSV were placed in a full-scale truck cab and then treated with one of eight randomly assigned disinfectant treatments. After application, surfaces were environmentally sampled with cotton gauze and tested for PEDV and PRRSV using qPCR duplex analysis. There was a disinfectant × surface interaction (p < 0.0001), indicating a detectable amount of PEDV or PRRSV RNA was impacted by disinfectant treatment and surface material. For rubber surfaces, 10% bleach application had lower detectable amounts of RNA compared to all other treatments (p < 0.05) except Intervention via misting fumigation, which was intermediate. In both fabric and plastic surfaces, there was no evidence (p > 0.05) of a difference in detectable RNA between disinfectant treatments. For disinfectant treatments, fabric surfaces with no chemical treatment had less detectable viral RNA compared to the corresponding plastic and rubber (p < 0.05). Intervention applied via pump sprayer to fabric surfaces had less detectable viral RNA than plastic (p < 0.05). Furthermore, 10% bleach applied via pump sprayer to fabric and rubber surfaces had less detectable viral RNA than plastic (p < 0.05). Also, a 10 h downtime, with no chemical application or gaseous fumigation for 10 h, applied to fabric surfaces had less detectable viral RNA than other surfaces (p < 0.05). Sixteen treatments were evaluated via swine bioassay, but all samples failed to produce infectivity. In summary, commercially available disinfectants successfully reduced detectable viral RNA on surfaces but did not eliminate viral genetic material, highlighting the importance of bioexclusion of pathogens of interest.

Genome stability assessment of PRRS vaccine strain with new ARTIC-style sequencing protocol.

A tiling amplicon sequencing protocol was developed to analyse the genome sequence stability of the modified live PRRSV vaccine strain, Porcilis MLV. The backbone of the ARTIC-style protocol was formed by 34 individual primer pairs, which were divided into two primer pools. Primer pairs were designed to amplify 532 to 588 bp fragments of the corresponding genomic region. The amplicons are suitable for sequencing on Illumina DNA sequencers with available 600-cycle sequencing kits. The concentration of primer pairs in the pools was optimized to obtain a balanced sequencing depth along the genome. Deep sequencing data of three vaccine batches were also analysed. All three vaccine batches were very similar to each other, although they also showed single nucleotide variations (SNVs) affecting less than 1 % of the genome. In the three vaccine strains, 113 to 122 SNV sites were identified; at these sites, the minority variants represented a frequency range of 1 to 48.7 percent. Additionally, the strains within the batches contained well-known length polymorphisms; the genomes of these minority deletion mutants were 135 to 222 bp shorter than the variant with the complete genome. Our results show the usefulness of ARTIC-style protocols in the evaluation of the genomic stability of PRRS MLV strains.