Enhancing the understanding of coinfection outcomes: Impact of natural atypical porcine pestivirus infection on porcine reproductive and respiratory syndrome in pigs.

Atypical porcine pestivirus (APPV) is a novel member of the Pestivirus genus detected in association with congenital tremor (CT) type A-II outbreaks and from apparently healthy pigs, both as singular infection and as part of multi-pathogen infections. 'Classical' pestiviruses are known to cause immunosuppression of their host, which can increase susceptibility to secondary infections, severely impacting health, welfare, and production. To investigate APPV's effect on the host's immune system and characterise disease outcomes, 12 piglets from a natural APPV CT type A-II outbreak were experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV), a significant porcine pathogen. Rectal temperatures indicating febrile responses, viremia and viral-specific humoral and cellular responses were assessed throughout the study. Pathological assessment of the lungs and APPV-PRRSV co-localisation within the lungs was performed at necropsy. Viral co-localisation and pathological assessment of the lungs (Immunohistochemistry, BaseScope in situ hybridisation) were performed post-mortem. APPV status did not impact virological or immunological differences in PRRSV-infected groups. However, significantly higher rectal temperatures were observed in the APPV+ve/PRRSV+ve group over four days, indicating APPV increased the febrile response. Significant differences in the lung consolidation of the apical and intermediate lobes were also present, suggesting that APPV co-infection may augment lung pathology.

Porcine reproductive and respiratory syndrome virus infects the reproductive system of male piglets and impairs development of the blood-testis barrier.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease that threatens the global swine industry. Recent studies have focused on the damage that PRRSV causes to the reproductive system of male pigs, although pathological research is lacking. Therefore, we examined the pathogenic mechanisms in male piglets infected with PRRSV. Gross and histopathological changes indicated that PRRSV affected the entire reproductive system, as confirmed via immunohistochemical analysis. PRRSV infected Sertoli cells and spermatogonia. To test the new hypothesis that PRRSV infection in piglets impairs blood - testis barrier (BTB) development, we investigated the pathology of PRRSV damage in the BTB. PRRSV infection significantly decreased the quantity and proliferative capacity of Sertoli cells constituting the BTB. Zonula occludens-1 and ß-catenin were downregulated in cell - cell junctions. Transcriptome analysis revealed that several crucial genes and signalling pathways involved in the growth and development of Leydig cells, Sertoli cells, and tight junctions in the testes were downregulated. Apoptosis, necroptosis, inflammatory, and oxidative stress-related pathways were activated, whereas hormone secretion-related pathways were inhibited. Many Sertoli cells and spermatogonia underwent apoptosis during early differentiation. Infected piglets exhibited disrupted androgen secretion, leading to significantly reduced testosterone and anti-Müllerian hormone levels. A cytokine storm occurred, notably upregulating cytokines such as tumour necrosis factor-α and interleukin-6. Markers of oxidative-stress damage (i.e. H2O2, malondialdehyde, and glutathione) were upregulated, whereas antioxidant-enzyme activities (i.e. superoxide dismutase, total antioxidant capacity, and catalase) were downregulated. Our results demonstrated that PRRSV infected multiple organs in the male reproductive system, which impaired growth in the BTB.

Quercetin alleviates inflammation induced by porcine reproductive and respiratory syndrome virus in MARC-145 cells through the regulation of arachidonic acid and glutamine metabolism.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe inflammatory response, respiratory disease and sow reproductive failure. Quercetin is among the widely occurring polypheno found abundantly in nature. Quercetin has anti-inflammatory, anti-oxidative and anti-viral properties. OBJECTIVES: This study aimed to explore the effect and mechanism of quercetin on PRRSV-induced inflammation in MARC-145 cells. METHODS: Observing the cytopathic effect and measurements of inflammatory markers in MARC-145 cells collectively demonstrate that quercetin elicits a curative effect on PRRSV-induced inflammation. Liquid chromatography-mass spectrometry was further used for a non-targeted metabolic analysis of the role of quercetin in the metabolic regulation of PRRSV inflammation in MARC-145 cells. RESULTS: It was shown that quercetin attenuated PRRSV-induced cytopathy in MARC-145 cells. Quercetin treatment inhibited PRRSV replication in MARC-145 cells in a dose-dependent manner. We also found that quercetin inhibited PRRSV-induced mRNA expression and secretion levels of tumour necrosis factor-α, interleukin 1ß and interleukin 6. Metabolomics analysis revealed that quercetin ameliorated PRRSV-induced inflammation. Pathway analysis results revealed that PRRSV-induced pathways including arachidonic acid metabolism, linoleic acid, glycerophospholipid and alanine, aspartate and glutamate metabolism were suppressed by quercetin. Moreover, we confirmed that quercetin inhibited the activation of NF-κB/p65 pathway, probably by attenuating PLA2, ALOX and COX mRNA expression. CONCLUSIONS: These results provide a crucial insight into the molecular mechanism of quercetin in alleviating PRRSV-induced inflammation.

Boosting PRRSV-Specific Cellular Immunity: The Immunological Profiling of an Fc-Fused Multi-CTL Epitope Vaccine in Mice.

The continuously evolving PRRSV has been plaguing pig farms worldwide for over 30 years, with conventional vaccines suffering from insufficient protection and biosecurity risks. To address these challenges, we identified 10 PRRSV-specific CTL epitopes through enzyme-linked immunospot assay (ELISPOT) and constructed a multi-epitope peptide (PTE) by linking them in tandem. This PTE was then fused with a modified porcine Fc molecule to create the recombinant protein pFc-PTE. Our findings indicate that pFc-PTE effectively stimulates PRRSV-infected specific splenic lymphocytes to secrete high levels of interferon-gamma (IFN-γ) and is predicted to be non-toxic and non-allergenic. Compared to PTE alone, pFc-PTE not only induced a comparable cellular immune response in mice but also extended the duration of the immune response to at least 10 weeks post-immunization. Additionally, pFc-PTE predominantly induced a Th1 immune response, suggesting its potential advantage in enhancing cellular immunity. Consequently, pFc-PTE holds promise as a novel, safe, and potent candidate vaccine for PRRSV and may also provide new perspectives for vaccine design against other viral diseases.

Fidelity Characterization of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and NADC30-like Strain.

The porcine reproductive and respiratory syndrome virus (PRRSV) has significantly impacted the global pork industry for over three decades. Its high mutation rates and frequent recombination greatly intensifies its epidemic and threat. To explore the fidelity characterization of Chinese highly pathogenic PRRSV JXwn06 and the NADC30-like strain CHsx1401, self-recombination and mutation in PAMs, MARC-145 cells, and pigs were assessed. In vitro, CHsx1401 displayed a higher frequency of recombination junctions and a greater diversity of junction types than JXwn06. In vivo, CHsx1401 exhibited fewer junction types yet maintained a higher junction frequency. Notably, JXwn06 showed more accumulation of mutations. To pinpoint the genomic regions influencing their fidelity, chimeric viruses were constructed, with the exchanged nsp9-10 regions between JXwn06 and CHsx1401. The SJn9n10 strain, which incorporates JXwn06's nsp9-10 into the CHsx1401 genome, demonstrated reduced sensitivity to nucleotide analogs compared to CHsx1401. Conversely, compared with JXwn06, the JSn9n10 strain showed increased sensitivity to these inhibitors. The swapped nsp9-10 also influences the junction frequency and accumulated mutations as their donor strains. The results indicate a propensity for different types of genetic variations between these two strains and further highlight the nsp9-10 region as a critical determinant of their fidelity.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

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.

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.].

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.

Frequency of PCV-2 viremia in nursery piglets from a Spanish swine integration system in 2020 and 2022 considering PRRSV infection status.

BACKGROUND: Porcine circovirus 2 (PCV-2) poses a significant economic threat for the swine industry, causing a range of diseases collectively referred to as porcine circovirus diseases (PCVDs). Despite PCV-2 vaccine effectiveness, the need for monitoring infectious pressure remains. PCV-2 coinfection with other pathogens like porcine reproductive and respiratory syndrome virus (PRRSV) can exacerbate disease severity and lead to PCV-2-systemic disease cases. Monitoring both PRRSV and PCV-2 in co-infected farms is crucial for an effective management and vaccination programs. The present cross-sectional study aimed to determine PCV-2 antibody levels in piglets at weaning and PCV-2 and PRRSV viremia in pooled serum samples at weaning (vaccination age) and at 6 and 9 weeks of age from a Spanish swine integration system in 2020 (48 farms) and in 2022 (28 out of the 48 analysed previously). RESULTS: The frequency of PCV-2 detection in pools of piglet sera was 2.1% (2020) and 7.1% (2022) at vaccination age but increased at the end of the nursery period (10.4% in 2020 and 39.3% in 2022) in both years. Co-infections between PCV-2 and PRRSV were detected in a significant proportion of PRRSV positive farms (15% in 2020, and 60% in 2022). PCV-2 antibody levels (ELISA S/P ratios) at weaning were lower in PCV-2 qPCR positive farms at different sampling time-points (0.361 in 2020 and 0.378 in 2022) compared to PCV-2 qPCR negative ones (0.587 in 2020 and 0.541 in 2022). The 28 farms tested both years were classified in four different epidemiological scenarios depending on their PCV-2 virological status. Those PCV-2 qPCR negative farms in 2020 that turned to be positive in 2022 had a statistically significant increase of PRRSV RT-qPCR detection and a PCV-2 antibody levels reduction, facts that were not observed in the rest of the scenarios. CONCLUSION: This epidemiological study in farms from the same integration system determined the occurrence, in 2020 and in 2022, of PCV-2 and PRRSV infections in piglets during the nursery period by using pooled serum samples.

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.

Research progress in porcine reproductive and respiratory syndrome virus and vaccine against it / 中国生物制品学杂志

@#Porcine reproductive and respiratory syndrome(PRRS),also known as blue ear disease,is an immunosuppressive disease caused by porcine reproductive and respiratory syndrome virus(PRRSV). PRRSV is an important infectious pathogen of porcine,which has the characteristics of easy recombination and mutation,high transmission ability,and has been widely spread in the world. At the same time,because of the characteristics of immunosuppression,immune evasion,and easy-to-cause antibody-dependent enhancement(ADE),the pathogen has brought serious challenges to the disease prevention and control of PRRS and the development of related vaccines. In this paper,the pathogenic process of PRRSV,immune evasion mechanism,and research progress in different types of PRRSV vaccines were reviewed,in order to provide ideas for the development of related vaccines in the future

Epidemiological investigation and pathogenicity of porcine reproductive and respiratory syndrome virus in Sichuan, China.

Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) lineage 8 was first detected in mainland China in 2006 and has since rapidly spread to become the primary epidemic strain in the country. In this study, samples such as lung tissue, hilar lymph nodes, abortion fetuses, and blood were collected from large-scale pig farms across 11 prefecture-level cities in Sichuan province between 2019 and 2020 for antigen detection and PRRS virus isolation. The antigen detection results indicated that the positive rate of HP-PRRSV (JXA1-Like strain) was 44.74% (51/114), NADC30-Like PRRSV was 17.54% (20/114), and classical PRRSV (VR2332-Like strain) was 37.72% (43/114). The predominant strain was HP-PRRSV. Positive samples were further inoculated into Marc-145 cells for virus isolation and identification, leading to the isolation of a new JXA1-Like PRRSV strain named SCSN2020. The strain was characterized by RT-qPCR, indirect immunofluorescence assay (IFA), plaque purification, electron microscopy, and whole genome sequencing. The total length of the viral genome was determined to be approximately 15,374 bp. A comparison of the SCSN2020 genome with VR2332 revealed that both strains had the same discontinuous 30-amino acid deletion on the Nsp2 gene. ORF5 genotyping classified the SCSN2020 strain as sublineage 8.7, with a whole genome sequence identity of 99.34% with JXA1. Furthermore, we evaluated the pathogenicity of the SCSN2020 strain in 28-day-old piglets and observed persistent fever from day 4 to day 10, weight loss started on day 7, dyspnea and severe lung lesions began started on day 14. The results of this study highlight the current PRRSV epidemic situation in Sichuan province and provide a scientific reference for subsequent prevention and control measures.

Production of a chimeric porcine reproductive and respiratory syndrome virus (PRRSV)-2 vaccine using a lab-scale packed-bed bioreactor CelCradle.

BACKGROUND: We developed a MARC-145 cell culture and porcine reproductive and respiratory syndrome (PRRS) vaccine production using a novel CelCradle bioreactor. CelCradle is a packed-bed bioreactor capable of both batch and perfusion culture, and the operating parameters are easy to optimize. RESULTS: In this study, CelCradle reached a maximum cell density of 8.94 × 105 cells/mL at 5 days post-seeding when seeded at 8.60 × 104 cells/mL (doubling time = 35.52 h). Inoculation of PRRS vaccine candidate, K418DM1.1, was performed at a multiplicity of infection (MOI) of 0.01 at 5 days post-seeding, which resulted in a high viral titer of 2.04 × 108 TCID50/mL and total viral load of 1.02 × 1011 TCID50/500 mL at 2 days post-infection (dpi). The multilayer cultivation system, BioFactory culture, yielded a higher doubling time (37.14 h) and lower viral titer (i.e., 8.15 × 107 TCID50/mL) compared to the CelCradle culture. Thus, the culture medium productivity of the CelCradle culture was 2-fold higher than that of the BioFactory culture. In the animal experiment, the CelCradle-produced vaccine induced high levels of neutralizing antibodies and effectively protected pigs against homologous challenge, as shown by the significantly lower levels of viremia at 1- and 7-days post-challenge (dpc) compared to the non-vaccinated pigs. CONCLUSIONS: Overall, this study demonstrates that the CelCradle system is an economical platform for PRRS vaccine production.

miR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been regarded as a persistent challenge for the swine farms worldwide. microRNAs (miRNAs) play key roles in regulating almost every important biological process, including virus-host interaction. In this study, we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection. Subsequently, we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages (PAMs). Through bioinformatic analysis, we found that there existed a potential binding site of miR-204 on the 3'UTR of microtubule associated protein 1 light chain 3B (MAP1LC3B, LC3B), a hallmark of autophagy. Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay, we demonstrated that miR-204 directly targeted LC3B, thereby downregulating autophagy. Meanwhile, we investigated the interplay between autophagy and PRRSV replication in PAMs, confirming that PRRSV infection induces autophagy, which in turn facilitates viral replication. Overall, we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs. These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.

Evasion strategies of porcine reproductive and respiratory syndrome virus.

During the co-evolution of viruses and their hosts, viruses have developed various strategies for overcoming host immunological defenses so that they can proliferate efficiently. Porcine reproductive and respiratory syndrome virus (PRRSV), a significant virus to the swine industry across the world, typically establishes prolonged infection via diverse and complicated mechanisms, which is one of the biggest obstacles for controlling the associated disease, porcine reproductive and respiratory syndrome (PRRS). In this review, we summarize the latest research on how PRRSV circumvents host antiviral responses from both the innate and adaptive immune systems and how this virus utilizes other evasion mechanisms, such as the manipulation of host apoptosis and microRNA. A thorough understanding of the exact mechanisms of PRRSV immune evasion will help with the development of novel antiviral strategies against PRRSV.

Comparative analysis of newly identified rodent arteriviruses and porcine reproductive and respiratory syndrome virus to characterize their evolutionary relationships.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses for the global pig industry, but its origins and evolution remain a mystery. In 2018, the genome sequences of seven arteriviruses isolated from rodents were determined, and here we publish new analysis showing that they may be ancestors of PRRSV. The sequence similarity of these viruses to PRRSV was ~60%, with shared genome organization and other characteristics, such as slippery sequences and C-rich motifs in nsp2, and a transactivated protein sequence in nsp1ß. Codon usage basis analysis showed that PRRSV was closer to these rodent arteriviruses than lactate dehydrogenase-elevating virus (LDV) and they were both under pressure of natural selection. Evolutionary analysis revealed that four of the rodent arteriviruses shared the same genus with PRRSV, and were more closely related to PRRSV-2 than PRRSV-1. In addition to this, they all appeared earlier than PRRSV according to evolutionary modeling, and we speculate that they represent an intermediate step in the origin of PRRSV by arterivirus transmission from rodents to swine. Our in-depth analysis furthers our understanding of arteriviruses, and will serve as the basis for subsequent exploration of the evolution of PRRSV and other arteriviruses.

Evaluating anti-viral effect of Tylvalosin tartrate on porcine reproductive and respiratory syndrome virus and analyzing the related gene regulation by transcriptomics.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen, characterized by its genetic and antigenic variation. The PRRSV vaccine is widely used, however, the unsatisfied heterologic protection and the risk of reverse virulence raise the requirement to find some new anti-PRRSV strategies for disease control. Tylvalosin tartrate is used to inhibit PRRSV in the field non-specifically, however, the mechanism is still less known. METHODS: The antiviral effects of Tylvalosin tartrates from three producers were evaluated in a cell inoculation model. Their safety and efficacy concentrations, and effecting stage during PRRSV infection were analyzed. And, the Tylvalosin tartrates regulated genes and pathways which are potentially related to the anti-viral effect were further explored by using transcriptomics analysis. Last, the transcription level of six anti-virus-related DEGs was selected to confirm by qPCR, and the expression level of HMOX1, a reported anti-PRRSV gene, was proved by western blot. RESULTS: The safety concentrations of Tylvalosin tartrates from three different producers were 40 µg/mL (Tyl A, Tyl B, and Tyl C) in MARC-145 cells and 20 µg/mL (Tyl A) or 40 µg/mL (Tyl B and Tyl C) in primary pulmonary alveolar macrophages (PAMs) respectively. Tylvalosin tartrate can inhibit PRRSV proliferation in a dose-dependent manner, causing more than 90% proliferation reduction at 40 µg/mL. But it shows no virucidal effect, and only achieves the antiviral effect via long-term action on the cells during the PRRSV proliferation. Furthermore, GO terms and KEGG pathway analysis was carried out based on the RNA sequencing and transcriptomic data. It was found that the Tylvalosin tartrates can regulate the signal transduction, proteolysis, and oxidation-reduction process, as well as some pathways such as protein digestion and absorption, PI3K-Akt signaling, FoxO signaling, and Ferroptosis pathways, which might relate to PRRSV proliferation or host innate immune response, but further studies still need to confirm it. Among them, six antivirus-related genes HMOX1, ATF3, FTH1, FTL, NR4A1, and CDKN1A were identified to be regulated by Tylvalosin tartrate, and the increased expression level of HMOX1 was further confirmed by western blot. CONCLUSIONS: Tylvalosin tartrate can inhibit PRRSV proliferation in vitro in a dose-dependent manner. The identified DEGs and pathways in transcriptomic data will provide valuable clues for further exploring the host cell restriction factors or anti-PRRSV target.

A novel NADC34-like porcine reproductive and respiratory syndrome virus 2 with complex genome recombination is highly pathogenic to piglets.

The NADC34-like porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) first emerged in China in 2017 and has the potential to become the dominant PRRSV strain in China. Here, a novel PRRSV-2, SCcd2020, was isolated from diseased piglets in Sichuan province, southwest China in 2020. The complete viral genome was determined and analyzed. An ORF5-based phylogenetic analysis showed that SCcd2020 clustered with NADC34-like strains, whereas the genome sequence clustered the isolate with NADC30-like viruses and it contains a discontinuous 131-aa deletion in NSP2 when compared to NADC30 strain. Notably, recombination analyses indicated that SCcd2020 is a multiple recombinant virus from NADC30-like, NADC34-like and JXA1-like strains, which is the first description of Chinese domestic HP-PRRSV involving the recombination event of an NADC34-like strain. Importantly, an animal challenge study in 4-week-old piglets showed that SCcd2020 causes high fever and severe hemorrhagic pneumonia with pulmonary consolidation and edema, and it has a high mortality rate (60%), which indicated that SCcd2020 is a highly pathogenic PRRSV strain. The study reports the emergence of a novel highly pathogenic NADC34-like recombinant strain, and it highlights the importance of monitoring newly emerging PRRSV strains in China.