Rearing pigs with play opportunities: the effects on disease resilience in pigs experimentally inoculated with PRRSV.

Positive emotions can reduce disease susceptibility during infectious challenges in humans, and emerging evidence suggests similar effects in farm animals. Because play behaviour may support a positive emotional state in pigs, this study investigates whether rearing pigs with regular intermittent play opportunities enhances disease resilience when challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Litters were assigned to either play (PLY; n = 5 L) or control (CON; n = 4 L) treatments at birth. In PLY, play was promoted with extra space and enrichment items for three hours daily from five days of age (doa). At weaning (25 ± 2 doa; mean ± SD), 28 pigs (14/treatment) were selected for a disease challenge, based on weight, sex, and sow. The pigs were transported to a disease containment facility and at 43 ± 2 doa (day 0 post-inoculation, DPI) inoculated with PRRSV. Skin lesions, blood, rectal temperature, clinical signs, body weight, and behaviour were collected pre- and post-inoculation. Play opportunities for PLY continued every other day until euthanasia of all pigs at 65 ± 2 doa (22 DPI). PLY pigs exhibited fewer skin lesions following transport and throughout the infection compared to CON. Although the viral load did not differ between treatments, PLY pigs had a lower probability of experiencing moderate and severe respiratory distress, with a shorter duration. PLY also performed better throughout the infection, showing higher ADG and greater feed efficiency. The immune response differed as well. PLY pigs had fewer monocytes on 8 DPI than CON, with levels returning to baseline by 21 DPI, whereas CON levels exceeded baseline. Regardless of day of infection, lymphocyte counts tended to be lower in PLY than in CON, and white blood cells and neutrophils were also lower, but only in slow-growing pigs. PLY pigs continued to play during the infection, demonstrating less sickness behaviour and emphasizing the rewarding properties of play. Results suggest that PLY pigs were less affected by PRRSV and developed increased resilience to PRRSV compared to CON. This study demonstrates that rearing pigs in an environment supporting positive experiences through provision of play opportunities can enhance resilience against common modern production challenges, underscoring the value of positive welfare in intensive pig farming.

NLRP12 inhibits PRRSV-2 replication by promoting GP2a degradation via MARCH8.

NLRP12, a member of the NLR family, has been shown to exert a vital function in orchestrating immune responses. Here, using the immunosuppressive porcine reproductive and respiratory syndrome virus (PRRSV) as a model, the role of NLRP12 in virus infection was deciphered. We demonstrated that overexpression of NLRP12 significantly restrained PRRSV replication, while NLRP12 silencing resulted in increased viral titer. Mechanistically, NLRP12 interacts with glycoprotein 2a (GP2a) through its LRR domain and recruits the membrane-associated RING-CH E3 ubiquitin ligase 8 (MARCH8) via the PYD domain. NLRP12 facilitates the lysine-48 (K48)-linked polyubiquitination of GP2a at K128 and induces its lysosome degradation via the MARCH8-NDP52 (nuclear dot protein 52 kDa) pathway. To counteract this, PRRSV Nsp2 effectively prevented the polyubiquitination of GP2a induced by NLRP12 by its deubiquitinating activity. Meanwhile, the overexpression of Nsp4 decreased the mRNA of endogenous NLRP12 and cleaved NLRP12 in a 3C-like protease activity-dependent manner, which collaboratively counteracts the antiviral function of NLRP12. Collectively, this study revealed the mechanisms of the NLRP12-MARCH8-NDP52 axis in the host defense against PRRSV, which might be harnessed for the development of anti-PRRSV therapies.

Molecular detection and genetic characteristics of porcine reproductive and respiratory syndrome virus in central China.

Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically devastating viral diseases in the global pork industry. To further clarify the epidemic characteristics of the virus, 365 clinical samples were collected from diseased pigs suffering from abortion and respiratory disease from 2018 to 2023 on 63 pig farms in Henan and Shanxi provinces, and screened for the presence of PRRSV using reverse transcription-polymerase chain reaction (RT-PCR). A total of 62 clinical samples (62/365, 16.99 %) were positive for PRRSV, and subsequently, full-length ORF5 gene sequences of 29 PRRSV strains and the complete genome sequence of one PRRSV HeN-HC isolate were obtained and analyzed. Phylogenetic analysis based on the ORF5 gene showed that 22 of the 29 PRRSV2 strains belonged to sublineage 1.8 (NADC30-like), 5 belonged to sublineage 8.5 (HP-PRRSV), and 2 belonged to sublineage 5.1 (VR-2332-like), indicating that both HP-PRRSV and NADC30-like strains were mainly circulating in Henan and Shanxi provinces. Compared to VR-2332 strain, different types of amino acid mutations were found in the GP5 protein of these 29 strains, and the amino acid deletions were displayed in the Nsp2 protein of the HeN-HC isolate, leading to the variation of protein structures. It is noteworthy that recombination events were identified in the HeN-Ping and HeN-B strains. In addition, a total of 60, 094 pig serum samples from Henan province were collected, and the positive rate of specific antibodies against PRRSV was 86.37 % from 2019 to 2022, and 86.66 %, 84.85 %, 87.54 % and 86.30 % in 2019, 2020, 2021 and 2022, respectively. Overall, this study provides valuable insights into the molecular epidemiology and evolution of PRRSV circulating in central China.

Host combats porcine reproductive and respiratory syndrome virus infection at non-coding RNAs level.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global swine industry. The emergence of new, highly virulent strains has precipitated recurrent outbreaks worldwide, underscoring the ongoing battle between host and virus. Thus, there is an imperative to formulate a more comprehensive and effective disease control strategy. Studies have shown that host non-coding RNA (ncRNA) is an important regulator of host - virus interactions in PRRSV infection. Hence, a thorough comprehension of the roles played by ncRNAs in PRRSV infection can augment our understanding of the pathogenic mechanisms underlying PRRSV infection. This review focuses on elucidating contemporary insights into the roles of host microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) in PRRSV infection, providing both theoretical foundations and fresh perspectives for ongoing research into the mechanisms driving PRRSV infection and its pathogenesis.

miR-191-5p suppresses PRRSV replication by targeting porcine EGFR to enhance interferon signaling.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major thread to the global swine industry, lack of effective control strategies. This study explores the regulatory role of a small non-coding RNA, miR-191-5p, in PRRSV infection. We observed that miR-191-5p significantly inhibits PRRSV in porcine alveolar macrophages (PAMs), contrasting with negligible effects in MARC-145 and HEK293-CD163 cells, suggesting a cell-specific antiviral effect. Further investigation unveiled that miR-191-5p directly targets the porcine epidermal growth factor receptor (EGFR), whose overexpression or EGF-induced activation suppresses type I interferon (IFN-I) signaling, promoting PRRSV replication. In contrast, siRNA-or miR-191-5p-induced EGFR downregulation or EGFR inhibitor boosts IFN-I signaling, reducing viral replication. Notably, this miRNA alleviates the suppressive effect of EGF on IFN-I signaling, underscoring its regulatory function. Further investigation revealed interconnections among miR-191-5p, EGFR and signal transducer and activator of transcription 3 (STAT3). Modulation of STAT3 activity influenced IFN-I signaling and PRRSV replication, with STAT3 knockdown countering EGFR activation-induced virus replication. Combination inhibition of STAT3 and miR-191-5p suggests that STAT3 acts downstream in EGFR's antiviral response. Furthermore, miR-191-5p's broad efficacy in restricting various PRRSV strains in PAMs was identified. Collectively, these findings elucidate a novel mechanism of miR-191-5p in activating host IFN-I signaling to inhibit PRRSV replication, highlighting its potential in therapeutic applications against PRRSV.

Tripartite motif 25 inhibits protein aggregate degradation during PRRSV infection by suppressing p62-mediated autophagy.

Viral infection causes endoplasmic reticulum stress and protein metabolism disorder, influencing protein aggregates formation or degradation that originate from misfolded proteins. The mechanism by which host proteins are involved in the above process remains largely unknown. The present study found that porcine reproductive and respiratory syndrome virus (PRRSV) infection promoted the degradation of intracellular ubiquitinated protein aggregates via activating autophagy. The host cell E3 ligase tripartite motif-containing (TRIM)25 promoted the recruitment and aggregation of polyubiquitinated proteins and impeded their degradation caused by PRRSV. TRIM25 interacted with ubiquitinated aggregates and was part of the aggregates complex. Next, the present study investigated the mechanisms by which TRIM25 inhibited the degradation of protein aggregates, and it was found that TRIM25 interacted with both Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor E2-related factor 2 (Nrf2), facilitated the nuclear translocation of Nrf2 by targeting KEAP1 for K48-linked ubiquitination and proteasome degradation, and activated Nrf2-mediated p62 expression. Further studies indicated that TRIM25 interacted with p62 and promoted its K63-linked ubiquitination via its E3 ligase activity and thus caused impairment of its oligomerization, aggregation, and recruitment for the autophagic protein LC3, leading to the suppression of autophagy activation. Besides, TRIM25 also suppressed the p62-mediated recruitment of ubiquitinated aggregates. Activation of autophagy decreased the accumulation of protein aggregates caused by TRIM25 overexpression, and inhibition of autophagy decreased the degradation of protein aggregates caused by TRIM25 knockdown. The current results also showed that TRIM25 inhibited PRRSV replication by inhibiting the KEAP1-Nrf2-p62 axis-mediated autophagy. Taken together, the present findings showed that the PRRSV replication restriction factor TRIM25 inhibited the degradation of ubiquitinated protein aggregates during viral infection by suppressing p62-mediated autophagy.IMPORTANCESequestration of protein aggregates and their subsequent degradation prevents proteostasis imbalance and cytotoxicity. The mechanisms controlling the turnover of protein aggregates during viral infection are mostly unknown. The present study found that porcine reproductive and respiratory syndrome virus (PRRSV) infection promoted the autophagic degradation of ubiquitinated protein aggregates, whereas tripartite motif-containing (TRIM)25 reversed this process. It was also found that TRIM25 promoted the expression of p62 by activating the Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor E2-related factor 2 (Nrf2) pathway and simultaneously prevented the oligomerization of p62 by promoting its K63-linked ubiquitination, thus suppressing its recruitment of the autophagic adaptor protein LC3 and ubiquitinated aggregates, leading to the inhibition of PRRSV-induced autophagy activation and the autophagic degradation of protein aggregates. The present study identified a new mechanism of protein aggregate turnover during viral infection and provided new insights for understanding the pathogenic mechanism of PRRSV.

Comparison of pathogenicity and host responses of emerging porcine reproductive and respiratory syndrome virus variants in piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly variable virus with genetic diversity. This study comparatively examines the pathogenicity and immunological impact of two emergent PRRSV strains, SD53 and HuN4, in piglets. Our results indicate that SD53 strain induces milder clinical syndromes and less severe tissue damage than HuN4, despite similar replication rates. Hematological tests showed less perturbations in peripheral blood cell profiles after SD53 infection, suggesting a less systemic impact. The neutrophil-to-lymphocyte ratio was notably lower in SD53-infected piglets, suggesting a less intense inflammatory reaction. Moreover, SD53 infection led to lower levels of pro-inflammatory cytokines, further supporting a less pronounced inflammatory profile. Both strains induced the production of PRRSV-specific antibodies. However, transcriptomic analysis of lung and lymph node tissues from infected piglets disclosed a more moderate up-regulation of core genes, including ISGs, in the SD53 group. Further analysis indicated that SD53 primarily enhanced immune-related signaling, particularly in T cell response modules, while HuN4 caused a more robust pro-inflammatory reaction and a dampening of T cell functionality. Flow cytometry analyses confirmed these findings, showing higher CD4/CD8 ratios and increased CD4+ T cell percentages in SD53-infected piglets, implying a more robust T cell response. Collectively, these findings broaden our comprehension of PRRSV pathogenesis and may inform the development of future therapeutic or prophylactic strategies for controlling PRRSV infections more effectively. IMPORTANCE: The high mutation rate of porcine reproductive and respiratory syndrome virus (PRRSV) poses significant challenges to its accurate diagnosis and the implementation of effective control measures. This research explores the pathogenic profiles of two emerging PRRSV stains: the NADC30-like strain SD53 and the highly pathogenic strain HuN4. Our investigation reveals that SD53 initiates distinct immunopathological responses in vivo compared with those provoked by HuN4. By conducting a transcriptome analysis of differential gene expression in the lungs and lymph nodes of infected piglets, we unveil the intricate molecular mechanisms underlying the contrasting pathogenicity of these two strains. The comprehensive insights yielded by this study are instrumental in advancing our understanding of the dominant NADC30-like PRRSV strain, which has become increasingly prevalent in China's swine industry.

Betulonic Acid Inhibits Type-2 Porcine Reproductive and Respiratory Syndrome Virus Replication by Downregulating Cellular ATP Production.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) infection, has been a serious threat to the pork industry worldwide and continues to bring significant economic loss. Current vaccination strategies offer limited protection against PRRSV transmission, highlighting the urgent need for novel antiviral approaches. In the present study, we reported for the first time that betulonic acid (BA), a widely available pentacyclic triterpenoids throughout the plant kingdom, exhibited potent inhibition on PRRSV infections in both Marc-145 cells and primary porcine alveolar macrophages (PAMs), with IC50 values ranging from 3.3 µM to 3.7 µM against three different type-2 PRRSV strains. Mechanistically, we showed that PRRSV replication relies on energy supply from cellular ATP production, and BA inhibits PRRSV infection by reducing cellular ATP production. Our findings indicate that controlling host ATP production could be a potential strategy to combat PRRSV infections, and that BA might be a promising therapeutic agent against PRRSV epidemics.

Linear epitopes of PRRSV-1 envelope proteins ectodomains are not correlated with broad neutralization.

BACKGROUND: Neutralizing antibodies against PRRSV are capable of conferring protection against viral reinfection, but they tend to be strain specific and usually have poor cross-reactivity. Nonetheless, it has been described that there are individuals capable of efficiently neutralizing viruses of different origin, so it is expected that there are conserved neutralizing epitopes relevant for broad neutralization. However, although immunodominant regions and neutralizing epitopes have been described in different envelope proteins, their role in broad neutralization is unknown. The main objective of this study was to determine whether the linear epitopes existing in the ectodomains of PRRSV envelope proteins play a role in cross-neutralization. RESULTS: A pepscan analysis was carried out using synthetic peptides against the ectodomains of PRRSV envelope proteins and PRRSV-hyperimmune sera of different cross-reactivity. The results obtained confirm the existence of antigenic regions in the ectodomains of the GP2, GP3, GP4 and GP5 that tend to be relatively conserved among different PRRSV isolates. Nonetheless, these antigenic regions have poor immunogenicity since they are only recognized by a limited number of sera. Furthermore, no differences were found between the reactivity of sera with broad cross-neutralization capacity and sera with poor heterologous neutralization activity, which indicate that linear epitopes existing in the ectodomains of PRRSV envelope proteins are not relevant for the development of broadly reactive neutralizing antibodies. Subsequently, some selected peptides were used in competition assays with the virus for binding to the cell receptors and in seroneutralization inhibition assays by incubation with hyperimmune sera. Firstly, some peptides that interfere with virus infectivity were identified in competition assays, but only in the case of one viral isolate, which points to the possible existence of a strain-dependent inhibition. However, the results of the seroneutralization inhibition assay indicate that, under the conditions of our study, none of the peptides used was capable of inhibiting virus neutralization by the hyperimmune sera. CONCLUSIONS: The results obtained indicate that the linear peptides analyzed in this study do not play a major role in the induction of broadly reactive neutralizing antibodies, which could probably depend on conformational neutralizing.

Investigation of the distribution and origin of porcine reproductive and respiratory syndrome virus 1 in the swine production chain: A retrospective study of three farms in Thailand.

Background and Aim: Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a global issue that affects Thai swine as well. In Thailand, PRRSV-2 predominates over PRRSV-1. The origin of PRRSV-1 transmission remains undiscovered. This study traced the source of infected pigs responsible for disease transmission among three pig-fattening farms and analyzed the spread of PRRSV-1. Materials and Methods: A total of 696 swine samples from breeding and pig-fattening farms in Thailand were screened for PRRSV using open reading frames (ORF7) reverse transcription polymerase chain reaction (RT-PCR). Positive samples were identified as PRRSV-1 using ORF5 RT-PCR. The analysis included the study of nucleotide homology, GP5 amino acid sequences, and N-linked glycosylation patterns to assess the spread of PRRSV-1 across these farms. Results: Genetic examination identified 28 PRRSV-1-positive samples, of which 13 were chosen as representatives. These strains were categorized into three groups based on breeding farm pig houses and showed distinct distribution patterns across pig-fattening farms. Group 1 included piglets transferred from pig house A to Nakhon Pathom, Chonburi, and Sa Kaeo. Groups 2 and 3 showed transfers from pig houses F and H to Chonburi and Sa Kaeo farms. All 13 PRRSV-1 strains were categorized into PRRSV-1 subtype 1/clade H. N-linked glycosylation analysis revealed that nearly all PRRSV-1 strains exhibited a conserved glycosylation pattern at amino acid positions N37, N46, and N53. This pattern is consistent with the glycosylation profile of the previous Thai PRRSV-1 subtype 1/clade H. Conclusion: The present study highlights the persistent presence of PRRSV-1 in Thai swine, which leads to sporadic outbreaks. The molecular genetic analysis identified three primary strain groups dispersed throughout the pig production system, emphasizing the importance of regular monitoring for new PRRSV strains in this herd. Understanding the PRRSV-1 distribution in swine farms is vital for veterinarians. This knowledge supports strategies for eradicating the virus and managing swine health effectively in Thailand.

Intermolecular disulfide bond of PRRSV GP5 and M facilitates VLPs secretion and cell binding.

Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of porcine reproductive and respiratory syndrome (PRRS), continues to significantly impact on the global swine industry. GP5 and M are the primary structural proteins of PRRSV, playing crucial roles in the processes of virus attachment, entry, assembly and budding. The co-expression of GP5 and M can result in the formation of virus-like particles (VLPs). However, the underlying mechanisms remain incompletely understood. This study investigated the role of GP5-M interaction in VLPs secretion and cell binding. VLPs were generated by co-expressing GP5 and M via recombinant baculoviruses in Sf9 cells and confirmed by transmission electron microscopy. The secretion of VLPs was modulated by the expression levels of GP5 and M. Using the BirA technique, the GP5-M interaction was confirmed in Sf9 cells. Disruption of the N-terminally intermolecular disulfide bond between GP5 and M weakened, but did not completely abolish, the interaction between the proteins, leading to reduced VLPs secretion. Notably, the absence of this intermolecular disulfide bond resulted in the loss of VLPs' ability to bind to MARC-145 cells. In summary, our findings reveal the critical function of the intermolecular disulfide bond in GP5-M interaction, which significantly contributes to VLPs secretion and cell binding, and suggest potential interaction sites between GP5 and M.

Prevalence and genetic diversity of PRRSV in Sichuan province of China from 2021 to 2023: Evidence of an ongoing epidemic transition.

Porcine reproductive and respiratory syndrome (PRRS) significantly impacts the global swine industry. Sichuan province, a key pig breeding center in China, has limited data on the molecular epidemiology of PRRS Virus (PRRSV). To address this, 1618 suspected PRRSV samples were collected from 2021 to 2023, with a prevalence rate of 39.74% (643/1618). Phylogenetic analysis showed PRRSV-2 as dominant (95.65%, 615/643), with PRRSV-1 at 4.35% (28/643). PRRSV-2 strains were further classified into NADC30-like (74.18%), NADC34-like (11.98%), C-PRRSV (5.44%), and HP-PRRSV (4.04%). The significant change in the proportions of different lineages indicates genomic divergence. NADC30-like strains exhibited significant amino acid mutations in ORF5, aiding immune evasion. Recombination analysis revealed complex patterns, primarily involving NADC30-like strains. This study highlights the genomic divergence of PRRSV in Sichuan, with NADC30-like strains becoming predominant and emerging strains like NADC34-like showing potential for further spread.

Virucidal effects of eucalyptus essential oil on porcine reproductive and respiratory syndrome virus.

Currently, the efficacy of vaccination for preventing and controlling PRRSV is insufficient. Therefore, there is an urgent need for novel effective preventive strategies. This study aimed to investigate the antiviral effect of Eucalyptus essential oil (EEO) against PRRSV in vitro. Marc-145 cells were infected with PRRSV (rJXA1-R), and the toxicity of EEO in the cells was measured using the Cell Counting Kit-8 method. Additionally, the antiviral effect of EEO on PRRSV-infected cells was assessed using three treatment methods: drug administration post-PRRSV inoculation (post-treatment), drug administration before PRRSV inoculation (pre-treatment), and simultaneous drug administration and PRRSV inoculation (co-treatment). The EEO could not inhibit virus adsorption and/or replication since post-treatment and pre-treatment did not prevent viral infectivity. However, EEO exerted a significant virucidal effect on PRRSV. When PRRSV-infected cells were treated with 0.0156, 0.0312, and 0.0625% EEO, the cell survival rates were 55.37, 118.96, and 121.67%, respectively, and the titer of progeny virions decreased from 5.77 Log10TCID50 to 5.21 Log10TCID50, 0.55 Log10TCID50, and less than 0.167 Log10TCID50, respectively (where TCID50 is the 50% tissue culture infected dose). The fluorescence intensity of the PRRSV N protein significantly decreased in the indirect immunofluorescence assay. When cells were co-treated with EEO (0.0625%) and PRRSV (1000 TCID50) for 15 min, the viral particles were inactivated, and PRRSV (1000 TCID50) particles loss infectivity when the co-treatment time reached 60 min. In a word, EEO has no obvious therapeutic effect on PRRSV infection, but it can effectively inactivate virus particles and make them lose the ability to infect cells. These findings provide insights for the development and use of EEO to treat PRRS.

Molecular mechanism of autophagy in porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV), a significant pathogen affecting the swine industry globally, has been shown to manipulate host cell processes, including autophagy, to facilitate its replication and survival within the host. Autophagy, an intracellular degradation process crucial for maintaining cellular homeostasis, can be hijacked by viruses for their own benefit. During PRRSV infection, autophagy plays a complex role, both as a defense mechanism of the host and as a tool exploited by the virus. This review explores the current understanding of the molecular mechanisms underlying autophagy induction under PRRSV infection, its impact on virus replication, and the potential implications for viral pathogenesis and antiviral strategies. By synthesizing the latest research findings, this article aims to enhance our understanding of the intricate relationship between autophagy and PRRSV, paving the way for novel therapeutic approaches against this swine pathogen.

The evolution and diversity of porcine reproductive and respiratory syndrome virus in China.

The porcine reproductive and respiratory syndrome virus (PRRSV) has emerged as a significant pathogen in the global pork industry since the late 1980s, causing substantial economic losses due to its high contagiousness and genetic variability. China, with its complex epidemiological landscape, has witnessed the emergence of four distinct lineages of PRRSV-2 (Lineages 1, 3, 5, and 8) and occasional occurrences of PRRSV-1. This review summarizes the historical context and epidemiological trends that have led to the diversification of PRRSV in China, discusses the evolutionary dynamics behind the establishment of diverse genetic variants, as well as the impact of recombination and modified live vaccines (MLVs) on the virus's rapid evolution. The implications for disease management, including strategies to reduce the complexity of PRRSV epidemics and improve prevention and control measures, are also suggested. Understanding the evolutionary pattern and factors contributing to PRRSV diversity is crucial for enhancing our knowledge, control capabilities, and prevention strategies, which could be integrated into swine health management practices.

Identification and biophysical characterization of epitope atlas of Porcine Reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) have been a critical threat to swine health since 1987 due to its high mutation rate and substantial economic loss over half a billion dollar in USA. The rapid mutation rate of PRRSV presents a significant challenge in developing an effective vaccine. Even though surveillance and intervention studies have recently (2019) unveiled utilization of PRRSV glycoprotein 5 (GP5; encoded by ORF5 gene) to induce immunogenic reaction and production of neutralizing antibodies in porcine populations, the future viral generations can accrue escape mutations. In this study we identify 63 porcine-PRRSV protein-protein interactions which play primary or ancillary roles in viral entry and infection. Using genome-proteome annotation, protein structure prediction, multiple docking experiments, and binding energy calculations, we identified a list of 75 epitope locations on PRRSV proteins crucial for infection. Additionally, using machine learning-based diffusion model, we designed 56 stable immunogen peptides that contain one or more of these epitopes with their native tertiary structures stabilized through optimized N- and C-terminus flank sequences and interspersed with appropriate linker regions. Our workflow successfully identified numerous known interactions and predicted several novel PRRSV-porcine interactions. By leveraging the structural and sequence insights, this study paves the way for more effective, high-avidity, multi-valent PRRSV vaccines, and leveraging neural networks for immunogen design.

The 5'UTR of porcine reproductive and respiratory syndrome virus strain JXwn06 harbors a uORF that regulates cellular inflammation.

The 5' untranslated region (5'UTR) of many positive-stranded RNA viruses contain functional regulatory sequences. Here, we show that the porcine reproductive and respiratory syndrome virus (PRRSV), a member of arteriviruses, harbors small upstream open reading frames (uORFs) in its 5'UTR. Bioinformatics analysis shows that this feature is relatively well conserved among PRRSV strains and Arteriviridae. We also identified a uORF, namely uORF2, in the PRRSV strain JXwn06, that possesses translational activity and exerts a suppressive effect on the expression of the primary ORF evidenced by in vitro reporter assays. We tested its importance via reverse genetics by introducing a point mutation into the PRRSV infectious cDNA clone to inactivate the start codon of uORF2. The recovered mutant virus Mut2 surprisingly replicated to the same level as the wild-type virus (WT), but induced a higher level of inflammatory cytokines (e.g., TNF-α, IL-1ß, and IL-6) both in vitro and in animal experiments, correlating well with more severe lung injury and higher death rate. In line with this, over-expression of uORF2 in transfected cells significantly inhibited poly(I:C)-induced expression of inflammatory cytokines. Together, our data support the idea that uORF2 encodes a novel, functional regulator of PRRSV virulence despite of its short size. IMPORTANCE: PRRSV has remained a major challenge to the world swine industry, but we still do not know much about its biology and pathogenesis. Here, we provide evidence to show that the 5'UTR of PRRSV strain JXwn06 harbors a functional uORF that has the coding capacity and regulates induction of inflammation as demonstrated by in vitro assays and animal experiment. The findings reveal a novel viral factor that regulates cellular inflammation and provide insight into the understanding of PRRSV pathogenesis.

Exploratory application of a cannulation model in recently weaned pigs to monitor longitudinal changes in the enteric microbiome across varied porcine reproductive and respiratory syndrome virus (PRRSV) infection statuses.

Introduction: The enteric microbiome and its possible modulation to improve feed conversion or vaccine efficacy is gaining more attention in pigs. Weaning pigs from their dam, along with many routine procedures, is stressful. A better understanding of the impact of this process on the microbiome may be important for improving pig production. The objective of this study was to develop a weaner pig cannulation model, thus allowing ileum content collection from the same pig over time for 16S rRNA sequencing under different porcine reproductive and respiratory syndrome virus (PRRSV) infection statuses. Methods: A total of 15 3-week-old pigs underwent abdominal surgery and were fitted with an ileum cannula, with ileum contents collected over time. In this pilot study, treatment groups included a NEG-CONTROL group (no vaccination, no PRRSV challenge), a POS-CONTROL group (no vaccination, challenged with PRRSV), a VAC-PRRSV group (vaccinated, challenged with PRRSV), a VAC-PRO-PRRSV group (vaccinated, supplemented with a probiotic, challenged with PRRSV), and a VAC-ANTI-PRRSV group (vaccinated, administered an antibiotic, challenged with PRRSV). We assessed the microbiome over time and measured anti-PRRSV serum antibodies, PRRSV load in serum and nasal samples, and the severity of lung lesions. Results: Vaccination was protective against PRRSV challenge, irrespective of other treatments. All vaccinated pigs mounted an immune response to PRRSV within 1 week after vaccination. A discernible impact of treatment on the diversity, structure, and taxonomic abundance of the enteric microbiome among the groups was not observed. Instead, significant influences on the ileum microbiome were observed in relation to time and treatment. Discussion: The cannulation model described in this pilot study has the potential to be useful in studying the impact of weaning, vaccination, disease challenge, and antimicrobial administration on the enteric microbiome and its impact on pig health and production. Remarkably, despite the cannulation procedures, all vaccinated pigs exhibited robust immune responses and remained protected against PRRSV challenge, as evidenced by the development of anti-PRRSV serum antibodies and viral shedding data.

Knock down of transforming growth factor beta improves expressions of co-stimulatory molecules, type I interferon-regulated genes, and pro-inflammatory cytokine in PRRSV-inoculated monocyte-derived macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFß1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFß1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFß1 mRNA expression and protein translation. Transfection of TGFßAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFß1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNß, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFßAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2'-5'-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFßAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFß1 (rTGFß1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFß expression. These findings also suggest TGFß as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.

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.