Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

Method validation for the recovery of the porcine respiratory and reproductive syndrome virus, a potential SARS-CoV-2 surrogate, from stainless steel.

Virus survival on fomites may represent a vehicle for transmission to humans. This study was conducted to optimize and validate a recovery method for the porcine respiratory and reproductive syndrome virus (PRRSV), a potential SARS-CoV-2 surrogate, from stainless steel. Coupons (1.5 × 1.5 cm) inoculated with ca. 7 logs TCID50 of PRRSV were dried for 15 min at room temperature, followed by incubation at 4°C and 35% relative humidity. After 1 h and 24 h, the coupons were processed by four different methods: vortex in DMEM media, vortex in DMEM media with beads, vortex in elution buffer, and shake in elution buffer. The rinsates were processed for titration using the TCID50 method in the MARC-145 cell line. All four methods were equally effective to recover the virus from the soiled SS surfaces (> 79% recovery). The amount of infectious virus recovered after 24 h was similar (P > 0.05) to that recovered after 1 h, indicating that the virus was stable at 4°C for up to 24 h. Using an elution buffer followed by shaking was the least labor-intensive and most economical method. Therefore, this method will be used for future experiments on PRRSV survival and transfer from food-contact surfaces.

A Serological Investigation of Porcine Reproductive and Respiratory Syndrome and Three Coronaviruses in the Campania Region, Southern Italy.

Porcine coronaviruses and reproductive and respiratory syndrome (PRRS) are responsible for severe outbreaks that cause huge economic losses worldwide. In Italy, three coronaviruses have been reported historically: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Although repeated outbreaks have been described, especially in northern Italy, where intensive pig farming is common, there is a worrying lack of information on the spread of these pathogens in Europe. In this work, we determined the seroprevalence of three porcine coronaviruses and PRRSV in the Campania region, southern Italy. A total of 443 samples were tested for the presence of antibodies against porcine coronaviruses and PRRSV using four different commercial ELISAs. Our results indicated that PEDV is the most prevalent among porcine coronaviruses, followed by TGEV, and finally PRCV. PRRSV appeared to be the most prevalent virus (16.7%). For coronaviruses, seroprevalence was higher in pigs raised in intensive farming systems. In terms of distribution, TGEV is more widespread in the province of Avellino, while PEDV and PRRSV are more prevalent in the province of Naples, emphasizing the epidemic nature of both infections. Interestingly, TGEV-positive animals are more common among growers, while seropositivity for PEDV and PRRSV was higher in adults. Our research provides new insights into the spread of swine coronaviruses and PRRSV in southern Italy, as well as a warning about the need for viral surveillance.

Effect of BIO-PLYTM, a Platelet-Rich Plasma Derived Biologic on PRRSV-2-Infected Macrophages.

Porcine Reproductive and Respiratory Syndrome (PRRS) is the one of the most devastating diseases impacting the swine industry worldwide. Control and prevention methods rely on biosafety measures and vaccination. As an RNA virus with a high rate of mutation, vaccines are only partially effective against circulating and newly emerging strains. To reduce the burden of this disease, research on alternative control methods is needed. Here, we assess the in vitro antiviral effect of a novel platelet-rich plasma-derived biologic termed BIO-PLYTM (for the BIOactive fraction of Platelet-rich plasma LYsate) from both swine and equine origin. Our results show that BIO-PLYTM significantly reduces the amount of PRRSV viral load determined by RT-qPCR and the number of infectious viral particles measured by TCID50 in infected porcine alveolar and parenchymal macrophages. This study also showed limited toxicity of BIO-PLYTM in vitro and aspects of its immunomodulatory capacity evaluating the regulation of reactive oxygen species and cytokines production in infected cells. Finally, this study presents promising data on the effect of BIO-PLYTM on other RNA viruses such as human A influenza viruses and coronavirus.

Integrated time-series transcriptomic and metabolomic analyses reveal different inflammatory and adaptive immune responses contributing to host resistance to PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious disease that affects the global pig industry. To understand mechanisms of susceptibility/resistance to PRRSV, this study profiled the time-serial white blood cells transcriptomic and serum metabolomic responses to PRRSV in piglets from a crossbred population of PRRSV-resistant Tongcheng pigs and PRRSV-susceptible Large White pigs. Gene set enrichment analysis (GSEA) illustrated that PRRSV infection up-regulated the expression levels of marker genes of dendritic cells, monocytes and neutrophils and inflammatory response, but down-regulated T cells, B cells and NK cells markers. CIBERSORT analysis confirmed the higher T cells proportion in resistant pigs during PRRSV infection. Resistant pigs showed a significantly higher level of T cell activation and lower expression levels of monocyte surface signatures post infection than susceptible pigs, corresponding to more severe suppression of T cell immunity and inflammatory response in susceptible pigs. Differentially expressed genes between resistant/susceptible pigs during the course of infection were significantly enriched in oxidative stress, innate immunity and humoral immunity, cell cycle, biotic stimulated cellular response, wounding response and behavior related pathways. Fourteen of these genes were distributed in 5 different QTL regions associated with PRRSV-related traits. Chemokine CXCL10 levels post PRRSV infection were differentially expressed between resistant pigs and susceptible pigs and can be a promising marker for susceptibility/resistance to PRRSV. Furthermore, the metabolomics dataset indicated differences in amino acid pathways and lipid metabolism between pre-infection/post-infection and resistant/susceptible pigs. The majority of metabolites levels were also down-regulated after PRRSV infection and were significantly positively correlated to the expression levels of marker genes in adaptive immune response. The integration of transcriptome and metabolome revealed concerted molecular events triggered by the infection, notably involving inflammatory response, adaptive immunity and G protein-coupled receptor downstream signaling. This study has increased our knowledge of the immune response differences induced by PRRSV infection and susceptibility differences at the transcriptomic and metabolomic levels, providing the basis for the PRRSV resistance mechanism and effective PRRS control.

Using Alphafold2 to Predict the Structure of the Gp5/M Dimer of Porcine Respiratory and Reproductive Syndrome Virus.

Porcine reproductive and respiratory syndrome virus is a positive-stranded RNA virus of the family Arteriviridae. The Gp5/M dimer, the major component of the viral envelope, is required for virus budding and is an antibody target. We used alphafold2, an artificial-intelligence-based system, to predict a credible structure of Gp5/M. The short disulfide-linked ectodomains lie flat on the membrane, with the exception of the erected N-terminal helix of Gp5, which contains the antibody epitopes and a hypervariable region with a changing number of carbohydrates. The core of the dimer consists of six curved and tilted transmembrane helices, and three are from each protein. The third transmembrane regions extend into the cytoplasm as amphiphilic helices containing the acylation sites. The endodomains of Gp5 and M are composed of seven ß-strands from each protein, which interact via ß-strand seven. The area under the membrane forms an open cavity with a positive surface charge. The M and Orf3a proteins of coronaviruses have a similar structure, suggesting that all four proteins are derived from the same ancestral gene. Orf3a, like Gp5/M, is acylated at membrane-proximal cysteines. The role of Gp5/M during virus replication is discussed, in particular the mechanisms of virus budding and models of antibody-dependent virus neutralization.

Combining epidemiology and economics to assess control of a viral endemic animal disease: Porcine Reproductive and Respiratory Syndrome (PRRS).

Porcine reproductive and respiratory syndrome (PRRS) is an extremely contagious disease that causes great damage to the U.S. pork industry. PRRS is not subject to official control in the U.S., but most producers adopt control strategies, including vaccination. However, the PRRS virus mutates frequently, facilitating its ability to infect even vaccinated animals. In this paper we analyze how increased vaccination on sow farms reduces PRRS losses and when vaccination is profitable. We develop a SIR model to simulate the spread of an outbreak between and within swine farms located in a region of Minnesota. Then, we estimate economic losses due to PRRS and calculate the benefits of vaccination. We find that increased vaccination of sow farms increases the private profitability of vaccination, and also transmits positive externalities to farms that do not vaccinate. Although vaccination reduces industry losses, a low to moderate vaccine efficacy implies that large PRRS losses remain, even on vaccinated farms. Our approach provides useful insight into the dynamics of an endemic animal disease and the benefits of different vaccination regimens.

Metagenomic analysis of endemic viruses in oral secretions from Chinese pigs.

BACKGROUND: Pigs are unique reservoirs for virus ecology. Despite the increased use of improved biosecurity measures, pig viruses readily circulate in Chinese swine farms. OBJECTIVES: The main objective of this study was to examine archived swine oral secretion samples with a panel of pan-species viral assays such that we might better describe the viral ecology of swine endemic viruses in Chinese farms. METHODOLOGY: Two hundred (n = 200) swine oral secretion samples, collected during 2015 and 2016 from healthy pigs on six swine farms in two provinces in China, were screened with molecular pan-species assays for coronaviruses (CoVs), adenoviruses (AdVs), enteroviruses (EVs), and paramyxoviruses (PMV). Samples were also screened for porcine circovirus (PCV) 3, porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV). RESULTS: Among 200 swine oral secretion samples, 152 (76.0%) were found to have at least one viral detection. Thirty-four samples (17%) were positive for more than one virus, including 24 (70.5%) with dual detection and 10 (29.5%) with triple detection. Seventy-eight (39.0%) samples were positive for porcine AdVs, 22 (11.0%) were positive for porcine CoVs, 21 (10.5%) were positive for IAVs, 13 (6.5%) were positive for PCV, 7 (3.5%) were positive for PMV, six (3.0%) were positive for PRRSV and five (2.5%) were positive for porcine EV. CONCLUSION: Our findings underscore the high prevalence of numerous viruses among production pigs in China and highlight the need for routine, periodic surveillance for novel virus emergence with the goal of protecting pigs.

Intranasal delivery of inactivated PRRSV loaded cationic nanoparticles coupled with enterotoxin subunit B induces PRRSV-specific immune responses in pigs.

This study was conducted to evaluate the induction of systemic and mucosal immune responses and protective efficacy following the intranasal administration of inactivated porcine reproductive and respiratory syndrome virus (PRRSV) loaded in polylactic acid (PLA) nanoparticles coupled with heat-labile enterotoxin subunit B (LTB) and dimethyldioctadecylammonium bromide (DDA). Here, 42- to 3-week-old PRRSV-free pigs were randomly allocated into 7 groups of 6 pigs each. Two groups represented the negative (nonvaccinated pigs/nonchallenged pigs, NoVacNoChal) and challenge (nonvaccinated/challenged, NoVacChal) controls. The pigs in the other 5 groups, namely, PLA nanoparticles/challenged (blank NPs), LTB-DDA coupled with PLA nanoparticles/challenged (adjuvant-blank NPs), PLA nanoparticles-encapsulating inactivated PRRSV/challenged (KNPs), LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV/challenged pigs (adjuvant-KNPs) and inactivated PRRSV/challenged pigs (inactivated PRRSV), were intranasally vaccinated with previously described vaccines at 0, 7 and 14 days post-vaccination (DPV). Serum and nasal swab samples were collected weekly and assayed by ELISA to detect the presence of IgG and IgA, respectively. Viral neutralizing titer (VNT) in sera, IFN-γ-producing cells and IL-10 secretion in stimulated peripheral blood mononuclear cells (PBMCs) were also measured. The pigs were intranasally challenged with PRRSV-2 at 28 DPV and necropsied at 35 DPV, and then macro- and microscopic lung lesions were evaluated. The results demonstrated that following vaccination, adjuvant-KNP-vaccinated pigs had significantly higher levels of IFN-γ-producing cells, VNT and IgG in sera, and IgA in nasal swab samples and significantly lower IL-10 levels than the other vaccinated groups. Following challenge, the adjuvant-KNP-vaccinated pigs had significantly lower PRRSV RNA and macro- and microscopic lung lesions than the other vaccinated groups. In conclusion, the results of the study demonstrated that adjuvant-KNPs are effective in eliciting immune responses against PRRSV and protecting against PRRSV infections over KNPs and inactivated PRRSV and can be used as an adjuvant for intranasal PRRSV vaccines.

A multiplex assay for the detection of antibodies to relevant swine pathogens in serum.

Livestock industry supports the livelihood of around 1.3 billion people in the world, with swine industry contributing with 30% of total livestock production worldwide. To maintain and guarantee this production, a pivotal point according to the OIE is addressing potential biohazards. To control them, permanent sero-surveillance is crucial to achieve more focused veterinary public health intervention and prevention strategies, to break the chains of transmission, and to enable fast responses against outbreaks. Within this context, multiplex assays are powerful tools with the potential to simplify surveillance programs, since they reduce time, labour, and variability within analysis. In the present work, we developed a multiplex bead-based assay for the detection of specific antibodies to six relevant pathogens affecting swine: ASFV, CSFV, PRRSV, SIV, TB and HEV. The most immunogenic target antigen of each pathogen was selected as the target protein to coat different microsphere regions in order to develop this multiplex assay. A total of 1544 serum samples from experimental infections as well as field samples were included in the analysis. The 6-plex assay exhibited credible diagnostic parameters with sensitivities ranging from 87.0% to 97.5% and specificities ranging from 87.9% to 100.0%, demonstrating it to be a potential high throughput tool for surveillance of infectious diseases in swine.

Differences in Humoral Immune Response against the Type 2 Porcine Reproductive and Respiratory Syndrome Virus via Different Immune Pathways.

The intramuscular vaccine is the principal strategy to protect pigs from porcine reproductive and respiratory syndrome virus (PRRSV), However, it is still difficult to control PRRSV effectively. This study infected piglets with PRRSV through intramuscular and intranasal inoculation. Subsequently, viral loads, anti-PRRSV antibody levels, and neutralizing antibodies (NAs) titers in both serum and saliva were monitored for 43 days. Meanwhile, tissues were obtained through necropsy at 43 days post-inoculation (dpi) to detect viral loads. The results indicated that viremia lasted from 3 to 31 dpi in both the inoculation groups, but the viruses survived in the lungs and lymph nodes after viremia clearance. The antibody response was detected from 11 dpi, but the response of NAs was delayed until 3-4 weeks. Furthermore, intranasal inoculation induced lower viral load levels than injection inoculation. In addition, positive SIgA and NAs levels were produced early, with higher levels through intranasal inoculation. Therefore, our data indicated that a more robust antibody response and lower virus loads could be induced by intranasal inoculation, and mucosal inoculation could be a suitable pathway for PRRSV vaccines.

A longitudinal study on PRRSV detection in swine herds with different demographics and PRRSV management strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the major health-related concerns in the swine production industry. Through its rapid transmission and mutation, the simultaneous circulation of multiple PRRSV strains can be a challenge in PRRSV diagnostic, control and surveillance. The objective of this longitudinal study was to describe the temporal detection of PRRSV in swine farms with different production types and PRRS management strategies. Tonsil scraping (n = 344) samples were collected from three breeding and two growing herds for approximately one year. In addition, processing fluids (n = 216) were obtained from piglet processing batches within the three breeding farms while pen-based oral fluids (n = 125) were collected in the two growing pig farms. Viral RNA extraction and reverse-transcription quantitative PCR (RT-qPCR) were conducted for all samples. The sample positivity threshold was set at quantification cycle (Cq) of ≤ 37. Statistical analyses were performed using generalized linear modelling and post hoc pairwise comparisons with Bonferroni adjustments using R statistical software. The results suggested a higher probability of detection in processing fluids compared to tonsil scraping specimens [odds ratio (OR) = 3.86; p = .096] in breeding farms whereas oral fluids were outperformed by tonsil scrapings (OR = 0.26; p < .01) in growing pig farms. The results described herein may lead to an improvement in PRRSV diagnostic and surveillance by selecting proper specimens.

Combined Subcutaneous-Intranasal Immunization With Epitope-Based Antigens Elicits Binding and Neutralizing Antibody Responses in Serum and Mucosae Against PRRSV-2 and SARS-CoV-2.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.

A candidate multi-epitope vaccine against porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae induces robust humoral and cellular response in mice.

Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) are two of the most common pathogens involved in the porcine respiratory disease complex (PRDC) resulting in significant economic losses worldwide. Vaccination is the most effective approach to disease prevention. Since PRRSV and Mhp co-infections are very common, an efficient dual vaccine against these pathogens is required for the global swine industry. Compared with traditional vaccines, multi-epitope vaccines have several advantages, they are comparatively easy to produce and construct, are chemically stable, and do not have an infectious potential. In this study, to develop a safe and effective vaccine, B cell and T cell epitopes of PRRSV-GP5, PRRSV-M, Mhp-P46, and Mhp-P65 protein had been screened to construct a recombinant epitope protein rEP-PM that has good hydrophilicity, strong antigenicity, and high surface accessibility, and each epitope is independent and complete. After immunization in mice, rEP-PM could induce the production of high levels of antibodies, and it had good immunoreactivity with anti-rEP-PM, anti-PRRSV, and anti-Mhp antibodies. The anti-rEP-PM antibody specifically recognizes proteins from PRRSV and Mhp. Moreover, rEP-PM induced a Th1-dominant cellular immune response in mice. Our results showed that the rEP-PM protein could be a potential candidate for the development of a safe and effective multi-epitope peptide combined vaccine to control PRRSV and Mhp infections.

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.

Establishment of a peptide-based enzyme-linked immunosorbent assay for detecting antibodies against PRRSV M protein.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry globally. Evaluation of antibody responses and neutralizing antibody titers is the most effective method for vaccine evaluation. In this study, the B cell line epitopes of PRRSV M protein were predicted, and two peptide ELISA assays were established (M-A110-129 ELISA, M-A148-174 ELISA) to detect antibodies against PRRSV M protein. Field serum samples collected from pig farms were used to validate the peptide ELISA and compare it with an indirect immunofluorescence assay. RESULTS: The sensitivity and specificity of M-A110-129 ELISA and M-A148-174 ELISA were (111/125) 88.80%, (69/70) 98.57% and (122/125) 97.60%, (70/70) 100%, relative to indirect immunofluorescence assay. This peptide ELISA could detect antibodies against different genotypes of PRRSV including type 1 PRRSV, classical PRRSV, HP-PRRSV, and NADC30 like PRRSV, but not antibodies against other common swine viruses. The results of ROC analysis showed that the area under the curve (AUC) of the M-A110-129 ELISA and M-A148-174 ELISA were 0.967 and 0.996, respectively. Compared the concordance of results using two peptide ELISA assays, the IDEXX PRRSV X3 Ab ELISA and a virus neutralization test, were assessed using a series of 147 sera from pigs vaccinated with the NADC30-like PRRSV inactivated vaccine. The M-A148-174 ELISA had the best consistency, with a Cohen's kappa coefficient of 0.8772. The concordance rates of the Hipra PRRSV ELISA kit, M-A110-129 ELISA and M-A148-174 ELISA in the field seropositive detection results were 91.08, 86.32 and 95.35%, relative to indirect immunofluorescence assay. CONCLUSIONS: In summary, compared with M-A110-129 ELISA, the PRRSV M-A148-174 ELISA is of value for detecting antibodies against PRRSV and the evaluation of the NADC30-like PRRSV inactivated vaccine, but the advantage is insufficient in serological early diagnosis.

Antiviral activity of a novel mixture of natural antimicrobials, in vitro, and in a chicken infection model in vivo.

The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

Development of a biosecurity assessment tool and the assessment of biosecurity levels by this tool on Japanese commercial swine farms.

It is well known that infectious diseases such as porcine reproductive and respiratory syndrome (PRRS) and porcine epidemic diarrhea (PED) decrease herd productivity and lead to economic loss. It is believed that biosecurity practices are effective for the prevention and control of such infectious diseases. Therefore, the objective of the present study was to investigate whether or not an association between biosecurity level and herd productivity, as well as disease status exists on Japanese commercial swine farms. The present study was conducted on 141 farms. Biosecurity in each farm was assessed by a biosecurity assessment tool named BioAsseT. BioAsseT has a full score of 100 and consists of three sections (external biosecurity, internal biosecurity and diagnostic monitoring). Production data for number of pigs weaned per sow per year (PWSY) and post-weaning mortality per year (PWM) were collected for data analysis. Regarding PRRS status, the farms were categorized into two groups: unknown or unstable and stable or negative. In addition, these farms were categorized based on their PED status, either positive or negative. The total BioAsseT score was associated with herd productivity: as total score increased by 1, PWSY increased by 0.104 pigs and PWM decreased by 0.051 % (P < 0.05). Herd productivity was associated with the score of external and internal biosecurity (P < 0.05), but did not correlate with the score of diagnostic monitoring. Regarding PRRS status, farms with an unknown or unstable status had lower total score than those with stable or negative status (P < 0.05). Similarly, PED positive farms had a lower total score compared to PED negative farms (P < 0.05). In conclusion, the present study provides evidence for the association between high biosecurity levels and increased herd productivity as well as a decreased risk for novel introductions of infectious diseases such as PED.

Therapeutic effects of saponin components on porcine reproductive and respiratory syndrome virus-infected piglets.

The present study aimed to evaluate the potential therapeutic effects of Anemoside B4 (AB4), Panax notoginseng saponins (PNS), Notoginsenoside R1 (SR1), Saikosaponin A (SSA) and Saikosaponin D (SSD) on piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 132 completely healthy piglets were randomly divided into 22 groups consisting of six animals each. Control piglets were intramuscularly injected with 2 ml of PRRSV (NJGC strain) solution containing 106  TCID50  virus/ml. For low-, middle- and high-dose saponin treatment groups, the piglets were initially administrated with the same volume of PRRSV solution, followed by intraperitoneal injection with AB4, PNS, SR1, SSA or SSD at 1, 5 or 10 mg/kg b.w. on day 3. The piglets in drug control group were intraperitoneally injected with 10 mg/kg b.w. of each saponin without prior PRRS challenge, while those in blank control group were injected with the same amount of normal saline. The results indicated that all the five saponin components could decrease the incidence and severity of PRRSV-induced immunopathological damages, including the elevated body temperature, weight loss, anaemia and internal inflammation. Moreover, the saponin components could enhance protein absorption and immune responses. Taken together, this study reveals that the saponin components are effective against PRRSV infection and strengthen the immune system and thus may serve as potential antiviral therapeutic agents.