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The aetiological agent of porcine reproductive and respiratory syndrome, a deadly disease that affects pigs and seriously jeopardises the global swine industry, is a porcine reproductive and respiratory syndrome virus (PRRSV). Tylvalosin tartrate, which is a macrolide antibiotic, is the active ingredient in Aivlosin. In recent years, tylvalosin tartrate has widely been used to control porcine reproductive and respiratory syndrome in swine herds in China. However, whether tylvalosin tartrate has exerts anti-PRRSV effects remains controversial. In the present study, tylvalosin tartrate exhibited no effect on PRRSV susceptibility but suppressed the replication of PRRSV and the activity of infecting Marc-145 cells. Next, the relationship between the replication cycle of PRRSV and the activity of tylvalosin tartrate was further assessed. Tylvalosin tartrate did not affect the attachment and release stages of PRRSV or act during the internalisation stage of the virus in HuN4; however, contrasting effects were noted for strains CH-1a and SDVD-HN21. Tylvalosin tartrate acted on the replication stage of PRRSV and was not strain-specific in the replication stage of the PRRSV life cycle. The study findings provide an initial clarification of the inhibitory effects of tylvalosin tartrate on PRRSV, providing new insights into the treatment of PRRS., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

Natural preservation materials have long been a focus of research in the quality control of fruits and vegetables. This study aimed to develop composite films with exceptional preservation properties by utilizing chitosan (CS) as the film-forming material and incorporating onion polysaccharide (ONP) as the active component. The CS-ONP composite films were prepared, and their performance and preservation effects were evaluated. The results demonstrated that increasing the ONP content significantly enhanced the shading, antimicrobial, and antioxidant capabilities of the CS-ONP composite films. Preservation experiments revealed that the CS-ONP composite films effectively delayed the quality decline of cherry tomatoes during storage. However, despite the improvements brought by ONP, certain drawbacks persisted, such as reduced mechanical properties and alterations in surface structure. In summary, the CS-ONP composite films exhibit promising potential as novel materials for fruit and vegetable preservation. PRACTICAL APPLICATION: The spoilage of fruits and vegetables can cause huge economic losses. This study addresses this challenge by using chitosan as the film-forming substrate and adding crude onion polysaccharide as the active ingredient to create composite films. The preservation effects of these films on cherry tomatoes were studied. Although only cherry tomatoes were tested in this study, the composite films demonstrated significant potential for broader applications in fruit and vegetable preservation., (© 2024 Institute of Food Technologists.)

Porcine reproductive and respiratory syndrome (PRRS) is one of the most widespread illnesses in the world's swine business. To detect the antibodies against PRRSV-2, a blocking enzyme-linked immunosorbent assay (B-ELISA) was developed, utilizing a PRRSV-2 N protein monoclonal antibody as the detection antibody. A checkerboard titration test was used to determine the optimal detection antibody dilution, tested pig serum dilution and purified PRRSV coated antigen concentration. After analyzing 174 negative pig sera and 451 positive pig sera, a cutoff value of 40 % was selected to distinguish between positive and negative sera using receiver operating characteristic curve analysis. The specificity and sensitivity of the assay were evaluated to equal 99.8 % and 96 %, respectively. The method had no cross-reaction with PCV2, PRV, PPV, CSFV, PEDV, TGEV, and PRRSV-1 serum antibodies, and the coefficients of variation of intra-batch and inter-batch repeatability experiments were both <10 %. A total of 215 clinical serum samples were tested, and the relative coincidence rate with commercial ELISA kit was 99.06 %, and the kappa value was 0.989, indicating that these two detection results exhibited high consistency. Overall, the B-ELISA should serve as an ideal method for large-scale serological investigation of PRRSV-2 antibodies in domestic pigs., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Since 2011, PRV has resurged in China and is characterized by a mutated strain with significant alterations in antigenicity and virulence. Therefore, we hypothesized that antibody detection kits based on classic PRV strains may have limitations in detecting PRV variants. For more sensitive antibody detection of PRV variants, two MABs targeting the gB and gE proteins were developed. IFA revealed that these MABs exhibited strong reactivity toward both classic and variant PRV strains. MAB-gE recognizes a novel conserved linear B-cell epitope ( 41 PSAEVWD 47 ), while MAB-gB recognizes a conformational B-cell epitope. The binding of both MABs was effectively inhibited in the PRV-positive pig blood samples. Accordingly, we established blocking-ELISAs to detect anti-PRV gB and gE antibodies, which achieved higher sensitivity than commercial kits. Moreover, the clinical serum samples results of our method and that of IFA were in high agreement, and our test results had a higher coincidence rate than that of a commercial kit. Assessing antibody levels by our methods at various times following immunization and challenge accurately reflected the trend of antibody-level changes and revealed the conversion to positive antibody status before the commercial kit. Our method is crucial for monitoring PRV infections, assessing immune responses, and controlling disease., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a serious threat to the pig industry in China. Our previous study demonstrated that PRRSV persists with local circulations and overseas imports in China and has formed a relatively stable epidemic pattern. However, the sudden African swine fever (ASF) outbreak in 2018 caused serious damage to China's pig industry structure, which resulted in about 40 per cent of pigs being slaughtered. The pig yields recovered by the end of 2019. Thus, whether the ASF outbreak reframed PRRSV evolution with changes in pig populations and further posed new threats to the pig industry becomes a matter of concern. For this purpose, we conducted genomic surveillance and recombination, NSP2 polymorphism, population dynamics, and geographical spread analysis of PRRSV-2, which is dominant in China. The results showed that the prevalence of ASF had no significant effects on genetic diversities like lineage composition, recombination patterns, and NSP2 insertion and deletion patterns but was likely to lead to changes in PRRSV-2 recombination frequency. As for circulation of the two major sub-lineages of Lineage 1, there was no apparent transmission of NADC30-like among provinces, while NADC34-like had obvious signs of inter-provincial transmission and foreign importation during the ASF epidemic. In addition, two suspected vaccine recombinant epidemic strains suggest a slight safety issue of vaccine use. Herein, the interference of ASF to the PRRSV-2 evolutionary pattern was evaluated and vaccine safety was analyzed, in order to monitor the potential threat of PRRSV-2 to China's pig industry in the post-epidemic era of ASF., Competing Interests: The author(s) declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press.)

Heavy metal contamination of soil commonly accompanies problems around gold mine tailings ponds. Fully investigating the distribution characteristics of heavy metals and the survival strategies of dominant plants in contaminated soils is crucial for effective pollution management and remediation. This study aims to investigate the contamination characteristics, sources of heavy metals (As, Cd, Pb, Hg, Cu, Zn, Cr, and Ni) in soils around gold mine tailings ponds areas (JHH and WZ) and to clarify the form distribution of heavy metals (As, Cd, Pb, Hg) in contaminated plots as well as their accumulation and translocation in native dominant plants. The results of the study showed that the concentrations of As, Pb, Cd, Cu, and Zn in soil exceeded the national limits at parts of the sampling sites in both study areas. The Nemerow pollution index showed that both study areas reached extreme high pollution levels. Spatial analysis showed that the main areas of contamination were concentrated around metallurgical plants and tailings ponds, with Cd exhibiting the most extensive area of contamination. In the JHH, As (74%), Cd (66%), Pb (77%), Zn (47%) were mainly from tailings releases, and Cu (52%) and Hg (51%) were mainly from gold ore smelting. In the WZ, As (42%), Cd (41%), Pb (73%), Cu (47%), and Zn (41%) were mainly from tailings releases. As, Cd, Pb, and Hg were mostly present in the residue state, and the proportion of water-soluble, ion-exchangeable, and carbonate-bound forms of Cd (19.93%) was significantly higher than that of other heavy metals. Artemisia L. and Amaranthus L. are the primary dominating plants, which exhibited superior accumulation of Cd compared to As, Pb, and Hg, and Artemisia L. demonstrated a robust translocation capacity for As, Pb, and Hg. Compared to the concentrations of other forms of soil heavy metals, the heavy metal content in Artemisia L correlates significantly better with the total soil heavy metal concentration. These results offer additional systematic data support and a deeper theoretical foundation to bolster pollution-control and ecological remediation efforts in mining areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the production and health of pigs and causes severe economic losses to the swine industry worldwide. Different pig breeds have been reported to have different levels of susceptibility to PRRSV, and different PRRSV strains may also influence the infectivity and pathogenicity of the virus. In this study, the susceptibility of Rongchang pigs (a prominent local pig breed in China) to PRRSV infection was thoroughly investigated. Rongchang piglets were exposed to two PRRSV strains: HuN4 (highly pathogenic PRRSV) and SD53-1603 (moderately virulent NADC30-like PRRSV). We observed that Rongchang pigs infected with HuN4 displayed significant clinical manifestations, including fever, reduced body weight, and interstitial pneumonia lesions. Routine blood tests revealed that HuN4-infected pigs exhibited slightly decreased levels of red blood cells, hemoglobin, reticulocytes, and a notable increase in monocytes than control pigs. Additionally, the Rongchang pigs exhibiting severe clinical signs presented a higher neutrophil-to-lymphocyte ratio and a lower lymphocyte-to-monocyte ratio. In contrast, SD53-1603 infection did not cause considerable harm to Rongchang pigs, only resulting in slightly elevated leukocytes and lymphocytes. Furthermore, these two PRRSV strains elicited divergent cytokine responses, such that SD53-1603 infection induced higher levels of TNF-α and IFN-γ, whereas HuN4 infection upregulated IL-1β. These dissimilarities in clinical symptoms, pathological changes, viremia, cytokine expression, and routine blood indices between HuN4 and SD53-1603 infections are critical in understanding the mechanisms of PRRSV infection and developing rational prevention and control strategies against PRRSV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Ma, Pan, Wang, Wang, Zhang, Gao, Zhang, Tian, Li, Chen, Xia and Wang.)

Due to the high incidence of PRRSV mutation and recombination, PRRSV infection is difficult to prevent and control in China and worldwide. Two species of PRRSV, Betaarterivirus suid 1 (PRRSV-1) and Betaarterivirus suid 2 (PRRSV-2), exist in China, and PRRSV-1 has always received less attention in China. However, the number of PRRSV-1 strains detected in China has increased recently. To date, PRRSV-1 has spread to more than 23 regions in China. Based on the phylogenetic analysis of ORF5 and the whole genome of PRRSV-1, Chinese PRRSV-1 can be divided into at least seven independent subgroups. Among them, BJEU06-1-like has become the mainstream subgroup in some regions of China. This subgroup of strains has a 5-aa (4 + 1) characteristic discontinuous deletion pattern at aa 357~aa 360 and aa 411 in Nsp2. Previous studies have indicated that the pathogenicity of PRRSV-1 in China is mild, but recent studies found that the pathogenicity of PRRSV-1 was enhanced in China. Therefore, the emergence of PRRSV-1 deserves attention, and the prevention and control of PRRSV-1 infection in China should be strengthened. PRRSV infection is usually prevented and controlled by a combination of virus monitoring, biosafety restrictions, herd management measures and vaccination. However, the use of PRRSV-1 vaccines is currently banned in China. Thus, we should strengthen the monitoring of PRRSV-1 and the biosafety management of pig herds in China. In this review, we summarize the prevalence of PRRSV-1 in China and clarify the genomic characteristics, pathogenicity, vaccine status, and prevention and control management system of PRRSV-1 in China. Consequently, the purpose of this review is to provide a basis for further development of prevention and control measures for PRRSV-1.

Since 2011, pseudorabies virus (PRV) has recurred in several vaccinated pig farms in China. PRV variants with high virulence were found to be the main cause of the outbreaks. In the face of the PRV epidemic, detection of the wild strain is as important as vaccine immunization, so we hoped to achieve differential diagnosis of PRV by obtaining a monoclonal antibody (mAB) that could be used to identify the wild strain. In this study, we used a novel immunization and screening strategy to prepare an mAB and obtained mAB 1H5 against the gE glycoprotein. An immunofluorescence assay (IFA) revealed that this mAB was specific to both classic and variant strains of PRV. Subsequently, we further identified the linear epitopes of B cells recognized using the mAB. The mAB 1H5 bound at 67 RRAG 70 , which is a novel epitope and is conserved in almost all PRV strains. These findings provide novel insight into the structure and function of PRV proteins, the analysis of antigenic epitope characteristics, and the establishment of antigen or antibody detection methods.

Porcine reproductive and respiratory syndrome (PRRS) is an important viral disease, causing significant economic losses to the swine industry worldwide. Atypical cases caused by highly pathogenic PRRS virus (HP-PRRSV) emerged in 2006 in China. The vaccine strain HuN4-F112 has been developed from the wild-type HP-PRRSV HuN4 through repeated passages on MARC-145 cells. However, the mechanisms of attenuation have yet to be defined. Previous studies have shown that the vaccine strain HuN4-F112 could not effectively replicate in porcine alveolar macrophages (PAMs). In the present study, a series of chimeric and mutant PRRSVs were constructed to investigate regions associated with the virus attenuation. Firstly, the corresponding genome regions (ORF1a, ORF1b and ORFs 2-7) were exchanged between two infectious clones of HuN4 and HuN4-F112, and then the influence of small regions in ORF1a and ORF2-7 was evaluated, then influence of specific amino acids on NSP2 was tested. NSP2 was determined to be the key gene that regulated infection efficiency on PAMs, and amino acids at 893 and 979 of NSP2 were the key amino acids. The results of in vivo study indicated that NSP2 was not only important for infection efficiency in vitro, but also influenced the virulence, which was indicated by the results of survival rate, temperature, viremia, lung score and tissue score.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is considered one of the most devastating swine diseases worldwide. PRRSV-1 was first isolated in China in 2006. However, there were few reports concerning the genetic characteristics of PRRSV-1 in China. In this study, three PRRSV-1 strains (HL85, HeB3, and HeB47) were detected by a general RT-qPCR method from clinical samples in 2018. HeB47 was identified as a recombinant between the BJEU06-1 and CReSA228-like strains. To further analyze the recombination and deletion features of PRRSV-1, all the available 88 complete genome sequences (isolated in 19 countries) from 1991 to 2018 in GenBank were analyzed. The high-frequency recombination regions were concentrated in NSP2 and GP2 to GP4. More importantly, phylogenetic analysis of PRRSV-1 revealed four independent introductions in China. Therefore, it is necessary to strengthen the important monitoring of breeding pigs and pork products and epidemiological surveys on pig farms to prevent the further spread of PRRSV-1.

In this study, the metal(loid) fractions in two alkaline iron tailings areas with similar physico-chemical properties and the enrichment ability of dominant plants in these areas were investigated. Additionally, high-throughput sequencing and metagenome analysis were used to examine the rhizosphere microbial community structures and their strategies and potential for carbon fixation, nitrogen metabolism, and metal(loid) resistance in mining areas. Results showed that Salsola collina, Setaria viridis, and Xanthium sibiricum have strong enrichment capacity for As, and the maximum transport factor for Mn can reach 4.01. The richness and diversity of bacteria were the highest in rhizosphere tailings, and the dominant phyla were Proteobacteria, Actinobacteria, Ascomycota, and Thaumarchaeota. The key taxa present in rhizosphere tailings were generally metal(loid) resistant, especially Sphingomonas, Pseudomonas, Nocardioides, and Microbacterium. The reductive citrate cycle was the main carbon fixation pathway of microorganisms in tailings. Rhizosphere microorganisms have evolved a series of survival strategies and can adapt to oligotrophic and metal(loid) polluted mining environments. The results of this study provide a basis for the potential application of plant-microbial in situ remediation of alkaline tailings., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A latex microsphere-based immunochromatographic strip (ICS) test was successfully developed for the rapid and sensitive detection of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV N protein-specific monoclonal antibody (mAb) 1H4 labeled with latex microspheres was dispensed on a conjugate pad for use as the detector. The same mAb and goat anti-mouse antibody were blotted on a nitrocellulose membrane to generate test and control lines, respectively. The limit of virus detection was approximately 5 × 10 2.0 median tissue culture infectious dose (TCID 50 )/ml, and the limit of N protein detection was approximately 15 ng/ml. Other common porcine viruses were tested to evaluate the specificity of the ICS, and positive results were observed for only North American-type PRRSV. A comparison of the strip with a standard diagnostic test (reverse transcriptase polymerase chain reaction, RT-PCR) was also performed, and the results showed that the ICS test exhibited relatively high specificity and sensitivity (90.32 and 73.91%, respectively) and relatively high positive predictive value (PPV) and negative predictive value (NPV; 85 and 82.35%, respectively). These results suggest that the ICS test can be used to rapidly and accurately detect PRRSV and can be suitable for diagnostic applications in the field., Competing Interests: ML was employed by Beijing IDEXX Yuanheng Laboratories, Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Li, Zhang, Li, Xu, Gong, Fu, Guo, Peng, Zhou, Tian and Wang.)

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) with enhanced replication capability emerged in China and has become dominant epidemic strain since 2006. Up to now, the replication-regulated genes of PRRSV have not been fully clarified. Here, by swapping the genes or elements between HP-PRRSV and classical PRRSV based on infectious clones, NSP1, NSP2, NSP7, NSP9 and 3'-UTR are found to contribute to the high replication efficiency of HP-PRRSV. Further study revealed that mutations at positions 117th or 119th in the 3'-UTR are significantly related to replication efficiency, and the nucleotide at position 120th is critical for viral rescue. The motif composed by 117-120th nucleotides was quite conservative within each lineage of PRRSV; mutations in the motif of HP-PRRSV and currently epidemic lineage 1 (L1) PRRSV showed higher synthesis ability of viral negative genomic RNA, suggesting that those mutations were beneficial for viral replication. RNA structure analysis revealed that this motif maybe involved into a pseudoknot in the 3'-UTR. The results discovered a novel motif, 117-120th nucleotide in the 3'-UTR, that is critical for replication of PRRSV-2, and mutations in the motif contribute to the enhanced replicative ability of HP-PRRSV or L1 PRRSV. Our findings will help to understand the molecular basis of PRRSV replication and find the potential factors resulting in an epidemic strain of PRRSV.

The Georgia-07-like genotype II African swine fever virus (ASFV) with high virulence has been prevalent in China since 2018. Here, we report that genotype I ASFVs have now also emerged in China. Two non-haemadsorbing genotype I ASFVs, HeN/ZZ-P1/21 and SD/DY-I/21, were isolated from pig farms in Henan and Shandong province, respectively. Phylogenetic analysis of the whole genome sequences suggested that both isolates share high similarity with NH/P68 and OURT88/3, two genotype I ASFVs isolated in Portugal in the last century. Animal challenge testing revealed that SD/DY-I/21 shows low virulence and efficient transmissibility in pigs, and causes mild onset of infection and chronic disease. SD/DY-I/21 was found to cause necrotic skin lesions and joint swelling. The emergence of genotype I ASFVs will present more problems and challenges for the control and prevention of African swine fever in China.

African swine fever virus (ASFV) has been circulating in China for more than two years, and it is not clear whether the biological properties of the virus have changed. Here, we report on our surveillance of ASFVs in seven provinces of China, from June to December, 2020. A total of 22 viruses were isolated and characterized as genotype II ASFVs, with mutations, deletions, insertions, or short-fragment replacement occurring in all isolates compared with Pig/HLJ/2018 (HLJ/18), the earliest isolate in China. Eleven isolates had four different types of natural mutations or deletion in the EP402R gene and displayed a non-hemadsorbing (non-HAD) phenotype. Four isolates were tested for virulence in pigs; two were found to be as highly lethal as HLJ/18. However, two non-HAD isolates showed lower virulence but were highly transmissible; infection with 10 6 TCID 50 dose was partially lethal and caused acute or sub-acute disease, whereas 10 3 TCID 50 dose caused non-lethal, sub-acute or chronic disease, and persistent infection. The emergence of lower virulent natural mutants brings greater difficulty to the early diagnosis of ASF and creates new challenges for ASFV control.

Torque teno sus virus (TTSuV) infection is common in China's pig herd. Although of uncertain pathogenicity, TTSuVs have been reported as a worsening factor of other porcine diseases, including porcine circovirus associated disease (PCVAD), porcine respiratory diseases complex (PRDC) or porcine dermatitis and nephropathy syndrome (PDNS). To better understand the genetic diversity in TTSuVs, the complete genomes of two newly emerged isolates, referred to as HeN1-A9 and HeN1-A11, collected from pig samples at a large-scale pig farm in China, were analyzed. Phylogenetic relationships of TTSuV sequences separated TTSuV1 and TTSuVk2a groups and divided TTSuV1 into two major subtypes, including TTSuV1a and TTSuV1b; HeN1-A9 and HeN1-A11 strains classified into the TTSuV1a subtype. Recombination analysis demonstrated HeN1-A9 and HeN1-A11 were generated via recombination in the overlapping ORF1/ORF3 region of TTSuV1a genome, which we report for the first time. Furthermore, we found that HeN1-A9 could be replicated in cultured MARC-145 cells for 18 passages. Our findings may be useful for elucidating the characteristics and epidemic status of TTSuVs in China., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Long noncoding RNA (lncRNA) is highly associated with inflammatory response and virus-induced interferon production. By far the majority of studies have focused on the immune-related lncRNAs of mice and humans, but the function of lncRNAs in porcine immune cells are poorly understood. Porcine reproductive and respiratory syndrome virus (PRRSV) impairs local immune responses in the lungs of nursery and growing pigs, whereas the virus triggers the inflammatory responses. Porcine alveolar macrophage (PAM) is the primary target cell of PRRSV, thus PRRSV is used as an in vitro model of inflammation. Here, we profiled lncRNA and mRNA repertories from PRRSV-infected PAMs to explore the underlying mechanism of porcine lncRNAs in regulating host immune responses., Results: In this study, a total of 350 annotated lncRNAs and 1792 novel lncRNAs in PAMs were identified through RNA-seq analysis. Among them 86 differentially expressed (DE) lncRNAs and 406 DE protein-coding mRNAs were identified upon PRRSV incubation. GO category and KEGG pathway enrichment analyses revealed that these DE lncRNAs and mRNAs were mainly involved in inflammation- and pathogen infection-induced pathways. The results of dynamic correlated expression networks between lncRNAs and their predicted target genes uncovered that numerous lncRNAs, such as XLOC-022175, XLOC-019295, and XLOC-017089, were correlated with innate immune genes. Further analysis validated that these three lncRNAs were positively correlated with their predicted target genes including CXCL2, IFI6, and CD163. This study suggests that porcine lncRNAs affect immune responses against PRRSV infection through regulating their target genes in PAMs., Conclusion: This study provides both transcriptomic and epigenetic status of porcine macrophages. In response to PRRSV infection, comprehensive DE lncRNAs and mRNAs were profiled from PAMs. Co-expression analysis demonstrated that lncRNAs are emerging as the important modulators of immune gene activities through their critical influence upon PRRSV infection in porcine macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pathogen in the swine industry worldwide. miRNAs are reported to be involved in virus-host interaction. Here, we used high-throughput sequencing and miRNA inhibitors to screen possible miRNAs that can inhibit PRRSV infection on its target cell, porcine alveolar macrophages. We observed that miR-218 was downregulated upon virus infection, and knockdown of miR-218 significantly enhanced PRRSV replication. Overexpression of miR-218 resulted in a decrease in PRRSV replication, and this overexpression did not alter viral genomic RNA levels, but rather increased antiviral interferon signaling. Further analysis revealed that miR-218 regulated PRRSV replication by directly targeting porcine suppressor of cytokine signaling 3 (SOCS3), a JAK2 kinase inhibitor. Knockdown of the endogenous SOCS3 expression led to augmentation of type I interferon genes and resulted in decreased PRRSV replication, and vice versa. During PRRSV infection in vivo and in vitro, cellular miR-218 expression was downregulated and SOCS3 expression was upregulated, further supporting the inverse correlation between miR-218 and SOCS3 expression. The data on SOCS3 depletion in combination with miR-218 inhibition suggested that the antiviral activity of miR-218 required the SOCS3-mediated signaling pathway. Similarly, miR-218 negatively regulated PRRSV replication in Marc-145 cells, as well as the replication of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in Vero and ST cells respectively. Taken together, these results demonstrate that PRRSV-induced miR-218 downregulation serves to inhibit the type I interferon response and may provide a novel therapeutic target for treatment of PRRSV and other viral infections., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mine tailing dumps represent significant threats to ecological environments due to the presence of toxic substances. The present work investigated the relationship among microbial activity, the community, antibiotic resistance genes (ARGs) and trace metals in soil surrounding gold mine tailings. Using microbial metabolic activity and high-throughput sequencing analysis, we found the trace metals Cd and Hg could be main factors influencing the microbial community. According to bacterial co-occurrence pattern analysis, the effects of total cadmium and total mercury on bacterial diversity are potentially mediated by influencing bacteria community in the keystone module II. Additionally, most of metal-resistant bacteria belong to Actinobacteria and Proteobacteria, and the metal tolerance suggested to be linked with various functions including replication, recombination and repair, as well as inorganic ion transport and metabolism based on PICRUSt2 analysis. We also found that metals generated by mining activity may trigger the co-selection of antibiotic resistance in the phyla Actinobacteria and Proteobacteria due to co-resistance or cross resistance. Additionally, PLS-PM analysis revealed that metals could indirectly affect ARGs by influencing bacterial diversity in gold mining areas., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Some metallic tailings from closed mines were scattered in upstream of the Miyun Reservoir, Beijing, threatening the ecological environment of rivers due to trace metals. The Liuli River, one of the main rivers affected, was investigated as a typical model in this work. In this study, we selected eight sites to assess interactions among the various geochemical factors especially between trace metals and sediment microbiota. Random forest predicted that low concentrations of Cu, Cd, Cr and Ni (lower than 61.8 mg/kg, 3.2 mg/kg, 173.2 mg/kg and 34.1 mg/kg, respectively) were able to enhance community diversity but generally, trace metals contamination impaired microbial diversity. Environmental factor correlation analysis showed that As, pH and available P were the major factors that shifted the distribution of the microbial communities. Metagenome sequencing revealed that Proteobacteria harbored the vast majority of heavy metal resistance genes followed by Actinobacteria and Bacteroidetes. Metal tolerance of Proteobacteria were achieved by exportation of metals by the corresponding transporters, by pumps and ion channels, or by their reduction via redox reactions. In addition, Proteobacteria harbored a greater ability to repair DNA damage via DNA recombination., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The migration of metals (e.g., Fe, Cd, Co, Cr, Cu, Mn, Ni, and Zn) in both of iron tailings under different pH leachates was studied by laboratory static leaching experiments. The results indicated that Fe showed the highest leaching concentration at an initial pH of 2, reaching 16.19 and 51.72 mg L -1 in the Qian'anling (Q0) and Majuanzi (M0) iron tailings, respectively. Metal ions manifested a strong pH dependence. In addition, the leaching behavior of Cd, Cr, Fe, and Cu for the two tailings was also evaluated under leaching by three low-molecular-weight organic acids (LMWOAs). The results indicated the leaching of Cd and Fe followed the order of citric acid > malic acid > oxalic acid and that the leaching order for Cr and Cu was citric acid > oxalic acid > malic acid. The concentration of Fe was low in 5 mM oxalic acid leaching for 20 days because of the hydrolysis precipitation of iron ions and the complexation with organic ligand. The crystal lattice on the tailings was significantly damaged after leaching. The CO 3 2- peak appeared in M0 with different treatments, and the proportion of COO- fitting peak areas increased markedly after leaching with LMWOAs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase in mammalian cells, is known to regulate the kinase-driven intracellular signaling pathways. Emerging evidences have shown that the PP2A phosphatase functions as a bona-fide therapeutic target for anticancer therapy, but it is unclear whether PP2A affects a porcine reproductive and respiratory syndrome virus infection. In the present study, we demonstrated for the first time that inhibition of PP2A activity by either inhibitor or small interfering RNA duplexes in target cells significantly reduced their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) infection. Further analysis revealed that inhibition of PP2A function resulted in augmented production of type I interferon (IFN). The mechanism is that inhibition of PP2A activity enhances the levels of phosphorylated interferon regulatory factor 3, which activates the transcription of IFN-stimulated genes. Moreover, inhibition of PP2A activity mainly blocked PRRSV replication in the early stage of viral life cycle, after virus entry but before virus release. Using type I IFN receptor 2 specific siRNA in combination with PP2A inhibitor, we confirmed that the effect of PP2A on viral replication within target cells was an interferon-dependent manner. Taken together, these findings demonstrate that PP2A serves as a negative regulator of host cells antiviral responses and provides a novel therapeutic target for virus infection.

Different strains of porcine reproductive and respiratory syndrome virus (PRRSV) have emerged and circulated in different regions of mainland China since 1996, particularly after 2006. In 2012, NADC30-like PRRSV was first isolated in Henan Province. By 2016, it had spread to most provinces in China. In the present study, the whole genomes (excluding the poly(A) tails) of 13 newly emerged NADC30-like PRRSV strains were sequenced and analyzed. Furthermore, the pathogenicity of SD53-1603, one of the 13 PRRSV strains, was assessed. Phylogenetic analysis showed that these 13 newly emerged NADC30-like PRRSV strains, together with some reference strains, formed a new subgroup (subgroup 5), characterized by a predicted 131-amino-acid deletion in the nonstructural protein (NSP) 2. However, low levels of whole-genome similarity and a wide variety of recombination patterns complicated the classification of the NADC30-like PRRSV isolates. Interestingly, almost all of the recombination breakpoints found in these 13 PRRSV isolates and other NADC30-like PRRSV isolates occurred in genes encoding NSPs and/or minor structural proteins. In addition, piglets infected with the newly emerged NADC30-like strain SD53-1603 displayed clear clinical respiratory symptoms and underwent typical pathological changes. The findings may be useful for elucidating the characteristics and epidemic status of NADC30-like PRRSV in China.

Porcine reproductive and respiratory syndrome (PRRS), a serious disease of swine caused by the PRRS virus (PRRSV), had a severe economic impact worldwide. As commonly used PRRS vaccines, the attenuated or inactivated vaccines, provide unsatisfactory immune protection, a new PRRS vaccine is urgently needed. In this study, a part of the PRRSV ORF6 gene (from 253 to 519 bp) encoding the hydrophilic domain of PRRSV M protein was integrated into two Listeria strains via homologous recombination to generate two PRRS vaccine candidates, namely LI-M' and LM-ΔactAplcB-M'. Both candidate vaccines showed similar growth rate as their parent strains in culture media, but presented different bacterial loads in target organs. As the integrated heterogenous gene was not expressed, LM-ΔactAplcB-M' was excluded from the immunological test. In a mouse model, LI-M' provoked both CD4+ and CD8+ T cell-mediated immunity. In addition, LI-M' boosting dramatically enhanced CD8+ T cell-mediated immunity without affecting the response intensity of CD4+ T cell-mediated immunity. All of these data suggest that LI-M' is a promising PRRS vaccine candidate., (© 2020 S. Karger AG, Basel.)

Background: Classical swine fever (CSF) is one of the most devastating and highly contagious viral diseases in the world. Since late 2014, outbreaks of a new sub-genotype 2.1d CSF virus (CSFV) had caused substantial economic losses in numbers of C-strain vaccinated swine farms in China. The objective of the present study was to explore the genomic characteristics and pathogenicity of the newly emerged CSFV isolates in China during 2014-2015., Results: All the new 8 CSFV isolates belonged to genetic sub-genotype 2.1d. Some genomic variations or deletions were found in the UTRs and E2 of these new isolates. In addition, the pathogenicity of HLJ1 was less than Shimen, suggesting the HLJ1 of sub-genotype 2.1d may be a moderated pathogenic isolate and the C-strain vaccine can supply complete protection., Conclusions: The new CSFV isolates with unique genomic characteristics and moderate pathogenicity can be epidemic in many large-scale C-strain vaccinated swine farms. This study provides the information should be merited special attention on establishing prevention and control policies for CSF.

Pseudorabies virus (PRV) is a neurotropic virus with the ability to infect peripheral sensory ganglia. The transport of PRV from the peripheral to the central nervous system can cause lethal encephalitis in young piglets. However, the pathogenicity of PRV in the cerebral cortex remains poorly understood. In the present study, we developed a porcine cerebral cortex primary culture system (PCCS) using cerebral cortex tissue dissected from a 3-day-old piglet to investigate the pathogenicity of wild-type (WT) and US2 deleted (ΔUS2) PRV in the CNS in vitro. Immunofluorescence assays revealed cell bodies and neurites as the cellular locations infected by PRV. Growth kinetic analysis showed a persistent increase in WT and ΔUS2 viral titers in PCCS from 4 to 24 h post infection (hpi), thus indicating that US2 deletion did not disrupt viral growth. However, the mean plaque size was significantly higher in ΔUS2 PRV than in WT PRV in infected Vero cells. The viral titers and DNA levels of ΔUS2 PRV were significantly higher at 8 hpi than at 4 hpi, whereas those of WT showed no significant difference between the two time points in PCCS. Morphological investigation revealed induction of massive amounts of bouton-like swellings (varicosities) along the axon shaft in both WT and ΔUS2 PRV-infected neurons in the PCCS. Our data suggest that PRV US2 gene deletion enhances viral titers in PCCS but does not affect the varicosities induced by the viral infection.

Introduction: Classical swine fever (CSF) has caused severe economic losses in pig production in many countries. Recent CSF outbreaks in China are mainly associated with sub-genotype 2.1 of CSF virus (CSFV). Although there is abundant information regarding 2.1 isolates, few data are available on whole-genome analysis., Material and Methods: The biological and genome characteristics of three recently emerged Chinese CSFV isolates, i . e . SD2014-1, SD2014-2, and SD2014-3, were fully analysed., Results: Sequence analysis showed that the isolates shared 83.4%-95.0% nucleotide identity with eight other CSFV isolates. In addition, the 5' untranslated region (5'UTR) and the non-structural (NS) proteins NS3, NS4A, and NS4B were more conserved than other regions of the genome. Phylogenetic analysis based on the complete genome sequences or full-length structural protein E2 gene sequences revealed that the three isolates belonged to sub-genotype 2.1b. In addition, several unique molecular characteristics of the 5'UTR, 3'UTR, and E2 were identified., Conclusion: The genomic variations of the three isolates will support further analysis of virulence determinants and the evolutionary trend of CSFV., Competing Interests: Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article.

Background: Currently, porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viral pathogens in swine in most countries, especially China. Two PRRSV attenuated live vaccine strains (HuN4-F112 and CH-1R) are currently widely used in China. Our previous study showed that HuN4-F112, but not CH-1R, induced high anti-nucleocapsid (N) antibody and neutralizing antibody (NA) titers. Additionally, sera from HuN4-F112 inoculated pigs induced low cross neutralization of CH-1R., Methods: In the present study, 6 chimeric viruses through exchanging 5' untranslated region (UTR) + open reading frame (ORF)1a, ORF1b, and ORF2-7 + 3'UTR between HuN4-F112 and CH-1R were constructed and rescued based on the infectious clones of rHuN4-F112 and rCH-1R. The characteristics of these viruses were investigated in vitro and vivo., Results: All the three fragments, 5'UTR + ORF1a, ORF1b, and ORF2-7 + 3'UTR, could affect the replication efficiencies of rHuN4-F112 and rCH-1R in vitro. Additionally, both 5'UTR + ORF1a and ORF2-7 + 3'UTR affected the anti-N antibody and NA responses targeting rHuN4-F112 and rCH-1R in piglets., Conclusions: The 5'UTR + ORF1a region of HuN4-F112 played a key role in inducing NAs in piglets. Furthermore, we confirmed for the first time that ORF1a contains a neutralization region. This study provides important information that can be used for further study of the generation of anti-PRRSV NAs.

Introduction: In 2014-2015, the epidemic of classical swine fever (CSF) occurred in many large-scale pig farms in different provinces of China, and a subgenotype 2.1d of CSF virus (CSFV) was newly identified., Material and Methods: The phylogenetic relationship, genetic diversity, and epidemic status of the 2014-2015 CSFV isolates, 18 new CSFV isolates collected in 2015, and 43 other strains isolated in 2014-2015 were fully analysed, together with 163 CSFV reference isolates., Results: Fifty-two 2014-2015 isolates belonged to subgenotype 2.1d and nine other isolates belonged to subgenotype 2.1b. The two subgenotype isolates showed unique molecular characteristics. Furthermore, the 2.1d isolates were found to possibly diverge from 2.1b isolates., Conclusion: This study suggests that the Chinese CSFVs will remain pandemic., Competing Interests: Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article. Financial Disclosure Statement: The research and the article were financed with the funds of the State Key Laboratory of Veterinary Biotechnology (No. SKLVBF201612), the National Natural Science Foundation of China (No. 31502097), the Key Programme Foundation of Higher Education of Educational Commission of Henan Province (No. 15A230026) and the Foundation of Nanyang Normal University (No. 15082). Animal Rights Statement: The authors declare that the experiments on animals were conducted in accordance with local Ethical Committee laws and regulations as regards care and use of laboratory animals.

Background: The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) continues to pose one of the greatest threats to the swine industry. M protein is the most conserved and important structural protein of PRRSV. However, information about the host cellular proteins that interact with M protein remains limited., Methods: Host cellular proteins that interact with the M protein of HP-PRRSV were immunoprecipitated from MARC-145 cells infected with PRRSV HuN4-F112 using the M monoclonal antibody (mAb). The differentially expressed proteins were identified by LC-MS/MS. The screened proteins were used for bioinformatics analysis including Gene Ontology, the interaction network, and the enriched KEGG pathways. Some interested cellular proteins were validated to interact with M protein by CO-IP., Results: The PRRSV HuN4-F112 infection group had 10 bands compared with the control group. The bands included 219 non-redundant cellular proteins that interact with M protein, which were identified by LC-MS/MS with high confidence. The gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway bioinformatic analyses indicated that the identified proteins could be assigned to several different subcellular locations and functional classes. Functional analysis of the interactome profile highlighted cellular pathways associated with protein translation, infectious disease, and signal transduction. Two interested cellular proteins-nuclear factor of activated T cells 45 kDa (NF45) and proliferating cell nuclear antigen (PCNA)-that could interact with M protein were validated by Co-IP and confocal analyses., Conclusions: The interactome data between PRRSV M protein and cellular proteins were identified and contribute to the understanding of the roles of M protein in the replication and pathogenesis of PRRSV. The interactome of M protein will aid studies of virus/host interactions and provide means to decrease the threat of PRRSV to the swine industry in the future.

A large number of studies have demonstrated that autophagy is involved in the infection processes of different pathogens. Autophagy is now recognized as an essential component of innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral mechanism. Pseudorabies virus (PRV) is a swine herpesvirus with a broad host range that causes devastating disease in infected pigs. In this study, we described the interaction between PRV and autophagy for the first time. PRV infection had a dual effect on the cell autophagy response; during the early period of infection, PRV virions induced autophagy without viral replication, and with viral protein expression, PRV reduced the basal level of autophagy in several permissive cells. We observed that inhibit the level of autophagy could increase the titer of infectious PRV. We also found that the conserved alphaherpesvirus US3 tegument protein may reduce the level of autophagy via activation of the AKT/mTOR pathways in PRV infected cells. These findings suggest that autophagy likely contributes to clearance of PRV, and that the virus has evolved strategies to antagonize this pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) has become an important pathogen for the swine industry, and has resulted in substantial economic losses. In 2006, highly pathogenic PRRSV (HP-PRRSV) belonging to genotype 2 was first identified in China. Here, the replication kinetics of genotype 2 PRRSV strains were estimated in vitro in MARC-145 cells and porcine alveolar macrophages (PAMs) using a TaqMan-based real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. The lower limit of detection was 10 copies/μL, and the assay was linear between 10(1) and 10(8) copies/μL. The intra-assay coefficients of variation were 0.81-1.36%, and the inter-assay coefficients of variation were 1.77-2.56%. Compared to the low pathogenicity CH-1a-F45 strain, the viral loads of the highly pathogenic HuN4-F45 strain were 10(0.5)-10(1.05) and 10(0.84)-10(1.35) times greater in MARC-145 cells and PAMs, respectively from 12 to 96h after infection (P<0.01). This study is the first to demonstrate that the HuN4-F45 strain replicated at higher levels than CH-1a-F45 in MARC-145 cells and PAMs, suggesting that HuN4-F45 has more robust virus amplification efficiency than CH-1a-F45 in vitro., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a series of inflammatory reactions in sites of infection, companied by the upregulation of key inflammatory factor TNFα. TNFα, which serves as a "master regulator" of inflammatory cytokine production, is mainly produced by macrophages at the early infection stage. Here, we showed that porcine alveolar macrophages produced a great amount of soluble TNFα upon PRRSV infection. Furthermore, we found that TNFα had great anti-PRRSV effect. Next, by using inhibitor and genetic modification methods, we addressed that porcine TNFα production was mediated by ADAM17. Lastly, we proved that the (78)Arg-Ser-Ser motif of porcine TNFα contained the essential information for efficient cleavage. Taken together, our findings provide the direct evidence that ADAM17 cleaves porcine TNFα, which represents a new view for identifying potential therapeutic targets in anti-PRRSV therapy.

Soluble CD16 (sCD16) is closely correlated with chronic diseases in humans. Here, plasma sCD16 levels in pigs were increased by infection with porcine reproductive and respiratory syndrome virus (PRRSV) but not with porcine epidemic diarrhea virus, porcine circovirus type 2 and pseudorabies virus. Of interest, PRRSV attached to blood neutrophils and reduced surface CD16 expression on neutrophils. In vitro data confirmed that PRRSV caused CD16 shedding in neutrophils. Further analyses revealed that ADAM17 was involved in porcine CD16 shedding. Thus, our findings suggest that increase in sCD16 levels may be an indicator of PRRSV infection.

The complete genome sequence of classic swine fever virus (CSFV) strain JSZL was determined in this study. JSZL was originally isolated from an immune pig farm in Jiangsu Province, China. JSZL is more closely related to subgenotype 2.1b than to 2.1a and 2.1c. Importantly, JSZL was classified into a new subgenotype, 2.1d., (Copyright © 2015 Zhang et al.)

The lapinized attenuated vaccine against classical swine fever (CSF) has been used in China for over half a century and has generally prevented large-scale outbreaks in recent years. However, since late 2014, a large number of new cases of CSF were detected in many immunized pig farms in China. Several of these CSV viruses were isolated and characterized. Phylogenetic and genomic sequence analyses indicate that these new isolates, as well as some reference isolates, form a new subgenotype named 2.1d, and share several consistent molecular characteristics. Since these new isolates emerged in disparate geographic regions within 5 months, this suggests that these isolates may be widespread. Given that current vaccines do not appear to provide effective protection against this new subgenotype, further investigation of these strains is urgently needed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The immunological effect of porcine reproductive and respiratory syndrome disease virus (PRRSV) vaccines is thought to be influenced by a variety of host factors, in which antibody-dependent enhancement (ADE) of infection is one crucial factor. Here, we assessed the mechanism of ADE of PRRSV infection. First, we found that subneutralizing serum could induce ADE of PRRSV infection in porcine alveolar macrophages (PAMs). Quantitative PCR, Western blotting and flow cytometry revealed that CD16 is the most abundant Fcγ receptor (FcγR) expressed on the surface of PAMs; thus, the role of CD16 in ADE of PRRSV infection was examined in PAMs. By using functional blocking antibodies, we demonstrated that CD16 is involved in enhanced virus production in PRRSV-antibody immune complex-infected PAMs. Because PAMs co-express different FcγR isoforms, we evaluated the effects of CD16 in FcγR-non-bearing cells by transfection. Using these engineered cells, we found that CD16 could specifically bind to the PRRSV-antibody immune complex and subsequently mediate internalization of the virus, resulting in the generation of progeny virus. We also showed that efficient expression of CD16 required association of the FcR γ-chain. Together, our findings provide significant new insights into PRRSV infection, which can be enhanced by CD16-mediated PRRSV-antibody immune complexes. This CD16-mediated ADE may induce a shift in PRRSV tropism towards CD16-expressing cells, distributing virus to more organs during virus infection.

The M protein, encoded by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF6 gene, is considered to be one of the most conserved PRRSV proteins. In recent decades, highly specific monoclonal antibodies (Mabs) have been exploited to provide reliable diagnoses for many diseases. In this study, two different Mab clones targeting the linear epitopes on the PRRSV M protein were generated and characterized. Both Mabs showed binding activity against the native PRRSV virion and recombinant M protein when analyzed by immunofluorescence assay (IFA) and Western blot. The targeted epitope of each Mab was mapped by serial truncation of the M protein to generate overlapping fragments. Fine epitope mapping was then performed using a panel of expressed polypeptides. The polypeptide sequences of the two epitopes recognized by Mabs 1C8 and 3F7 were (3)SSLD(6) and (155)VLGGRKAVK(163), respectively, with the former being a newly identified epitope on the M protein. In both cases, these two epitopes were finely mapped for the first time. Alignments of Mab epitope sequences revealed that the two epitopes on the M protein were highly conserved between the North American-type strains. These Mabs, along with their mapped epitopes, are useful for the development of diagnostic and research tools, including immunofluorescence, ELISA and Western blot.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is a member of the genus Arterivirus within the family Arteriviridae. N and GP3 proteins are the immunodominance regions of the PRRSV viral proteins. To identify the B-cell linear antigenic epitopes within HP-PRRSV N and GP3 proteins, two monoclonal antibodies (mAbs) against N and GP3 proteins were generated and characterized, designated as 3D7 and 1F10 respectively. The mAb 3D7 recognized only HuN4-F112 not the corresponding virulent strain (HuN4-F5). It also recognized two other commercial vaccines (JXA1-R and TJM-F92), but not two other HP-PRRSV strains (HNZJJ-F1 and HLJMZ-F2). The B-cell epitope recognized by the mAb 3D7 was localized to N protein amino acids 7-33. Western blot showed that the only difference amino acid between HuN4-F112-N and HuN4-F5-N did not change the mAb 3D7 recognization to N protein. The epitope targeted by the mAb 1F10 was mapped by truncated proteins. We found a new epitope (68-76aa) can be recognized by the mAb. However, the epitope could not be recognized by the positive sera, suggesting the epitope could not induce antibody in pigs. These results should extend our understanding of the antigenic structure of the N protein and antigen-antibody reactions of the GP3 protein in different species.

Unlabelled: As a consequence of their effects on ectodomain shedding, members of the A disintegrin and metalloprotease (ADAM) family have been implicated in the control of various cellular processes. Although ADAM family members are also involved in cancer, inflammation, and other pathologies, it is unclear whether they affect porcine reproductive and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine alveolar macrophages (PAMs). We also demonstrate that the inhibition of ADAM17 upregulates membrane CD163 expression, a putative PRRSV receptor that is exogenously expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore, overexpression of ADAM17 induced downregulation of CD163 expression and a reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific small interfering RNA resulted in upregulation of CD163 expression with a corresponding increase in PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV nonpermissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate that ADAM17 downregulates CD163 expression and hinders PRRSV entry. Hence, downregulation of ADAM17 particular substrates may be an additional component of the anti-infection defenses., Importance: ADAM17 is one of the important membrane-associated metalloproteases that mediate various cellular events, as well as inflammation, cancer, and other pathologies. Here, we investigate for the first time the role of the metalloprotease ADAM17 in PRRSV infection. By using inhibitor and genetic modification methods, we demonstrate that ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More specifically, ADAM 17 mediates the downregulation of the PRRSV cellular receptor CD163. The reduction in CD163 expression represents another component of the anti-infection response initiated by ADAM17., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Non-structural protein 9 (Nsp9), a RNA-dependent RNA polymerase (RdRp) of the porcine reproductive and respiratory syndrome virus (PRRSV), is necessary for PRRSV replication. However, the binding partners of Nsp9 have not been identified. In this study, seven host proteins were identified as Nsp9-binding proteins using yeast two-hybrid (Y2H). Among of them, we confirmed the interaction of Nsp9 with Annexin A2 (ANXA2) using Y2H, Co-immunoprecipitation (Co-IP), GST pulldown and immunofluorescence assay (IFA). We found that only full-length ANXA2 could bind with Nsp9 in vitro and Nsp9 interacted with endogenous ANXA2 in PRRSV-infected MARC-145 cells. In addition, we found that the Nsp9-ANXA2 interaction was partially reduced by RNase A treatment. Furthermore, PRRSV growth was significantly hindered in ANXA2-knockdown MARC-145 cells. Taken together, these results indicate that Nsp9 binding partner ANXA2 is beneficial for PRRSV replication., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Haptoglobin (Hp) is one of the acute phase proteins (APPs) that help to alleviate the immune oxidative damage. The present study expressed a truncated porcine Hp in Escherichia coli and produced rabbit and mouse antisera to the recombinant protein, in order to investigate Hp levels in sera from piglets infected with porcine reproduction and respiratory syndrome virus (PRRSV). Antisera prepared revealed both chains of porcine Hp in Western blot, and mouse antisera showed stronger binding activities than the rabbit antisera. With the combination of Hp monoclonal antibodies, this study confirmed that serum Hp was increased in piglets infected with PRRSV and offered a tool to know about subunit levels of Hp in porcine serum. But Hp itself could not be used as a specific biomarker for PRRSV infection, for elevated Hp levels were also obtained from pigs infected with other pathogens., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Since 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has become the major pathogen attributed to the prevalent porcine reproductive and respiratory syndrome (PRRS) in China. The present study aims to identify serum proteins modified in response to infection of HuN4, a HP-PRRSV strain isolated from a farm in 2006. 2-D DIGE analysis allowed for the detection of 19 differentially expressed protein spots, of which 18 were identified by MALDI-TOF/TOF MS. These 18 spots represented for a total of 9 proteins (6 up-regulated and 3 down-regulated), most of which belonged to the acute phase proteins in swine and showed a trend of regression in the late phase of the experiment. One of a series of AGP spots was identified for the first time to be decreased in acute phase of PRRSV infection in swine. But the whole level of the protein in the serum did not show significant changes by Western blot. The rising tendency of Hp was confirmed by Western blot and ELISA. These altered proteins were probably involved in the inflammatory process triggered by HuN4 and in alleviating the oxidative damage occurring in the process. In summary, these results may provide new insights into understanding the mechanisms of HP-PRRSV infection., (Copyright © 2012. Published by Elsevier B.V.)

Two severe porcine infectious diseases, pseudorabies (PR) and transmissible gastroenteritis (TGE) caused by pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV) respectively often result in serious economic loss in animal husbandry worldwide. Vaccination is the important prevention means against both infections. To achieve a PRV genome-based virus live vector, aiming at further TGEV/PRV bivalent vaccine development, a recombinant plasmid pUG was constructed via inserting partial PK and full-length gG genes of PRV strain Bartha K-61 amplified into pUC119 vector. In parallel, another recombinant pHS was generated by introducing a fragment designated S1 encoding the major antigen sites of S gene from TGEV strain TH-98 into a prokaryotic expression vector pP(RO)EX HTc. The SV40 polyA sequence was then inserted into the downstream of S1 fragment of pHS. The continuous region containing S1fragment, SV40 polyA and four single restriction enzyme sites digested from pHS was subcloned into the downstream of gG promoter of pUG. In addition, a LacZ reporter gene was introduced into the universal transfer vector named pUGS-LacZ. Subsequently, a PRV genome-based virus live vector was generated via homologous recombination. The functionally effective vector was purified and partially characterized. Moreover, the potential advantages of this system are discussed.