Porcine reproductive and respiratory syndrome (PRRS), caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is one of the major diseases threatening the swine industry. This study aims to rationally design and optimize natural antimicrobial peptides to identify antiviral candidates with potent inhibitory activity against PRRSV, thereby establishing a foundation for the development of novel preventive and therapeutic agents targeting PRRS. In this study, with cecropin D (CD) as the parent peptide, three derivatives (CD-2, CD-3, and CD-4) were designed through amino acid substitutions. CD and derived peptides were obtained by solid-phase peptide synthesis. MS and reversed-phase (RP)-HPLC were employed for sequence identification, purification, and purity analysis. The secondary structures of the peptides were investigated by circular dichroism spectroscopy. CellTiter 96^® AQueous one solution cell proliferation assay was used to evaluate the cytotoxicity of the peptides. The inhibitory activities and mechanisms of the peptides against PRRSV were studied by Western blotting, RT-qPCR, and indirect immunofluorescence assay. The MS and RP-HPLC results showed that CD and derived peptides were successfully synthesized, with the purity reaching up to 95%. Circular dichroism analysis revealed that the CD derivatives exhibited more stable and abundant α-helices in a cell membrane-mimicking environment. The MTS assay indicated that all tested peptides at 100 μg/mL had negligible cytotoxicity. The experimental results of the action phase of the peptide against PRRSV demonstrated that the derived peptides significantly enhanced antiviral activities at the viral entry stage compared with the parent peptide. This enhancement was attributed to the introduction of lysine, tryptophan, and phenylalanine, which increased the hydrophobicity and positive charge of the peptides. These findings provide a theoretical basis for the application and structural optimization of antiviral peptides and may offer a new strategy for preventing and controlling PRRSV.
Porcine respiratory and reproductive syndrome virus/physiology* ; Animals ; Swine ; Antiviral Agents/chemistry* ; Porcine Reproductive and Respiratory Syndrome/virology* ; Virus Internalization/drug effects* ; Antimicrobial Peptides/chemistry*

This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine alveolar macrophages (PAMs). The changes of glucose metabolism, PRRSV protein levels, PRRSV titers, and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA, qPCR, virus titration, and Western blotting after PRRSV infection. The effect of hypoxia-inducible factor-1α (HIF-1α) on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α. The results showed that PRRSV infection enhanced glycolysis, elevated the levels of glucose uptake and lactate in the supernatant (P<0.05 and 0.01, respectively), reduced ATP production (P<0.05), and up-regulated the expression of hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), and pyruvate kinase isozyme type M2 (PKM2) in PAMs (P<0.05 and 0.01, respectively). Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers (P<0.01). The knockdown of HIF-1α by siRNAs inhibited glycolysis and lowered PRRSV titers (P<0.05). In addition, the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis. These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.
Porcine respiratory and reproductive syndrome virus/physiology* ; Glycolysis ; Swine ; Animals ; Virus Replication ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Macrophages, Alveolar/metabolism* ; Porcine Reproductive and Respiratory Syndrome/virology* ; Cells, Cultured ; RNA, Small Interfering/genetics*

The porcine reproductive and respiratory syndrome virus (PRRSV) can cause reproductive barriers in breeding pigs and respiratory symptoms in piglets. In this review, we summarize research progress of the infection and replication mechanisms of the PRRSV. We also review the virulence determinants of the PRRSV. All these fundamental studies are important for the control and elimination of the PRRSV.
Animals ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; pathogenicity ; physiology ; Swine ; Virulence ; Virus Replication

The objectives of the present study were to evaluate the anatomic localization of porcine reproductive and respiratory syndrome virus (PRRSV) in naturally infected pigs and to determine whether oral fluid could be used to detect the virus in infected animals. Two sows, seven 2-month-old grower pigs, and 70 6-month-old gilts were included in this study. PRRSV in sera and oral fluid were identified by nested reverse transcription PCR (nRT-PCR) while lung, tonsil, and tissue associated with oral cavity were subjected to nRT-PCR, immunohistochemistry, and in situ hybridization. In sows, PRRSV was identified in oral fluid and tonsils. PRRSV was also detected in oral fluid, tonsils, salivary glands, oral mucosa, and lungs of all seven grower pigs. However, viremia was observed in only two grower pigs. Double staining revealed that PRRSV was distributed in macrophages within and adjacent to the tonsillar crypt epithelium. In gilts, the North American type PRRSV field strain was detected 3 to 8 weeks after introducing these animals onto the farm. These results confirm previous findings that PRRSV primarily replicates in tonsils and is then shed into oral fluid. Therefore, oral fluid sampling may be effective for the surveillance of PRRSV in breeding herds.
Animals ; Female ; In Situ Hybridization/veterinary ; Lung/virology ; Male ; Palatine Tonsil/virology ; Polymerase Chain Reaction/veterinary ; Porcine Reproductive and Respiratory Syndrome/*virology ; Porcine respiratory and reproductive syndrome virus/*physiology ; Saliva/*virology ; Salivary Glands/virology ; Swine/virology ; Virus Replication/physiology

This study aims to express pig nuclear transcription factor-kappaB (NF-kappaB) p65/p50 fusion protein in E. coli Rosetta, and study its impacts on PRRSV proliferation in vitro. The p65 ORF and mature p50 encoding gene were amplified by RT-PCR, the products were cloned into the pET-21a(+) vector, then transformed into Escherichia coli Rosetta, recombinant fusion protein was expressed by IPTG induction, the expressed product was identified by SDS-PAGE and Western-Blot. The purified and re-folded p65/p50 was added to the 2% FBS DMEM, and the cytotoxicity on Marc145 was observed to select the optimum concentration. The effects of optimum concentration of p65/p50 on PRRSV proliferation activity were investigated by detecting PRRSV infection phase in the culture supernatant using real-time FQ-PCR method and drawing PRRSV one-step growth curve. The results showed the p65/p50-pET21a(+) prokaryotic expression vector were successfully constructed , recombinant p50 and p65 fusion protein was expressed abundantly in the form of inclusion body with molecular weight of 70kD, Western-Blot results showed that the rabbit anti-human p50 polyclonal serum, rabbit anti-human p65 purified antibody could bind specifically to p50 and p65 respectively. The optimum concentration of p65/p50 was 0.4 microg/mL. The real-time FQ-PCR results indicated that NF-kappaB p65/p50 could promote CPE appearance and PRRSV proliferation before CPE appeared, and suppress PRRSV proliferation after CPE appeared, and lower the virus titer levels significantly(P < 0.05). These results will provide some new insight of the pathogenic mechanism and treatment strategies of PRRS.
Animals ; Cell Line ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; NF-kappa B p50 Subunit ; genetics ; metabolism ; Porcine Reproductive and Respiratory Syndrome ; metabolism ; Porcine respiratory and reproductive syndrome virus ; genetics ; physiology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Swine ; Transcription Factor RelA ; genetics ; metabolism ; Virus Replication

To clone porcine bone marrow stromal antigen-2 (BST-2) gene, construct its recombinant eukaryotic expression plasmid and induce the expression of the fusion antiviral protein, we amplified BST-2 gene by RT-PCR from the total RNA extracted from PK15 cells. The recombinant expression plasmid pcDNA-BST-2 was constructed and then was transfected into HEK293T cells to expresse the BST-2 fusion protein. Western blot and indirect immunofluorescence assay (IFA) were performed, and the biological activity was detected. The results showed that the construction of recombinant plasmid pcDNA-BST-2 was confirmed by restriction enzyme digestion and sequencing. The expressed product had antiviral activity against Vesicular stomatitis virus (VSV), Avian influenza virus (AIV) and Porcine reproductive and respiratory syndrome virus (PRRSV). In conclusion, the research paves the way for further research on bioactivity assayand antiviral medication.
Animals ; Antigens, CD ; genetics ; immunology ; Cell Line ; Chickens ; Cloning, Molecular ; Gene Expression ; Humans ; Influenza in Birds ; immunology ; virology ; Orthomyxoviridae ; physiology ; Porcine Reproductive and Respiratory Syndrome ; immunology ; virology ; Porcine respiratory and reproductive syndrome virus ; physiology ; Swine ; Vesicular Stomatitis ; immunology ; virology ; Vesicular stomatitis Indiana virus ; physiology ; Virus Replication

The objective of this study was to investigate the function of vimentin in PRRSV infection. Vimentin gene from Marc-145 cells was amplified by RT-PCR, cloned into pET-28a vector and expressed in Escherichia coli BL21(DE3). The expressed vimentin was confirmed by Western blot and purified which was used to immunize BALB/c mice for the production of antibodies. Vimentin and antibodies were tested for blocking PRRSV infection of Marc-145 cells. The binding of vimentin to PRRSV N and GP5 proteins were tested by the ELISA. The results showed that vimentin gene was amplified successfully and expressed as identified by SDS-PAGE and Western blot. Mouse anti-vimentin antibodies were produced with the titer of 10(5). PRRSV infection of Marc-145 cells was blocked partially by vimentin while blocked completely by the antibobies. In addition, vimentin was bound N protein, but not GP5. These results provide additional information on PRRSV entry into Marc-145 cells.
Animals ; Antibodies ; immunology ; metabolism ; Cell Line ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred BALB C ; Porcine Reproductive and Respiratory Syndrome ; genetics ; metabolism ; virology ; Porcine respiratory and reproductive syndrome virus ; physiology ; Protein Binding ; physiology ; Recombinant Proteins ; genetics ; immunology ; isolation & purification ; metabolism ; Swine ; Vimentin ; genetics ; immunology ; isolation & purification ; metabolism ; Viral Proteins ; metabolism

A porcine reproductive and respiratory syndrome virus (PRRSV) was obtained from clinic samples. Genes 5 and 6 encoding for the viral glycoprotein 5 and a membrane protein of the PRRSV designated as HH08 were amplified by reverse transcription-PCR. These sequences were compared with reference sequences derived from different geographical locations. The results indicated that the virus belongs to the North American type rather than European. Comparative analyses of the genetic diversity between the PRRSV isolate HH08 and other Chinese as well as foreign reference strains of PRRSV were discussed based on the sequence comparison and the topology of phylogenetic trees constructed in this study.
Animals ; Base Sequence ; China/epidemiology ; Gene Expression Regulation, Viral/physiology ; Genetic Variation ; Molecular Sequence Data ; *Phylogeny ; Porcine Reproductive and Respiratory Syndrome/epidemiology/virology ; Porcine respiratory and reproductive syndrome virus/*genetics/*metabolism ; Sequence Alignment ; Swine ; Viral Envelope Proteins/genetics/*metabolism ; Viral Matrix Proteins/genetics/*metabolism

For constructing pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6, the ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified by RT-PCR, and transiently transfected into 293T cells by calciumphosphate co-precipitation. After 48 h, 293T cells were collected and surveyed by flow cytometry examination. The result indicated that the expression level of the E protein that mediated by the M protein was higher than that of the E protein expressed independently. Then pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6 were respectivly co-transfected into 293T cells with pHIT60 (include MuLV structural genes,namely gag and pol) and pHIT111 (retroviral genome, containing LacZ as a reporter). The supernatants were harvested 48 h post-transfection,and the analysis of the characteristic of the pseudotyping virions was performed by Western blot and infection test. The result indicated that the E proteins were expressed on the virions, and incorporated into the retroviral virions. Infection test were performed on Marc-145 and PAM, all the cells infected were Lac Z positive. These results indicated the pseudotype virions of MuLV-E and MuLV-E/M were infectious, and higher infectivity was achieved by MuLV-E/M.
Flow Cytometry ; Leukemia Virus, Murine ; genetics ; Plasmids ; Porcine respiratory and reproductive syndrome virus ; genetics ; Viral Envelope Proteins ; genetics ; physiology ; Viral Matrix Proteins ; genetics ; physiology ; Virion ; genetics

Animals ; Genome, Viral ; Porcine respiratory and reproductive syndrome virus ; genetics ; pathogenicity ; Swine ; Viral Nonstructural Proteins ; genetics ; physiology ; Viral Structural Proteins ; genetics ; physiology ; Virulence ; genetics