Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are very two important pathogens that have coursed huge economic losses in swine production in worldwide. In this study,a vector pCMV-TJM containing the full-length cDNA clone of PRRSV attenuated strain TJM-F92 was firstly constructed by PCR method. Then a gene sequence containing Afl II/Mlu I e restriction enzyme sites and a transcription regulatory sequence for ORF6 (TRS6) was inserted be- tween ORF7 and 3'UTR, yielding a expression vector pCMV-TJM-TRS. Subsequently, a plasmid pCMV-TJM-Cap was constructed by cloning of PCV2 ORF2 gene into the unique sites Afl II /Mlu I of pCMV- TJM-TRS plasmid DNA. Then three recombinant PRRSV, rTJM, rTJM/TRS and rTJM/Cap, were rescued by transfection of pCMV-TJM, pCMV-TJM-TRS and pCMV-TJM-Cap into Marc-145 cells, respectively,and confirmed by the genome sequence, restriction enzyme digestion, Western Blot and IFA. They all had the molecular markers which was different from the parent virus. The growth characteristics of the rescued viruses were similar to that of parent virus. rTJM/Cap could also express efficiently PCV2 Cap protein in Marc-145 cells. At passage 8, it still had PCV2 ORF2 gene which examined by RT-PCR. It indicated that the full-length cDNA clone of PRRSV attenuated strain TJM-F92 and recombinant PRRSV rTJM/Cap expressing PCV2 Cap protein were successfully constructed. It made an important foundation for studying on the pathogenic mechanisms of PRRSV and PRRSV-PCV2 vaccine in the future.
Animals ; Capsid Proteins ; genetics ; immunology ; Cell Line ; Circoviridae Infections ; veterinary ; virology ; Circovirus ; classification ; genetics ; metabolism ; Gene Expression ; Open Reading Frames ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; metabolism ; Recombination, Genetic ; Swine ; Swine Diseases ; virology ; Viral Vaccines ; genetics ; immunology

Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-gamma in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-gamma secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future.
Administration, Oral ; Animals ; Antibodies, Viral/*immunology ; B-Lymphocytes/immunology/virology ; Enzyme-Linked Immunosorbent Assay ; *Immunity, Cellular ; *Immunity, Mucosal ; Injections, Subcutaneous ; Kluyveromyces/genetics ; Mice ; Mice, Inbred BALB C ; Porcine respiratory and reproductive syndrome virus/*immunology ; Recombinant Proteins/genetics/immunology ; T-Lymphocytes/immunology/virology ; Viral Envelope Proteins/*genetics/*immunology ; Viral Vaccines/administration & dosage/*pharmacology

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).
Animals ; Cell Line ; Escherichia coli/genetics ; Haplorhini ; Immunity, Cellular ; Interleukin-2/genetics/*metabolism ; Interleukin-4/genetics/*metabolism ; Neutralization Tests/veterinary ; Plasmids ; Porcine Reproductive and Respiratory Syndrome/*prevention & control ; Porcine respiratory and reproductive syndrome virus/*immunology ; Recombinant Proteins/genetics/metabolism ; Swine ; Vaccines, DNA/immunology ; Viral Envelope Proteins/*genetics/metabolism ; Viral Vaccines/*immunology

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.
Animals ; Genotype ; Porcine respiratory and reproductive syndrome virus ; classification ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Swine ; Viral Nonstructural Proteins ; biosynthesis ; immunology

Establishment of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) with co-expression E2 Epitope of Classical Swine Fever virus (CSFV) is a crucial step to develop a genetic engineered vaccine against PRRSV and CSFV. Reverse genetic manipulation could be adopted as a com monly used technique. In this study, we focus on using nonessential regions of NSP2 (aa480-532 and aa508-532) as viral vector to express E2 Epitope of CSFV. A neutralizing epitope of classical swine fever virus (CSFV) E2 protein was inserted into the two nonessential region of nsp2 by the method of mutant PCR, basing on the infectious clone of HuN4-F112 vaccine strain. The co-expressed full-length cDNA clones (psk-HuN4-F112-delta508-532 + E2 and psk-HuN4-F112-delta480-532 + E2) were assembled by cloning and splice of the gene fragments. The completely assembled full-length cDNA clones were confirmed by sequence and Swa I enzyme digestion. Capped RNAs were transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into BHK-21 cells by liposome to acquire the rescued virus. The rescued recombinant viruses were passaged on MARC-145 cells. The successfully rescued viruses were tested by RT-PCR, digestion, and genome sequence. The results showed that these rescued viruses could be distinguished from the parental virus (HuN4-F112) with the mutant genetic marker (Mlu I enzyme site of virual genome at 14 667nt was created by synonymous mutation) and the inserted nsp2 gene region. The results of IFA showed that the inserted E2 epitope could be expressed by the recombinant viruses and the E2 epitope gene was stable during the viral serial passage. The results of plaque assay and viral growth curve showed that the recovery viruses possessed similar characterses of viral growth to those of the parental virus. In summary, the full-length infectious cDNA clones containing the marker gene were constructed and the marker recombinant viruses were rescued. The results suggested that these stable infectious clones could be used as an important tool for development of novel vaccine against PRRSV.
Base Sequence ; Cysteine Endopeptidases ; genetics ; Epitopes ; genetics ; Molecular Sequence Data ; Porcine respiratory and reproductive syndrome virus ; genetics ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; immunology

Using mutation PCR, we cloned the target gene containing 421-480nt (141-160aa) and 598-639nt (200-213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508-532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by transfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50 = -log10(-6.75)/0.1 mL) was similar to its direct parental virus rHuN4-F112-delta508-532, but higher than rHuN4-F112.
Animals ; Antigens, Viral ; immunology ; Base Sequence ; Capsid Proteins ; immunology ; Cell Line ; Cysteine Endopeptidases ; genetics ; Epitopes ; genetics ; Foot-and-Mouth Disease ; immunology ; prevention & control ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Molecular Sequence Data ; Mutation ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, Attenuated ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, causing economically significant impacts on the swine industry worldwide. Unfortunately, the traditional control strategies and conventional vaccines fail to provide sustainable disease control, in particular against genetically diverse strains, as they suffer from both antigenic heterogeneity and various immune evasion strategies of PRRSV. In this paper, latest research progress in immunology and immune evasion of PRRSVis summarized to provide a referenc for PRSSV prevention and control as well as the design of new vaccines.
Animals ; Immune Evasion ; Porcine Reproductive and Respiratory Syndrome ; immunology ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Swine ; Viral Proteins ; genetics ; immunology

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

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.
Animals ; Antibodies, Viral/analysis/immunology ; Blotting, Western/veterinary ; Cell Line ; Cricetinae ; Female ; Genotype ; Nucleocapsid Proteins/genetics/*immunology ; Porcine Reproductive and Respiratory Syndrome/diagnosis/*immunology ; Porcine respiratory and reproductive syndrome virus/genetics/*immunology ; Swine ; Transfection/veterinary