Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5' and 3' cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5'-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3'-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5' cap structure, 3' poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.
Arteriviridae ; Arterivirus ; DNA, Complementary ; Genome ; Humans ; Mental Competency ; Nucleotides ; Polyadenylation ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; RNA* ; Swine

Porcine reproductive and respiratory syndrome virus (PRRSV) is a small enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. It causes the porcine reproductive and respiratory syndrome in swine. The virus has 7 structural proteins: Of the seven, the N protein is the nucleocapsid that comprises a core of the virus particle. We have expressed the N protein of PRRSV PL97-1/LP1 strain using a heterologous gene expression vector derived from Sindbis virus, called pSinrep5. Immunofluorescence analysis showed that the N proteins were mainly found in the cytoplasm as well as in the nucleus of BHK-21 cells transfected with pSinrep5-N-derived RNA. Moreover, expression of the N protein did not change the incompetence of RNA replication of Mutant/nt14900 that lacks a 3' cis-acting replication element and the efficiency of RNA replication of Mutant/nt14800 that has a low level of RNA replication. Overall, our findings are consistent with previous results and help to understand a role of the N protein in PRRSV biology.
Arteriviridae ; Cytoplasm ; Fluorescent Antibody Technique ; Gene Expression ; Humans ; Nucleocapsid ; Porcine Reproductive and Respiratory Syndrome ; Porcine respiratory and reproductive syndrome virus ; Proteins ; RNA ; RNA Viruses ; Sindbis Virus ; Sprains and Strains ; Swine ; Virion ; Viruses

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important animal pathogens in swine industry worldwide. In this study, we isolated the large-plaque forming variant virus designated PL97-1/LP1 from the parental strain PL97-1, the first Korean strain of PRRSV, isolated from the serum of an infected pig in 1997. We found that the 15411-nucleotide genome of PL97-1/LP1 consisted of a 189-nucleotide 5' untranslated region (UTR), a 15071-nucleotide protein-coding region, and a 151-nucleotide 3'UTR, followed by a poly (A) tail of about 50~60 nucleotides in size. The 5'-end of PL97-1/LP1 began with ATGACGTAT. Comparison of the PL97-1/LP1 genome with the 11 fully sequenced PRRSV genomes currently available revealed sequence similarity from 99.6~99.7% (the North American VR-2332 and two VR-2332-derived vaccine strains MLV RespPRRS/Repro and RespPRRS MLV) to 62.0% (the Dutch Lelystad strain). Phylogenetc analysis revealed that PL97-1/LP1 is most closely related to the North American genotype VR-2332, two VR-2332-derived vaccine strains, and Chinese BJ-4. It is distantly related to the European genotype Lelystad. The entire nucleotide sequence of PL97-1/LP1 was identical to that of the parental virus PL97-1 except for three silent nucleotide substitutions, one in ORF1a (U4230C), one in ORF1b (C10977U), and one in ORF5 (U13976A). This nucleotide sequence has been submitted to the GenBank database under the accession number AY612613.
3' Untranslated Regions ; 5' Untranslated Regions ; Animals ; Arterivirus ; Asian Continental Ancestry Group ; Base Sequence* ; Databases, Nucleic Acid ; Genome ; Genotype ; Humans ; Nucleotides ; Parents ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; RNA* ; Swine

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic variation. In this study, we characterized the genetic variation and evolutionary relationships among circulating PRRSV strains in southern China. We analyzed 29 NSP2 strains and 150 ORF5 strains from clinical samples collected in southern China during 2007–2014. The alignment results showed that the nucleotide identity similarities of the two genes among these strains were 80.5%–99.7% and 80.9%–100%, respectively. Phylogenetic analysis based on the NSP2 gene showed that highly pathogenic (HP)-PRRSV was still the dominant virus in southern China from 2013 to 2014. Compared with reference strains CH-1a and VR-2332, the field strain 131101-GD-SHC, which shared high homology with JXA1-P170, had a novel 12 amino acid deletion at position 499–510. Phylogenetic analysis based on the ORF5 gene showed that HP-PRRSV, VR2332-like strains, and QYYZ-like strains were simultaneously circulating in southern China from 2007 to 2014, suggesting that, in recent years, the type 2 PRRSV was more diverse in southern China. In conclusion, mutations in the decoy epitope and primary neutralizing epitope could be markers of viral evolution and used to study evolutionary relationships among PRRSV strains in China.
China ; Genetic Variation* ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus*

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

PURPOSE: Porcine reproductive and respiratory syndrome virus (PRRSV) leads to major economic losses in the swine industry. Vaccination is the most effective method to control the disease by PRRSV. MATERIALS AND METHODS: In this study, the efficacy of a glycoprotein (GP) 5-modified inactivated vaccine was investigated in pigs. The study was performed in three farms: farm A, which was porcine reproductive and respiratory syndrome (PRRS)-negative, farm B (PRRS-active), which showed clinical signs of PRRS but had not used vaccines, and farm C (PRRS-stable), which had a history of endemic PRRS over the past years, but showed no more clinical signs after periodic administration of modified live virus vaccine. RESULTS: The inactivated vaccine induced great enhancement in serum neutralizing antibody titer, which was sufficient to protect pigs from further infections of PRRSV in a farm where pre-existing virus was circulating. CONCLUSION: These results indicated that vaccination with the inactivated vaccine composed of viruses possessing deglycosylated GP5 would provide enhanced protection to pigs from farms suffering from endemic PRRSV.
Antibodies, Neutralizing ; Glycoproteins ; Neutralization Tests ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; Swine* ; Vaccination ; Vaccines ; Vaccines, Inactivated

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases worldwide. In the present study, a new virulent strain of PRRS virus (PRRSV), GDsg, was isolated in Guangdong province, China, and caused high fever, high morbidity, and high mortality in sows and piglets. The genome of this new strain was 15,413 nucleotides (nt) long, and comparative analysis revealed that GDsg shared 82.4% to 94% identity with type 2 PRRSV strains, but only 61.5% identity with type 1 PRRSV Lelystad virus strain. Phylogenetic analysis indicated that type 2 PRRSV isolates include five subgenotypes (I, II, III, IV, and V), which are represented by NADC30, VR-2332, GM2, CH-1a, and HuN4, respectively. Moreover, GDsg belongs to a newly emerging type 2 PRRSV subgenotype III. More interestingly, the newly isolated GDsg strain has multiple discontinuous nt deletions, 131 (19 + 18 + 94) at position 1404–1540 and a 107 nt insertion in the NSP2 region. Most importantly, the GDsg strain was identified as a virus recombined between low pathogenic field strain QYYZ and vaccine strain JXA1-P80. In conclusion, a new independent subgenotype and recombinant PRRSV strain has emerged in China and could be a new threat to the swine industry of China.
China ; Fever ; Genome ; Mortality ; Nucleotides ; Porcine Reproductive and Respiratory Syndrome ; Porcine respiratory and reproductive syndrome virus ; Swine ; Swine Diseases

Two strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in 2006 and 2016 and designated as FZ06A and FZ16A, respectively. Inoculation experiments showed that FZ06A caused 100% morbidity and 60% mortality, while FZ16A caused 100% morbidity without death. By using genomic sequence and phylogenetic analyses, close relationships between a Chinese highly pathogenic PRRSV strain and the FZ06A and FZ16A strains were observed. Based on the achieved results, multiple genomic variations in Nsp2, a unique N-glycosylation site (N³³→K³³), and a K151 amino acid (AA) substitution for virulence in the GP5 of FZ16A were detected; except the 30 AA deletion in the Nsp2-coding region. Inoculation experiments were conducted and weaker virulence of FZ16A than FZ06A was observed. Based on our results, a 30 AA deletion in the Nsp2-coding region is an unreliable genomic indicator of a high virulence PRRSV strain. The Nsp2 and GP5 differences, in addition to the virulence difference between these two highly pathogenic PRRSV strains, have the potential to be used to establish a basis for further study of PRRSV virulence determinants and to provide data useful in the development of vaccines against this economically devastating disease.
Asian Continental Ancestry Group ; China ; Genomics ; Humans ; Mortality ; Phylogeny ; Porcine Reproductive and Respiratory Syndrome ; Porcine respiratory and reproductive syndrome virus ; Vaccines ; Virulence

Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most common and economically significant infectious agents in the swine industry worldwide. The virus causes mild to severe reproductive failure in sows and gilts and respiratory problems in piglets. In this study, the author expressed the full-length PRRSV Nsp1a protein as a Glutathion-S-Transferase (GST)-Nsp1a fusion protein in E. coli BL-21. The full-length Nsp1a coding region of the Korean PRRSV isolate PL97-1 was amplified by RT-PCR from the genomic RNA and the cDNA amplicons were cloned into the pGEX4T-1 E. coli expression vector. The recombinant GST-Nsp1a fusion protein was used for immunization in rabbits. After a 5-shot immunization schedule, the PRRSV Nsp1a-specific antiserum was obtained. Using the PRRSV-infected MARC-145 cell lysates, immunoblotting analyses showed that these antisera specifically reacted with ~18 kDa of the PRRSV Nsp1a protein. The rabbit antisera raised against the PRRSV Nsp1a recombinant protein will provide a useful reagent to investigate the role of this protein in PRRSV replication and to diagnose infection.
Clinical Coding ; Clone Cells ; DNA, Complementary ; Immune Sera ; Immunization ; Immunization Schedule ; Immunoblotting ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; Rabbits ; RNA ; Staphylococcal Protein A ; Swine

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.
Antibodies* ; Collodion ; Colloids ; Diagnostic Tests, Routine ; Enzyme-Linked Immunosorbent Assay ; Gold Colloid ; Immunochromatography ; Membranes ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; Sensitivity and Specificity ; Staphylococcal Protein A ; Swine