Genetic analysis of two porcine reproductive and respiratory syndrome viruses with different virulence isolated in China

The S1 and SY0608 strains of porcine reproductive and respiratory syndrome virus (PRRSV) were individually isolated and had different pathogenicity in pigs in 1997 and 2006. In order to understand their genomic characteristics, the full-length genome of S1 and SY0608 isolates were sequenced and analyzed. The results indicated that their genome composition differed significantly and shared only 88.5% nucleotide identity with each other. The genetic variation and amino acid substitutions were not randomly distributed in the genome, and mainly focused on ORF1a, ORF3 and ORF5. The SY0608 strain, with high pathogenicity, had a 30-amino-acid deletion at amino acid positions 480 and 532-560 in comparison with the S1 strain. The alignment of amino acid sequence of Nsp1-Nsp8, GP2-GP5, M and N of S1 and SY0608 with other PRRSV isolates demonstrated that variation was mainly found in the Nsp2, GP3 and GP5 proteins. In comparison with the S1 strain, the SY0608 strain showed some potential glycosylation site mutations in GP5 at amino acid positions between 26 and 39, which might be associated with viral antigenicity. Phylogenetic analysis showed that the two strains belonged to two different branches that do not indicate differences in pathogenicity. Interestingly, the deletion strains isolated recently in China formed a new minor branch, revealing the same evolutionary trend.     

Two natural recombinant highly pathogenic porcine reproductive and respiratory syndrome viruses with different pathogenicities

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is a variant of type 2 PRRSV with high virulence. Genetic and pathogenic characteristics of HP-PRRSV vary rapidly during the evolution. In this study, we determined the complete genome of a HP-PRRSV isolate designated 10FUJ-2, which shared 98.34 % nucleotide identity with HP-PRRSV reference strain JXA1. Genomic analyses by phylogenetic tree and recombination detection program confirmed 10FUJ-2 to be a recombinant with 09JS and JXA1 as potential parental viruses. Furthermore, we identified that 10FUJ-2 has high virulence as similar as the parental viruses by animal challenge study. In addition, we found that SY0909 was also a recombination virus probably from JXA1 and NT0801, which has been reported to be low pathogenic. Recombination analysis also revealed that Glycoproteins GP2 to GP5 of HP-PRRSV might contain major virulence determinants. Identification of two natural recombinants with different virulence supports the notion that recombination is a driving force affecting HP-PRRSV pathogenicity and a common mechanism contributing to HP-PRRSV evolution.     

Recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo

Porcine reproductive and respiratory syndrome virus (PRRSV) is characteristic of genetically extensive variation. In this study, five SPF pigs were co-infected with two strains of PRRSV (JXwn06-81c and HB-1/3.9c), and 352 viruses were cloned by plaque assay from the sera of the infected pigs on days 3, 5, 7, 10, 14, 21 postinfection (pi), and the recombinant events between the two viruses were systematically investigated by sequencing the ORF5, ORF3 and Nsp2 genes of each cloned virus and using SimPlot and Genetic Algorithm for Recombination Detection (GARD) analysis. Totally, 133 recombinant viruses out of the plaque viruses were acquired from four of five infected pigs during days 7-21pi upon co-infection with JXwn06-81c and HB-1/3.9c. The intragenic recombination and intergenic fragment exchange of the ORF5, ORF3 and Nsp2 genes between the two viruses exhibited different patterns, and the recombination for ORF5 gene and Nsp2 occurred as early as on day 7pi. The recombination between the ORF5, ORF3 or Nsp2 gene resulted in the generation of chimeric GP5, GP3 or Nsp2. Of the three genes, Nsp2 gene exhibited more complicated recombination situation. Meanwhile, the putative recombination breakpoints and hotspots for the three genes were analyzed. Our findings not only provide valuable evidences for understanding that recombination is an important genetic mechanism contributing to the variation and evolution of PRRSV, but also suggest that extensive use of attenuated vaccine of PRRSV undoubtedly contributes to the increased diversity of PRRSV in field.     

Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in china

Recently, NADC30-like porcine reproductive and respiratory syndrome viruses (PRRSVs), which are genetically similar to the NADC30 strain isolated in the United States of America in 2008, have become prevalent in China. Here, a novel variant PRRSV strain named HNhx was successfully isolated on porcine alveolar macrophages from Henan province and the full-length genome sequence was determined. Phylogenetic analysis indicated that HNhx strain was classified into the NADC30-like PRRSV subgroup, in which all the strains had the unique discontinuous 131-amino acid deletion relative to that of the nonstructural protein 2 (Nsp2) of the VR2332 strain. Genetically, HNhx shared 92.9% nucleotide similarity to NADC30. Furthermore, HNhx strain contained extensive amino acid mutations in GP5. In particular, the S32H, N33D, D34N, and S36G variations resulted in that HNhx lost all the putative N-linked glycosylation sites at amino acid positions 30, 32, 33, 34, and 35. Recombination analysis revealed that HNhx was the result of recombination between the NADC30 strain and the highly pathogenic PRRSV vaccine strain circulating in China in Nsp4 (nt 5261) to Nsp9 (nt 7911). The novel genome data of HNhx will be helpful for understanding the evolution and epidemiology of PRRSV in China.     

Recombination between North American strains of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), a recently discovered arterivirus swine pathogen, was shown to undergo homologous recombination. Co-infection of MA-104 cells with two culture-adapted North American PRRSV strains resulted in recombinant viral particles containing chimeric ORF 3 and ORF 4 proteins. Nucleotide sequence analysis of cloned recombinant PCR products, encompassing 1182 bases of the 15.4 kb viral genome, revealed six independent recombination events. Recombinant products persisted in culture for at least three passages, indicating continuous formation of recombinant viruses, growth of recombinant viruses in competition with parental viruses, or both. The frequency of recombination was estimated from <2% up to 10% in the 1182 b fragment analyzed, which is similar to recombination frequencies observed in coronaviruses. An apparent example of natural ORF 5 recombination between naturally occurring wild type viruses was also found, indicating that recombination is likely an important genetic mechanism contributing to PRRSV evolution.     

Genomic analysis of two porcine encephalomyocarditis virus strains isolated in China

Summary. Two strains of encephalomyocarditis virus (EMCV), designated BJC3 and HB1, were isolated from an aborted fetus and the heart tissue of a dead piglet that had pericardial fluid, respectively. The complete genomic sequences of the two viruses were determined and analyzed. The size of the genomes of BJC3 and HB1 were 7746 and 7735 nucleotides, respectively, including poly(A) tails. Comparative analysis with the genomic sequences of other EMCV strains showed that BJC3 and HB1 shared higher identity (92.5–99.6%) with BEL-2887A/91, EMCV-R and PV21, but lower identity (83.3–84.6%) with EMC-B, EMC-D and D variants, and only 81.0% with Mengo virus. Two amino acid mutations in the leader protein of the two viruses and one amino acid substitution in VP1 of BJC3 were found in comparison to other EMCV strains Phylogenetic analysis based on the amino acid sequences of the entire ORF revealed that the two Chinese isolates BJC3 and HB1 clustered together with the strains BEL-2887/91, EMCV-R and PV21, which belong to the same genetic subgroup as EMCV-30. Our results provide genomic information for EMCV isolated in China.     

Typing of porcine reproductive and respiratory syndrome viruses by a multiplex PCR assay.

A rapid multiplex PCR assay was developed to distinguish between North American and European genotypes of porcine reproductive and respiratory syndrome (PRRS) virus after a portion of the polymerase gene (open reading frame 1b) was sequenced for two North American PRRS virus strains. DNA products with unique sizes characteristic of each genotype were obtained.     

Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains

Two full-length porcine reproductive and respiratory syndrome virus (PRRSV) genomes, strain VR-2332 and its cell culture passaged descendent RespPRRS vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. Of the 44 nucleotide changes, 13 resulted in conservative changes and 18 produced non-conservative changes. The results suggest that key amino acids in ORF1 may contribute to the phenotype of RespPRRS, which includes increased growth rate on MA-104 cells and decreased virulence in swine. The results provide a genetic basis for future manipulation of a PRRSV reverse genetics system.     

Genetic diversity and evolutionary characterization of Chinese porcine reproductive and respiratory syndrome viruses based on NSP2 and ORF5

To more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the Nsp2 and ORF5 gene sequences of 35 representative PRRSV isolates from 2008 to 2012. Sequence analysis revealed that the Nsp2 and ORF5 genes have undergone genetic variation. Furthermore, the isolate FJLYDX04 contains five insertions at positions 599 to 603 and is the first isolate from China reported to have an insertion in Nsp2. Our results suggest that the highly pathogenic PRRSV has become the dominant strain in China and that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines.     

Mutagenesis analysis of porcine reproductive and respiratory syndrome virus nonstructural protein 7

Nonstructural protein 7 (nsp7), which is flanked by nsp6 and nsp8, is one of the most conserved nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein (nsp)-specific antibodies are produced in high titers in response to virus replication, especially against nsp1a, nsp1b, nsp2, and nsp7. However, many regional aspects of nsp7 are still veiled, such as its impact on viral replication and virulence or the immunological mechanism between virus and host. Based on the structure of the predicted nsp7 domain, we have constructed a series of large mutations and deletions. We ultimately demonstrated all mutations (nsp7, nsp7α/nspβ) and the majority of substitutions of nsp7 affected the PRRSV replicative cycle in some ways and were fatal for viral recovery, which indicates that these are significant to structure or function of the nsp7. What’s more, the mutant vOKXH-nsp7 (F40A) indeed caused some of the variation compared with the parental virus vOKXH-GD, which shortens the amount of time needed to reach its highest viral titer, and decreases the concentration of the highest viral titer, obstructing viral mRNA and protein synthesis. Consequently, these valuable results possibly provide the first direct evidence that the nsp7 is really a critical protein domain for the RNA synthesis and the translation of viral protein of PRRSV.     

Evolutionary and recombination analysis of porcine reproductive and respiratory syndrome isolates in China

Seven strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated from 2014 to 2017 in the Shandong province of China and their genomes were sequenced and analyzed. Results showed that all seven of the isolates belong to PRRSV 2, and are clustered into four lineages (lineage 1, 3, 5 and 8) based on comparisons of the ORF5 gene. Comparative analysis of genomes and specific amino acid sites revealed that three of the strains (SDwh1402, SDwh1602 and SDwh1701) have evolved directly from modified live virus (MLV) JXA1-P80, TJM-F92 and IngelvacPRRS. Further recombination analysis revealed that two of the strains (SDyt1401 and SDwh1601) were the result of a recombination event between MLVs JXA1-P80 and NADC30 while two other strains (SDwh1403 and SDqd1501) were the result of recombination between MLVs IngelvacPRRS and NADC30 and HP-PRRSV and QYYZ, respectively. Our results add to the data on MLV evolution and PRRSV recombination and provide a better understanding of the epidemiology of PRRSV in China.

     

Different biological characteristics of wild-type porcine reproductive and respiratory syndrome viruses and vaccine viruses and identification of the corresponding genetic determinants

Two attenuated vaccines, Ingelvac PRRS MLV and Ingelvac PRRS ATP, derived from VR2332 and JA142, respectively, have been used to control porcine reproductive and respiratory syndrome (PRRS) virus. However, there have been several field reports concerning the reversion of the vaccine virus to virulence. Furthermore, viruses genetically indistinguishable from the vaccines and wild-type parental viruses have been detected in clinical PRRS cases, raising the need for a better differential tool. As the vaccine viruses replicated better and produced bigger plaques in MARC-145 cells than did the wild-type parental strains, the following study was conducted to determine if the growth difference in MARC-145 cells can be utilized to differentiate a vaccine-like virus (VLV) from a wild-type virus and to identify genetic markers corresponding to such phenotype of the vaccine viruses. The relatedness of 83 field isolates collected between 1996 and 2005 to VR2332 and JA142 was classified genetically and antigenically. Thirteen of 25 VR2332-related viruses and 9 of 10 JA142-related viruses were determined as VLVs, since those viruses produced plaques similar to those by the vaccine viruses. Four unique amino acids each were identified throughout structural genes for MLV and ATP. Among those, F(10) in open reading frame 2 (ORF2) of MLV and E(85) and Y(165) in ORF3 of ATP were stable during pig passages. When the sequences unique for MLV were incorporated into an infectious clone constructed based on VR2332, the virus growth and resultant plaque size in MARC-145 cells were increased, suggesting that these sequences can be used as genetic markers for VLVs.     

Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States

Recently, isolates of porcine reproductive and respiratory syndrome virus (PRRSV) that possess nucleotide sequences similar to European isolates have been reported in United States herds. The origin, diversity and prevalence of European-like North American PRRSV isolates in the U.S. remain unknown. Nucleotide sequence analysis of the 12 kb ORF1 of a North American isolate, SDPRRS 01-08 (01-08), showed 93.7% identity with Lelystad virus (LV), the prototypic European isolate, but only 58% identity with VR-2332, the prototypic North American isolate. Comparisons between LV and 01-08 at the peptide sequence level of the predicted non-structural proteins (Nsp) showed that Nsp9 (98.9% amino acid identity) was the most conserved and the least conserved was Nsp2 at 90.6% identity. For the purpose of comparison, GP5, the principal envelope structural protein, showed a 93.5% identity between 01-08 and LV. The most dramatic differences between the Nsp2 proteins of LV and 01-08 were a single 17 amino acid deletion between residues 734 and 750, as well as several amino acid differences. The same deletion was identified in the Nsp2 in five of seven other EuroPRRSV isolates submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory. The remaining two isolates contained small deletions, but in other regions of Nsp2. Peptide sequence diversity in the form of hypervariability and deletions in Nsp2 demonstrate that European-like PRRSV isolates in the USA represent a heterogeneous group. Furthermore, areas in Nsp2 with deletions and amino acid hypervariability localize to regions that are predicted to be immunologically important.     

Antigenic structure analysis of glycosylated protein 3 of porcine reproductive and respiratory syndrome virus

The function of the glycosylated protein 3 (GP3), a porcine reproductive and respiratory syndrome virus (PRRSV) associated protein is poorly known. In the present study, the gene encoding GP3 (ORF3), lacking the highly hydrophobic domain in the N- and C-termini was expressed as GST-fusion proteins in E. coli. Monoclonal antibodies (MAbs) against GP3 were developed and used to probe a series of GP3 peptides using ELISA. After precise analysis by sequential deletion of the terminal amino acid residues from each peptide, the minimal epitopes recognized by the MAbs were localized to W(74)CRIGHDRCGED(85) and Y(67)EPGRSLW(74). The epitope sequences were well conserved among most of the North American-type isolates, with the exception of two amino acid mutations in both epitopes in a few of these isolates. Mutational analysis revealed that these mutants were not recognized by any of the five MAbs, indicating that genetic variation could lead to altered antigenicity. Eight out of nine peptide fragments, 58-72aa, 73-87aa, 88-101aa, 102-115aa, 50-65aa, 66-81aa, 80-95aa and 94-109aa were recognized by PRRSV-positive pig serum as determined by Western blot analysis. The results herein may elucidate partially the antigenic structure of GP3 and variations of PRRSV.     

Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1beta, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66-70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324-434, 486 and 505-523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].     

Evolution of ORF5 of Spanish porcine reproductive and respiratory syndrome virus strains from 1991 to 2005

ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) were analysed to determine genetic diversity, codon usage, positive and negative selection sites and potential changes in the predicted glycoprotein 5 (GP5). A hypothetical GP5 containing all selected sites was constructed to determine its characteristics. These sequences corresponded to isolates obtained 10 years apart (1991-1995, 18 strains) and a second set (n = 46) from 2000 to 2005. Similarity to Lelystad virus (LV) decreased from 95.5% in 1991-1995 to 89.5% in 2000-2005. Three highly variable regions were found in ORF5. Codon usage was different in both sets for leucine, glutamine, serine and proline. Thus, 2000-2005 sequences used codons more similar to those present in highly expressed pig genes compared to the 1991-1995 set. Twenty four sites of positive selection and 20 sites of negative selection were found in GP5, most of them in transmembrane regions. Additional glycosylation in N37 of GP5 was common in 2000-2005 but some sequences lack a glycosylation site in N46. The hypothetical GP5 was only 88.1% similar to LV and was less hydrophobic. Taking together these results suggest that PRRSV is still adapting to pig cells.     

Genetic variation of the prevailing porcine respiratory and reproductive syndrome viruses occurring on a pig farm upon vaccination

Summary. Recurrence of porcine respiratory and reproductive syndrome (PRRS) was observed on a pig farm after introducing two PRRS live virus vaccines to combat preceding outbreaks. The phylogenetic analysis of the nucleotide sequence encoding the GP5 envelope glycoprotein and the nucleocapsid protein coding sequences (ORF5 and ORF7, respectively) showed a close genetic relationship between the new outbreak-related and one of the vaccine viruses, while the prevailing PRRS virus genetic variants disappeared from the farm. These findings, supported by the epidemiological data, indicate that the new variant PRRS viruses might originate from a vaccine virus and demonstrate the limited efficacy of modified live vaccines against heterologous PRRS virus strains.     

Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.