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.     

Molecular characterization and recombination analysis of porcine reproductive and respiratory syndrome virus emerged in southwestern China during 2012–2016

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen causing tremendous economic losses to the swine industry. To investigate the prevalence of PRRSV of genotype 2 (North American type, NA-type) in southwestern China, the Nsp2 hypervariable region (Nsp2 HV) and ORF5 of 61 PRRS viruses collected during 2012–2016 were sequenced and analyzed. All the virus detected clustered into the JXA1-like (52/61), VR-2332-like (7/61), and NADC30-like (2/61) sub-genotypes. Five deletions in Nsp2 HV were detected in addition to the typical 30aa discontinuous deletion in HP-PRRSV, and two of these five were not reported previously. Strikingly, two PRRS virus (SCnj16 and SCcd16) isolated in 2016 contained the classic HP-PRRSV molecular marker in the Nsp2-coding region, but belonged to the NADC30-like sub-genotype on the ORF5 gene. Further recombination and phylogenetic analysis on the two complete genomic sequences revealed that they may have originated from recombination events between the NADC30 and Chinese HP-PRRSV strains. The present study suggests that the endemic PRRSVs in the region have continuously evolved and new vaccine strategies are necessary for more efficient control of the virus.     

Epidemiology and evolutionary characteristics of the porcine reproductive and respiratory syndrome virus in China between 2006 and 2010

In 2006, an emerging highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, broke out in mainland China and spread rapidly to neighboring countries. To examine the epidemiology and evolutionary characteristics of Chinese PRRSV after the 2006 outbreak, we tested 2,981 clinical samples collected from 2006 to 2010 in China, determined 153 Nsp2 sequences and 249 ORF5 sequences, and analyzed the epidemiology and genetic diversity of Chinese PRRSV. Our results showed that the percentage of PRRSV-positive specimens collected from sick pigs averaged 60.85% in the past 5 years and that the highly pathogenic PRRSV has become the dominant strain in China. Furthermore, a reemerging strain which apparently evolved from the highly pathogenic PRRSV strain in 2006 appeared to be widely prevalent in China from 2009 onwards. Sequence analyses revealed that the hypervariable region of Nsp2 in most of the isolates contained a discontinuous deletion equivalent to 30 amino acids, along with other types of deletions. Extensive amino acid substitutions in the GP5 sequence translated from ORF5 were found, particularly in the potential neutralization epitope and the N-glycosylation sites. Our results suggest that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines. Information from this study will help in understanding the evolutionary characteristics of Chinese PRRSV and assist ongoing efforts to develop and use PRRSV vaccines in the future.     

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.

     

Molecular epidemiology of PRRSV from China’s Guangxi Province between 2007 and 2009

Porcine reproductive and respiratory syndrome (PRRS) is considered to be one of the most important infectious diseases affecting livestock. This study used gene sequence analysis of ORF5 and Nsp2 to determine the molecular epidemiology of PRRSV in different parts of the Guangxi province of China. These genes were selected due to their extensive variation within the genome. Out of 189 samples from animals suspected to have PRRS, 145 were PRRSV RNA positive. ORF5 and Nsp2 gene sequence analysis of 31 of these samples showed that all of the Guangxi isolates were of type 2. A phylogenetic tree analysis based on ORF5 showed that the Guangxi isolates were divided into two groups. Most of these were closely related to highly pathogenic strains, showing a 30 amino acid deletion at positions 481 and 533–561 of Nsp2, but an additional unique isolate (GXNN06) possessed a further four amino acid deletion at positions 485–488 of Nsp2.     

Complete genome characterization of a East European Type 1 subtype 3 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of major economic importance that causes reproductive and respiratory problems in pigs. PRRSV strains are divided into European (Type 1) and North-American (Type 2) genotypes. Within the European PRRSV genotype, three subtypes have been delineated. Full genome sequences for North American and European subtype 1 strains have been described. Here, the first complete genomic characterization of a European subtype 3 strain (Lena) is described. Amplification of Orf1a and Orf1b fragments was achieved using a set of degenerate oligonucleotides. Using RT-PCR with Lena-specific primers, the full length sequence (15001 nt) was obtained. Alignment of Lena with European subtype 1 reference strain Lelystad showed variation over the entire length (84% identity/89% similarity at amino acid level) with the most variation in Orf1a (Nsp2/NSP2) with a deletion of 29 amino acids. Phylogenetic relationships using different Orfs supported Lena’s genetic distinction from European subtype 1 strains. The availability of the European subtype 3 PRRSV full genome may be important for the understanding of PRRSV evolution and the more pronounced pathogenic nature of Lena.     

Genomic analysis of two Chinese strains of porcine reproductive and respiratory syndrome viruses with different virulence

Two strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated, designated GDQJ and GDBY1. Experimental inoculation showed that GDBY1, caused 100% morbidity and 67% mortality, while GDQJ, caused 100% morbidity but no death. Full-length genomes were sequenced. Homologic and phylogenetic analyses indicated that these two strains were closely related to Chinese highly pathogenic PRRSV strains. Surprisingly, identical 30 amino acids (aa) deletion in the NSP2-coding region, a presumed high virulence marker, was present in low virulent strain GDQJ. Further comprehensive analysis of GDQJ genome in comparison with Chinese highly pathogenic PRRSV strains revealed multiple genomic variations, distributing in 5′ UTR, NSP1b, NSP2, NSP3, NSP5, NSP7, NSP9, NSP10, GP5, and N regions. Data present in this article confirm that the 30 aa deletion in the NSP2-coding region alone is not a reliable genomic indicator for the high virulence of PRRSV strains emerged in China. The genomic variations of GDQJ strain provided the basis for further studies of virulence determinants for PRRSVs.     

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.     

Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype

Summary.  Although live-attenuated vaccines have been used for some time to control clinical symptoms of the porcine reproductive and respiratory syndrome (PRRS), the molecular bases for the attenuated phenotype remain unclear. We had previously determined the genomic sequence of the pathogenic PRRSV 16244B. Limited comparisons of the structural protein coding sequence of an attenuated vaccine strain have shown 98% homology to the pathogenic 16244B. Here we have confirmed the attenuated phenotype and determined the genomic sequence of that attenuated PRRSV vaccine and compared it to its parental VR-2332 and the 16244B strains. The attenuated vaccine sequence was colinear with that of the strain 16244B sequence containing no gaps and 212 substitutions over 15,374 determined nucleotide sequence. We identified nine amino acid changes distributed in Nsp1β, Nsp2, Nsp10, ORF2, ORF3, ORF5 and ORF6. These changes may provide the molecular bases for the observed attenuated phenotype. Received August 28, 1999 Accepted December 16, 1999     

Genetic variation and pathogenicity of highly virulent porcine reproductive and respiratory syndrome virus emerging in China

A highly pathogenic swine disease designated as ‘porcine high fever disease (PHFD)’ appeared recently in China. Porcine reproductive and respiratory syndrome virus (PRRSV) was identified as an agent associated with PHFD, and two discontiguous sequence deletions were identified as a genetic marker in the Nsp2 region of the viral genome. To examine PHFD in Shandong province, a total of 10 PRRSV isolates were recovered from pig herds that had never been vaccinated for PRRS. Sequence analysis of open reading frame 5 (ORF5) showed that the level of identity among the 10 isolates ranged between 88.2 and 99.2%. For the non-structural protein 2 (Nsp2) gene, three isolates shared high sequence identity with VR-2332, the prototype virus of the North American genotype, while the remaining seven isolates exhibited two discontiguous sequence deletions that were identical to those of PHFD: a one-amino-acid (phenylalanine) deletion at position 482 and a 29-amino-acid deletion at positions 533–561 of Nsp2. Experimental infection of pigs with SD-JN, which was one of the seven isolates containing such deletions, resulted in severe clinical symptoms characterized by red discoloration on the body and hemorrhages in the lungs, kidneys, and inguinal lymph nodes, accompanied by higher mortality and longer duration of viremia. These symptoms were similar to those of PHFD observed in the field. Our results show that VR2332-like PRRSV coexists with PHFD-associated atypical PRRSV in pig herds in the Shandong area, and different PRRSV isolates differ greatly in their pathogenesis and virulence in pigs.     

Recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the Nsp2-encoding region

Non-structural protein 2 (Nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the most variable region and postulated to play an important role in cell and tissue tropism of PRRSV. To investigate the role of Nsp2 in the viability and growth of PRRSV in cells in vitro, two cDNA clones were constructed containing a deletion of 63 consecutive nucleotides (pWSK-DCBAd63) or 117 nucleotides (pWSK-DCBAd117) within the Nsp2-encoding region of PRRSV (BJ-4). The clone pWSK-DCBAd63 was infectious and produced viable recombinant virus, whereas clone pWSK-DCBAd117 could not be rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 cells and was stably propagated during sequential in vitro cell passages, like the virus recovered from the full-length cDNA clone of PRRSV BJ-4. In comparison to the parental virus (BJ-4) and the virus recovered from the full-length cDNA clone of the BJ-4 strain, the rescued virus from pWSK-DCBAd63 exhibited enhanced growth kinetics, reaching the peak progeny virus titer by 48 h postinfection. These observations suggest that the Nsp2-encoding region is necessary for productive virus infection, and partial deletion does not influence the viability and propagation of PRRSV in cell culture, which may provide a way to insert a foreign gene into the viral genome as a marker for differentiation. Hanchun Yang and Kyoung-Jin Yoon are equal contribution corresponding authors.     

Monoclonal antibody and porcine antisera recognized B-cell epitopes of Nsp2 protein of a Chinese strain of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for swine industry. The non-structural protein 2 (Nsp2) is considered to be one of the immunogenic proteins of PRRSV. In this study, the B-cell epitopes of the Nsp2 protein of a North American type Chinese strain PRRSV BJ-4 were identified on a prokaryotic expressed Nsp2 fragment (73-567aa). A total of six monoclonal antibodies (mAbs) recognizing different epitopes on the expressed protein were prepared. All six mAbs exhibited immunoreactivity with the denatured Nsp2 protein in Western blotting and produced strong perinuclear staining in PRRSV infected MARC-145 cells in an immunofluorescence assay. Pepscan analysis revealed six distinct linear epitopes for the six mAbs, respectively, and of which four were identified to be novel linear Nsp2 B-cell epitopes: T(73)LPERVRPPDDWAT(86), D(385)ELKDQMEED(394), P(452)VPAPRRKVGSDCGS(466), and P(467)VSLGGDVPNS(477). All of the six mAb specific peptides could be recognized by porcine PRRSV antiserum, indicating that the epitopes involving these synthetic peptides were immunogenic and immunodominant during PRRSV infection in pigs. Our results provided valuable information for developing novel PRRSV vaccines using the Nsp2 epitopes as potential serological markers.     

Identification of new defective interfering RNA species associated with porcine reproductive and respiratory syndrome virus infection

In the present study, seven new defective interfering (DI) RNA species of porcine reproductive and respiratory syndrome virus (PRRSV) were identified. RT-PCR, Northern blot and sequence analyses indicated that these DI RNA specie have deletions of 8513-9176 nucleotides located between Nsp1/Nsp2 and Nsp10. Compared with the previous DI RNAs of PRRSV reported, they have three distinct characteristics: much smaller deletion sizes; different nucleotide repeats (2-12nt) used in the junction sites and in-frame deletions. The results further suggested that the similarity-assisted RNA recombination may be the main cause of generation of DI RNAs in PRRSV and probably in other arteriviruses.     

Concurrent porcine circovirus type 2a (PCV2a) or PCV2b infection increases the rate of amino acid mutations of porcine reproductive and respiratory syndrome virus (PRRSV) during serial passages in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic and antigenic variability. The purpose of this study was to determine if porcine circovirus type 2 (PCV2) infection increases genetic variability of PRRSV during serial passages in pigs and to determine if there is a difference in the PRRSV mutation rate between pigs concurrently infected with PCV2a or PCV2b. After 8 consecutive passages of PRRSV alone (group 1), PRRSV with PCV2a (group 2), or PCV2b (group 3) in pigs, the sequences of PRRSV structural genes for open reading frame (ORF) 5, ORF6, ORF7 and the partial non-structural protein gene (Nsp) 2 were determined. The total number of identified amino acid mutations in ORF5, ORF6, ORF7 and Nsp2 sequences was 30 for PRRSV infection only, 63 for PRRSV/PCV2a concurrent infection, and 77 for PRRSV/PCV2b concurrent infection when compared with the original VR2385 virus used to infect the passage 1 pigs. Compared to what occurred in pigs infected with PRRSV only, the mutation rates in ORF5 and ORF6 were significantly higher for concurrent PRRSV/PCV2b infected pigs. The PRRSV/PCV2a pigs had a significantly higher mutation rate in ORF7. The results from this study indicated that, besides ORF5 and Nsp2, the PRRSV structural genes ORF6 and ORF7 were shown to mutate at various degrees when the PRRSV was passaged over time in vivo. Furthermore, a significantly higher mutation rate of PRRSV was observed when pigs were co-infected with PCV2 highlighting the importance of concurrent infections on PRRSV evolution and control.     

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.     

Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-β production by porcine reproductive and respiratory syndrome virus

Induction of type I interferon (IFN-α/β) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1α (Nsp1α) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-β production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1α suppressed the activation of nuclear factor (NF)-κB when stimulated with dsRNA or tumor necrosis factor (TNF)-α, and NF-κB suppression was RIG-I-dependent. The suppression of NF-κB activation was associated with the poor production of IFN-β during PRRSV infection. The C-terminal 14 amino acids of the Nsp1α subunit were critical in maintaining immunosuppressive activity of Nsp1α for both IFN-β and NF-κB, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1α inhibited IκB phosphorylation and as a consequence NF-κB translocation to the nucleus was blocked, leading to the inhibition of NF-κB stimulated gene expression. Our results suggest that PRRSV Nsp1α is a multifunctional nuclear protein participating in the modulation of the host IFN system.     

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].     

The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332.

GP5, the principal envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV), contains a hypervariable region within the ectodomain which is responsible for generating diversity in field isolates. The purpose of this study was to gain insight into the possible origin of this diversity by following GP5 sequence changes in pigs exposed to PRRSV strain VR-2332 in utero. A region of the PRRS virus genome containing portions of ORF4 and ORF5 was amplified directly from tissues of infected pigs from birth to 132 days of age. We observed the emergence of a new PRRSV population, identified by a single nucleotide change in the ectodomain. The Asp to Asn change at amino acid 34 was also found as a minor component in pigs that expressed the wild-type sequence. The results from this study suggest that the variability in the ectodomain of ORF5 is the result of positive or negative selection, of which the mechanism remains to be determined.     

Molecular characterization of PL97-1, the first Korean isolate of the porcine reproductive and respiratory syndrome virus

We determined the complete nucleotide and predicted amino acid sequence of the genomic RNA of PL97-1, the first Korean strain of porcine reproductive and respiratory syndrome virus (PRRSV), which was isolated from the serum of an infected pig in 1997. We found that the 15411-nucleotide genome of PL97-1 consisted of a 189-nucleotide 5' noncoding region (NCR), a 15071-nucleotide protein-coding region, and a 151-nucleotide 3'NCR, followed by a poly (A) tail. The 5'-end of PL97-1 began with 1ATG ACG TAT AGG12. Comparison of the PL97-1 genome with the 11 fully sequenced PRRSV genomes currently available revealed sequence divergence ranging from 0.3% (the VR-2332-derived vaccine MLV RespPRRS/Repro strain) to 38% (the Dutch Lelystad strain). To better understand the genetic relationships between these different strains, phylogenetic analyses were performed on the full-length PRRSV genomes. Significantly, the phylogenetic tree based on the ORF1b or ORF7 genes most closely resembled the tree based on the full-length genomes. Thus, these single genes will be the most useful in revealing the genetic relationships between the different strains relative to their geographical distribution. Extensive phylogenetic analyses using the ORF7 sequences of 111 PRRSV isolates available revealed that PL97-1 is most closely related to the North American genotype VR-2332, a VR-2332-derived vaccine strain, and Chinese BJ-4. It is distantly related to the European genotype Lelystad. This study provides the largest full-length genome phylogenetic analysis of PRRSV that has been published to date, and supports an earlier genetic grouping of the many temporally and geographically diverse PRRSV strains currently isolated.