Emergence of mosaic recombinant strains potentially associated with vaccine JXA1-R and predominant circulating strains of porcine reproductive and respiratory syndrome virus in different provinces of China
© The Author(s). 2017
Received: 17 November 2016
Accepted: 23 March 2017
Published: 4 April 2017
Porcine reproductive and respiratory syndrome virus (PRRSV) has caused several outbreaks in China since 2006. However, the genetic diversity of PRRSV in China has greatly increased by rapid evolution or recombination events. Modified live-attenuated vaccines are widely used to control this disease worldwide. Although the risk and inefficacy of the vaccine has been reported, the genetic diversity between epidemic field strains and vaccine strains in China has not been completely elucidated.
A total of 293 clinical samples were collected from 72 pig farms in 16 provinces of China in 2015 for PRRSV detection. A total of 28 infected samples collected from 24 pig farms in nine provinces were further selected for immunohistochemical analysis and whole genome sequencing of PRRSV. Phylogenetic analysis and recombination screening were performed with the full genome sequences of the 28 strains and other 623 reference sequences of PRRSV.
Of 293 clinical samples, 117 (39.93%) were positive for PRRSV by RT-PCR. Phylogenetic results showed that the 28 strains were nested into sublineage 10.5 (classic highly pathogenic [HP]-PRRSV), sublineage 10.6 (HP-PRRSV-like strains and related recombinants), sublineage 10.7 (potential vaccine JXA1-R-like strains), and lineage 9 (NADC30-like strains and recombinants of NADC30-like strains), respectively, suggesting that multiple subgenotypes of PRRSV currently circulate in China. Recombination analyses showed that nine of 28 isolates and one isolate from other laboratory were potential complicated recombinants between the vaccine JXA1-R-like strains and predominant circulating strains.
These results indicated an increase in recombination rates of PRRSV under current vaccination pressure and a more pressing situation for PRRSV eradication and control in China.
KeywordsPRRSV Vaccine JXA1-R Predominant circulating strains Recombination
Porcine reproductive and respiratory syndrome (PRRS) is an economically critical swine disease worldwide that causes severe reproductive failure in sows, poor viability of piglets, and respiratory disease with secondary infection in growing pigs. The agent of PRRS, PRRS virus (PRRSV), belongs to the Arterivirus genus and has a single-stranded positive-sense RNA genome of approximately 15 kb, which is organized into nine overlapping open reading frames (ORFs) [1–4]: ORF1a, ORF1b, ORF2a, ORF2b, and ORFs 3–7. ORF1a and ORF1b, which comprise 80% of the genome, encode the viral nonstructural proteins involved in genome transcription and replication , whereas ORF2a, ORF2b, and ORFs 3–7 encode the viral structural proteins GP2, E, GP3, GP4, GP5, M, and N, respectively [6–8]. The two genotypes of PRRSV, the European type (Type 1) and North American type (Type 2), share only about 60% nucleotide similarities at the genomic level and are represented by Lelystad virus and VR-2332, respectively [9–11].
The North American type PRRSV has been a major viral genotype in China [12–14], but was not a major concern until 2006, when highly pathogenic PRRSV (HP-PRRSV), characterized by a 30-aa deletion in Nsp2, emerged . Having evolved from a less-pathogenic variant in China, HP-PRRSV has widely spread and continues to cause high mortality and huge economic losses to the swine industry in China [15, 16]. In a second major outbreak in China and other Southeast Asian countries in 2009 and 2010, the HP-PRRSV strain was later shown to be a recombinant between two Chinese PRRSV strains [17, 18]. NADC30 was isolated in the US in 2008 and in China in 2013 . Associated with the importation of NADC30-like strains or recombination events between NADC30-like and domestic strains in China, a third outbreak occurred from 2013 to 2015 [19–21].
Imported and domestic modified live-attenuated vaccines (MLVs) have been widely used to safeguard against PRRSVs since the outbreaks in China. However, evidence of the risk and inefficacy of MLVs to prevent the spread of the latest PRRSV strain has begun to accumulate [21–24]. In order to investigate the epidemiological and evolutionary characteristics of PRRSV in 2015, a total of 293 clinical samples were collected from pigs in 16 provinces in China and the full-length sequence of 28 PRRSV isolates were sequenced and analyzed.
A total of 293 clinical lung samples were collected from pigs from different swine herds that experienced high fever and reproductive and respiratory syndrome in 16 provinces of China in 2015. A portion of each sample was homogenized for RNA extraction and stored at −70 °C. Meanwhile, the clinical symptoms, morbidity, mortality, and changes in autopsy tissues of pigs were examined.
RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA was extracted from tissue homogenates using TRIzol reagent (Life Technologies, New York, NY, USA). cDNA was constructed from 7 μL of RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Two microliters of cDNA was used as a template for subsequent PCR analysis. Specific PRRSV nucleotide sequences of 143 bp were amplified with the primers 5′-AGC TGT GCC ARA TGY TGG-3′ and 5′-GGT RAA GTG ATG YCT GAC-3′ targeting ORF7. The cycling conditions were 95 °C for 1 min, followed by 35 cycles at 95 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 2 min.
Immunohistochemical (IHC) analysis
The presence of PRRSV antigens in lung tissues was examined by IHC analysis. Briefly, the lung tissues were fixed in 10% neutral buffered formalin for 48 h, sectioned, and stained with monoclonal antibodies specific for the N protein of PRRSV for detection of antigen-positive cells, as previously described .
Full-length sequencing of PRRSV
Information of the 28 PRRSV isolates in China
Vaccination history of MLV
A total of 651 genomic sequences, which included 28 sequences from the present study and all available sequences of type 2 PRRSVs (n = 623) retrieved from the GenBank database in March 2017 (https://www.ncbi.nlm.nih.gov/genbank/) were aligned using the MUSCLE multiple alignment program (v3.8.31)  under default settings with minor manual adjustments afterward. Phylogenetic trees were constructed using the neighbor-joining algorithm with the Kimura 2-parameter substitution model and bootstrap tests of 1000 replicates using the MEGA6 sequence alignment tool . Interactive tree of life (http://itol.embl.de)  was employed to display and annotate the phylogenetic trees. The genetic distances of intra- and inter- clusters were computed using MEGA 6 with Kimura 2-parameter model and 1000 bootstrap replicates. Sub-data sets with a diversity level of >10% were further divided into smaller monophyletic clusters and sublineages within lineages were identified with a cut off of 1.4%.
To accurately identify potential recombinants, sequences of the 28 strains identified in this study were combined with up to 623 isolate sequences for recombination screening. Recombination events were only considered significant when supported by at least three of the following five methods: RDP , GENECONV , MAXCHI , CHIMAERA , and 3SEQ  implemented in RDP v4.16 . In order to check the recombination signals and estimate the breakpoint locations, sequences of potential recombinants isolated in this study or similar sequences of the parental lineage and those retrieved from the GenBank database were chosen for similarity plot analysis implemented in SIMPLOT v3.5.1 , with a window of 500 bp and step size of 10 bp. Furthermore, recombination events were confirmed with a neighbor-joining phylogenetic tree.
Only the strains that fit all of the following three criteria, as modified from a previous report , were recognized as JXA1-R (JXA1 P80)-like strains: (1) nested in the phylogenetic tree within the same cluster as the vaccine strain JXA1-R, (2) sharing the highest sequence identity with the JXA1-R strain (>99%), and (3) presenting amino acids (aa) unique to JXA1-R strain derivatives.
Detection of PRRSV in the clinical samples
The full length sequences of 28 PRRSV strains from 24 pig farms in nine provinces were amplified and sequenced for further analysis. Strain names, areas, accession numbers, and clinical backgrounds are summarized in Table 1.
Pairwise interlineage genetic distance comparisons for complete genome sequences of type 2 PRRSVs
% Difference for lineage
Seven strains (15LN1, 15ZJ1, 15HEN4, 15JX1, 15HEN1, 15SC3, and 15LN3) in this study were nested in lineage 9 together with NADC30-like strains and its recombinants. A total of 350 isolates mainly from China were clustered into lineage 10, which was divided into eight sublineages (10.1–10.8).
CH-1a (isolated in 1996) was located in sublineage 10.1 and HB-1(sh)/2002 (isolated in 2002) was nested in sublineage 10.2. 15HEB1 isolated in this study and recombinant SH1211 were nested within sublineage 10.3. The early HP-PRRSV isolates [15, 26] were clustered in sublineage 10.5 and 10.8. Four strains (15SN1, 15SN2, 15SN3, and 15GD4) isolated in this study and BJsy06 (isolated in 2006) were located in sublineage 10.5. HP-PRRSV JXA1, isolated in the first outbreak in 2006 , was clustered in subgroup 10.8. The isolates identified in this study (15GD2, 15GD3, 15HUN1, and 15HUN2) were potential recombinants associated with 2009–2010 HP-PRRSV-like strains and were located in sublineage 10.6. Sublineage 10.7 consisted of JXA1 derivatives by serial passaging in MARC-145 cells [38, 39] and potential vaccine JXA1-R (JXA1 P80)-like strains. In sublineage 10.7, 10 isolates (15ZJ2, 15ZJ3, 15HUN3, 15LN2, 15JX2, 15JX3, 15JX4, 15HEN3, 15SC1, and 15SC2) from the present study were identified as potential vaccine JXA1-R-like strains with a 30-aa deletion in Nsp2.
These results indicated that multiple subgenotypes of PRRSV co-existed in China in 2015, which included classic HP-PRRSV (sublineages 10.5 and 10.8), 2009–2010 HP-PRRSV-like strains and its recombinants (sublineage 10.6), potential vaccine JXA1-R-like strains (sublineage 10.7), and NADC30-like strains and its recombinants strains (lineage 9).
Distribution of JXA1-R vaccine-like strains in China
PRRSV recombination events documented in this study
Breakpoint position (nt)
Set1 ( innerlineages )
( SL10.7 ± 10.6 )
15GD1 + JXA1-R
1310, 4895, 8716
15GD1 + JXA1-R
2421, 4895, 8716
10HD-GD + JXA1-R
7942, 9254, 14692
Set2 ( interlineages )
( L9 ± SL10.7 )
NADC30 + JXA1-R
NADC30 + JXA1-R
NADC30 + JXA1-R
2004, 7758, 8024
Set3 ( inner & interlineages )
( L9 ± SL10.7 ± 10.6 )
NADC30 + JXA1-R + 10-10JX
2639, 3318, 12171
NADC30 + JXA1-R + 10-10JX
684, 1318, 2020, 5666, 8743
PRRSV is among the most economically important viruses in the swine industry worldwide [40, 41]. In 2006, a HP-PRRSV with a unique discontinuous 30-aa deletion in Nsp2 emerged and caused the most severe viral diseases in swine in China to date . The genetic diversity of this HP-PRRSV has greatly increased by rapid evolution and recombination events [14, 17–19]. The current commercial PRRSV vaccines cannot provide complete protection against this disease . In this study, 117 (39.93%) of 293 clinical samples were positive for PRRSV by RT-PCR, similar to that reported in 2010 (45.2%) . Full-length sequences of 28 PRRSV isolates showed that they belonged to two lineages together with HP-PRRSV isolated in China. Among them, 10 JXA1-R-like isolates and 44 other Chinese strains were possibly derived from JXA1-R strains by recombination events of different strains. These findings indicated that the mosaic recombinant strains associated with the vaccine strains JXA1-R and HP-PRRSV were the predominant circulating strains of PRRSV in China.
The virulence reversion of the vaccine strain JXA1-R has been described [23, 27], but no recombination of this strain has yet been reported. Recombination analyses showed that 10 strains, which included nine strains from the present study and one isolate retrieved from the GenBank database, evolved through complicated recombination events between vaccine JXA1-R-like strains and predominant circulating strains in China. Phylogenetic analysis showed that the nine strains isolated in this study were positioned within major clusters with recombinants from other laboratories (Fig. 2). Furthermore, all breakpoints of the recombinants isolated in this study were not adjacent to the corresponding primer binding sites, suggesting that all of the recombinant strains isolated in this study are circulating recombinant strains rather than laboratory artifacts.
Most of the breakpoints of recombination events in this study were located in Nsp2 or Nsp9. Similarly, Liu et al.  conducted in vivo experiments to identify recombination events with Nsp2, ORF3, and ORF5, which demonstrated that Nsp2 was the most complicated region for recombination. Furthermore, all breakpoints of the recombinant strain Em2007 (lineage 10) were located in Nsp2 or Nsp9 , as were two of the three breakpoints of 2009–2010 HP-PRRSV . Thus, it is possible that Nsp2 or Nsp9 is the hot region for breakpoint of recombination events.
Time of emergence of vaccine JXA1-R-like strains in China
GX1001, NVDC-HeB1-2011, 11NZ-GD, 11SH1-GD, 11SH-GD, 11XX-GD
NT1, NT2, NT3, JL-04/12, NVDC-BJ3-2012, NVDC-BJ4-2012, NVDC-BJ5-2012, NVDC-BJ6-2012, NVDC-BJ9-2012, NVDC-SD2-2012
NVDC-SDXX-2013, NVDC-HBCZ-2013, NVDC-SXJC-2013, HNyc13, HEB-2013, HEB 20130008-14, NVDC-BJPG-2013
HNxa14, HUN-2014, HB2014001, HENZK-1, NVDC-shh01-2014, NVDC-SHH02-2014, NVDC-HuNCS-2014, NVDC-13SXJC-2014
HENPDS-2, 15ZJ2, 15ZJ3, 15HUN3, 15LN2, 15JX2, 15JX3, 15JX4, 15HEN3, 15SC1, 15SC2
GenBank, this study
Recombination events between field PRRSV strains could result in greater PRRSV pathogenicity, such as strain JL580 from the recombination between NADC30 and HP-PRRSV 09HEN1, which has been confirmed as highly pathogenic . In this study, the PRRSV-positive piglets exhibited severe clinical symptoms and gross lesions typical of HP-PRRS. Morbidity (63.6%–94.7%) and mortality (48.8%–90.8%) rates were relatively high among piglets infected the recombinant strains (Table 1, Additional file 6: Figure S3), suggesting that the recombinant strains associated with JXA1-R might be highly pathogenic. Of course, the pathogenicity of these recombinant JXA1-R-like strains should be examined in future studies.
In order to control this disease, at least seven MLV PRRSV vaccine strains (JXA1-R, HuN4-F112, TJM-F92, GDr180, RespPRRS/Repro, CH-1R, and R98) have been widely used in China [38, 42, 43]. In order to determine the original of these isolates, we compared 28 sequences identified in this study with all available genomic sequences (n = 623) of type 2 PRRSV in the GenBank database. The sublineage 10.7 include up to 65 field samples with a wide geographical range, in which the intralineage diversity was the lowest (0.8%), and found nine JXA1-R-like isolates out of the 28 isolates based one the three criteria reported previously . Of course, it was possible that these recombinants might be also derived from other HP-PPRSV vaccine strains, such as HuN4-F112, TJM-F92, and GDr180, because those vaccine strains are derived from HP-PRRSV and there is no published complete sequences of those vaccine strains. The improper use of vaccines may be associated with the emergence of mosaic recombinant strains . Hence, immunization schedules should be optimized to prevent future outbreaks of this infectious disease.
The findings of this study demonstrated that HP-PRRSV vaccine like strains (JXA1-R-like strains) are widely circulated in China and mosaic recombinant strains associated with the vaccine strains and predominant circulating strains of PRRSV emerged in different provinces of China under current vaccination pressure. PRRSV eradication and control is a pressing situation in China.
Highly pathogenic porcine reproductive and respiratory syndrome virus
Modified live-attenuated vaccines
Open reading frames
Polymerase chain reaction
Porcine reproductive and respiratory syndrome
Porcine reproductive and respiratory syndrome virus
Reverse transcription polymerase chain reaction
This work was mainly supported by the National Natural Science Foundation (31230071,31672565), a grant from the Ministry of Agriculture (CARS-36) for development of techniques to control swine diseases, and the priority academic program development of Jiangsu higher education institutions (PAPD).
HZ and PJ designed the study. HZ, QH, LZ, and ZZ performed the experiments. HZ, QH, HS, YW, and PJ analyzed the data. HZ and PJ wrote the manuscript. All authors have read and approved the submitted manuscript.
The authors declare that they have no competing interests.
All animal protocols were approved by the Animal Care and Ethics Committee of Nanjing Agricultural University (permit number: IACECNAU 20121001) and followed the Guiding Principles for Biomedical Research Involving Animals.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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