Analysis of Recombinant Characteristics Based on 949 PRRSV-2 Genomic Sequences Obtained from 1991 to 2021 Shows That Viral Multiplication Ability Contributes to Dominant Recombination

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases affecting the pig-raising industry. The PRRS virus (PRRSV) has high genetic diversity, partly owing to viral recombination. Some individual recombinant type 2 PRRSV (PRRSV-2) strains have been detected; however, the sequence composition characteristics of recombination hot spots and potential driving forces for recombinant PRRSV-2 are still unreported. Therefore, all available genomic sequences of PRRSV-2 (n = 949, including 29 genomes sequenced in this study) from 11 countries from 1991 to 2021 were collected and analyzed. The results revealed that the dominant major recombinant parent has been converted from lineage 3 (L3) to L1 since 2012. The recombination hot spots were located at nucleotides (nt) 7900 to 8200 (in NSP9, encoding viral RNA-dependent RNA polymerase) and nt 12500 to nt 13300 (in ORF2-ORF4, mean ORF2 to ORF4); no AU-rich characteristics were found in the recombination hot spots. Based on infectious clones of L1 and L8 PRRSV-2, recombinant PRRSVs were generated by switching complete or partial NSP9 (harboring the recombination hot spot). The results showed that recombinant PRRSVs based on the L1 backbone, but not the L8 backbone, acquired a higher replication capacity in pig primary alveolar macrophages. These findings will help to understand the reason behind the dominance of L1-based recombination in PRRSV-2 strains and provide new clues for an in-depth study of the recombination mechanism of PRRSV-2. IMPORTANCE Recombination is an important driver of the genetic shifts that are tightly linked to the evolution of RNA viruses. Viral recombination contributes substantially to the emergence of new variants, alterations in virulence, and pathogenesis. PRRSV is genetically diverse, partly because of extensive recombination. In this study, we analyzed interlineage recombination based on available genomic sequences of PRRSV-2 from 1991 to 2021. The study revealed the temporal and geographical distribution of recombinant PRRSVs and the recombination hot spot's location and showed that artificially constructed recombinant PRRSVs (harboring a high-frequency region) had more viral genomic copies than their parental virus, indicating that dominant recombination was shaped by a tendency to benefit viral replication. This finding will enrich our understanding of PRRSV recombination and provide new clues for an in-depth study of the recombination mechanism.

Keywords: PRRSV-2; recombination; recombination hot spots; viral multiplication.

FIG 1

Geographical and temporal distribution of the 949 PRRSV-2 genomes from 1991 to 2021. (A) Geographical distribution of the 949 PRRSV-2 strains collected from 11 countries. (B) Annual number of 949 PRRSV-2 strains. Different colors indicate different countries, including China (n = 612), the United States (n = 213), South Korea (n = 11), Denmark (n = 13), Japan (n = 4), Viet Nam (n = 2), Thailand (n = 1), Laos (n = 8), Hungary (n = 1), India (n = 5), and Canada (n = 1). The strains lacking collection date information are included in the “na” column.

FIG 2

Lineage classification of the 949 PRRSV-2 strains. (A) Phylogenetic tree based on ORF5 sequence of 949 PRRSV-2 strains. An ML-tree was generated using general time-reversible substitution as a model, and reliability was assessed by bootstrap analysis of 1,000 replications. The twenty-nine PRRSV-2 strains sequenced in this study are marked by red dots. (B) Distribution of PRRSV lineages in different countries, based on the 949 PRRSV-2 strains.

FIG 3

Interlineage recombination analysis of PRRSV-2 strains. (A) Map of major or minor parental lineages of recombinant PRRSV-2 strains collected from 2019 to 2021. Different colors indicate different lineages. The positions are in accordance with the PRRSV VR-2332 strain. (B) Comparison of the topological structure of phylogenetic trees of NSP9 and NSP10. A line connects genes from the same strains. The lines crossed with a large span indicate potential recombination. (C) Simplot analysis of a representative PRRSV-2 HB94 strain. The major parent is JXA1, and the minor parent is NADC30; the HB94 genome is divided by two breakpoints, and the recombinant region is shaded pink. (D) Phylogenic trees were constructed based on the regions from nt 5386 to 7987 and nt 7988 to 9988.

FIG 4

Distribution of recombinant PRRSV-2 strains. (A) Annual proportion of the recombinant PRRSVs from different lineages to the total number of PRRSVs. (B) Proportions of the recombinant PRRSVs in each lineage out of the total PRRSVs in each lineage. (C) Proportions of the recombinant PRRSVs in each lineage out of the total number of PRRSVs. (D) Numbers of recombinant and total PRRSV-2 strains in China and the United States. (E and F) Geographical distribution of recombinant PRRSV-2 strains in different provinces/states in China and the United States, respectively.

FIG 5

Temporal distribution of PRRSV-2 recombinants and PRRSV-2 vaccines in China and the United States. The annual number of recombinant PRRSVs and the year of approval of PRRS vaccines in China (A) and the United States (B) are indicated.

FIG 6

Characteristic of recombination hot spots. (A and B) Frequency of left or right breakpoints in recombinant hot spots in NSP9, respectively. The recombinant hot spot is mainly concentrated at the region between nt 7800 and nt 8200. (C and D) Frequency of left or right breakpoints in recombinant hot spots in ORF2-ORF4, respectively. The detailed position is mainly concentrated between nt 12500 and nt 13300. (E and F) Location of residues Cys228 to Tyr267, the recombination hot spot in RdRp. PRRSV RdRp structure was predicted with the Swiss-Model server, and the RdRp structure (PDB ID 6xqb, chain A) of SARS-CoV-2 was the template for building PRRSV RdRp model. (G) AU contents in the high frequency left or right breakpoints. NSP9-L, nt 7800 to 8000; NSP9-R, nt 8100 to 8400; ORF2-L, nt 12200 to 12900; ORF4-R, nt 13100 to 13400. The numbers of recombinant and nonrecombinant strains are 323 and 603, respectively. (H) GC contents in high-frequency left or right breakpoints.

FIG 7

Generation and characterization of artificially recombinant PRRSVs. (A) Schematic diagram for constructing recombinant plasmids. (B) Virus detection by immunofluorescence. Recombinant and parental plasmids (4 μg) were transfected into Marc-145 cells. At 5 days posttransfection, the transfected cells were fixed and stained with monoclonal antibody 3F7 against PRRSV, followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG. Scale bar, 400 μm. The growth kinetics of the recombinant (rHeB108-HuN4NSP9 and rHeB108-HuN4NSP9p) and parental viruses in PAMs (C and D) and Marc-145 cells (E and F) are represented. Each data point represents the mean value of three replicates with the SD. *, Significant difference between rHeB108 and rHeB108-HuN4NSP9 (*, P < 0.05; **, P < 0.01; ***, P < 0.001); #, significant difference between rHeB108 and rHeB108-HuN4NSP9p (#, P < 0.05; ##, P < 0.01; ###, P < 0.001); δ, significant difference between rHuN4 and rHeB108-HuN4NSP9 (δ, P < 0.05; δδ, P < 0.01; δδδ, P < 0.001); ψ, significant difference between rHuN4 and rHeB108-HuN4NSP9p (ψ, P < 0.05; ψψ, P < 0.01; ψψψ, P < 0.001).