Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes

Abstract

In this study, comprehensive analysis of 82,237 global porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) open reading frame 5 sequences spanning from 1989 to 2021 refined PRRSV-2 genetic classification system, which defines 11 lineages and 21 sublineages and provides flexibility for growth if additional lineages, sublineages, or more granular classifications are needed in the future. Geographic distribution and temporal changes of PRRSV-2 were investigated in detail. This is a thorough study describing the molecular epidemiology of global PRRSV-2. In addition, the reference sequences based on the refined genetic classification system are made available to the public for future epidemiological and diagnostic applications worldwide. The data from this study will facilitate global standardization and application of PRRSV-2 genetic classification.

Keywords: ORF5; PRRSV-2; genetic classification; lineage; molecular epidemiology; restriction fragment length polymorphism analysis; sublineage.

Fig 1

Phylogenetic tree showing the newly proposed PRRSV-2 lineages L1 to L11 based on global ORF5 sequences. Tip points are presented in different colors according to lineages. Bootstrap values are shown for the major clades. Six commercial PRRSV-2 modified live virus vaccines Ingelvac PRRS MLV, Ingelvac PRRS ATP, Fostera PRRS, Prime Pac PRRS RR, Prevacent PRRS, and PRRSGard are classified in the sublineages L5A, L8A, L8C, L7, L1D, and L1F, respectively.

Fig 2

Phylogenetic trees showing PRRSV-2 ORF5-based sublineages within lineages L1, L5, L8, and L9. (a) Nine sublineages, L1A, L1B, L1C, L1D, L1E, L1F, L1H, L1I, and L1J, in lineage L1. (b) Two sublineages, L5A and L5B, in lineage L5. (c) Five sublineages, L8A, L8B, L8C, L8D, and L8E, in lineage L8. (d) Five sublineages, L9A, L9B, L9C, L9D, and L9E, in lineage L9. Tip points are presented in different colors according to sublineages classified in a particular lineage.

Fig 3

Phylogenetic tree and temporal dynamics of ORF5 sequences classified in sublineage L1C. (a) Phylogenetic tree of ORF5 sequences classified in groups L1C.1 to L1C.5. (b) Average pairwise genetic distance (% difference) between and within the five groups L1C.1 to L1C.5 in sublineage L1C. (c) Temporal dynamics of US PRRSV-2 ORF5 sequences classified in L1C.1–L1C.5 during 2009–2021. Total number of L1C sequences is shown in the table below the graph and percent of sequences classified in L1C.1–L1C.5 are indicated in the graph. Sequences not classified in L1C.1–L1C.5 were defined as L1C-Others.

Fig 4

Discriminant analysis of principal components (DAPC) of different sublineages and RFLP of PRRSV-2. (a) DAPC of sequences in sublineage L1A. Eighty-six distinct RFLP patterns were detected in 16,625 sequences classified in L1A with 3.64% average pairwise genetic distance. Some of the most frequently detected RFLPs and their percentage in L1A are shown. (b) DAPC of sequences in sublineage L1E. (c) DAPC of sequences with RFLP 1-4-4. Among 10,726 sequences with RFLP 1-4-4 classified into 8 lineages and 17 sublineages, the top five frequently detected sublineages and their percentage are shown. (d) DAPC of sequences with RFLP 1-8-4. (e) DAPC of sequences with RFLP 1-4-2. (f) DAPC of sequences with RFLP 1-3-2.

Fig 5

Detection frequency and distribution of PRRSV-2 ORF5-based lineages and sublineages in representative countries based on the data set in this study. The number and percentage of sequences belonging to the major PRRSV-2 lineages and sublineages in Thailand, Vietnam, mainland China, South Korea, Canada, and Mexico are shown in (a) to (f). The number and percentage of sequences at the lineage level in the USA are shown in (g) and at the sublineage level in the USA are shown in (h) to (k). Two L5 sequences with undefined sublineage are not shown in (i). One L8E sequence is not shown in (j).

Fig 6

Temporal dynamics at the lineage level of 73,092 PRRSV-2 ORF5 sequences with samples collected in the USA during 1989–2021. Percent distribution of each lineage is indicated in the graph and the total number of sequences reported in a particular year is indicated in the table below the graph.

Fig 7

Temporal dynamics at the sublineage level of US PRRSV-2 ORF5 sequences classified in lineage L1 during 1998–2021. Percent of each sublineage is shown in the graph and the total number of sequences in the lineage reported in a particular year is indicated in the table below the graph. Percentages of sequences in L1D with <95%, 95% to <98%, and ≥98% ORF5 nt identity to Prevacent PRRS vaccine were calculated against the total number of sequences in L1 in a particular year and the data are indicated in the table below the graph.

Fig 8

Temporal dynamics at the sublineage level of US PRRSV-2 ORF5 sequences classified in lineage L5 or L9 during 1989–2021. Number of sequences in each sublineage is shown in the graph and the total number of sequences in the lineage reported in a particular year is indicated in the table below the graph. (a) Temporal dynamics of sublineages in lineage L5. (b) Among all L5A sequences in the USA, percentages of L5A sequences with <95%, 95% to <98%, and ≥98% ORF5 nt identity to Ingelvac PRRS MLV vaccine in a particular year are shown. (c) Temporal dynamics of sublineages in lineage L9.

Fig 9

Temporal dynamics at the sublineage level of US PRRSV-2 ORF5 sequences classified in lineage L8 during 1993–2021. (a) Percent of each sublineage is shown in the graph and total number of sequences in the lineage reported in a particular year is indicated in the table below the graph. Among all L8A sequences in the USA from 1993 to 2021, percentages of L8A sequences with <95%, 95% to <98%, and ≥98% ORF5 nt identity to Ingelvac PRRS ATP vaccine in a particular year or period are shown in (b). Among all L8C sequences in the USA from 2002 to 2021, percentages of L8C sequences with <95%, 95% to <98%, and ≥98% ORF5 nt identity to Fostera PRRS vaccine in a particular year or period are shown in (c).

Fig 10

Temporal dynamics of US PRRSV-2 ORF5 sequences classified in lineages and sublineages in L1 reported in eight US states during 2015–2021. Eight states are represented by two-letter state codes and the number of sequences reported in each state during 2015–2021 is indicated below the graph. (a) Temporal dynamics of lineages. The percentages were calculated against the total number of sequences during 2015–2021 for each state, respectively. (b) Temporal dynamics of sublineages in L1. The percentages were calculated against the total number of L1 sequences during 2015–2021 for each state, respectively.