Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs

Highlights

• The NADC30-like PRRSV strains have been circulating and prevalent in Central China.

• The classic HP-PRRSV strains are still circulating in pig farms of Central China and relatively conserved in recent years characterized by the highly similarity between each other.

• The NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs of GP5, such as major neutralizing epitope, potential N-glycosylation and signal peptides, which might be involved in viral virulence and immune escape.

• Our data would contribute to epidemiology investigation of PRRSV and have some implications for vaccine development.

Abstract

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders in piglets and reproductive failure in sows, is still the great threat of swine industry. Recently, Emergence of the novel NADC30-like PRRS viruses (PRRSVs) has caused widespread outbreaks of PRRS. To investigate the epidemic characteristics of PRRSVs in Central China since 2014, 6372 clinical serum samples were tested by ELISA, 250 tissue samples were tested by RT-PCR, and among these, 30 ORF5 and 17 Nsp2 genes sequences were analyzed. Phylogenetic tree based on ORF5 revealed that, 17 isolates were clustered into subgroup 1, represented by the NADC30. And for the Nsp2, The strains which had a discontinuous 131-amino-acid deletion in Nsp2, called NADC30-like strains, were clustered into subgroup 2. Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains. In addition, amino acid variation analysis of GP5 revealed that the amino acid sequences of NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs, such as potential neutralization epitope and the N-glycosylation sites. In summary, our data would provide a large amount of detailed information on molecular variation and genetic diversity of PRRSV in central China.

Introduction

Porcine respiratory and reproductive syndrome (PRRS) is one of the most economically devastating diseases affecting the global swine industry, which causes huge economic losses every year [11], [21], [27]. The causative agent is porcine reproductive and respiratory syndrome virus (PRRSV), which belongs to the family Arteriviridae, order Nidovirales.

Since the initial outbreak in China in 1995, PRRS rapidly spread to pig-producing areas of China and brought great losses to the Chinese pig production, particularly following the emergence of the highly pathogenic PRRS in 2006 [10], [25]. The main symptoms caused by the classic highly pathogenic-PRRSV (HP-PRRSV) are continuous high fever, high morbidity and high mortality in pigs of all ages. And the striking characteristic of HP-PRRSV genome is discontinuous deletion of 30-amino-acid in the Nsp2 regions [25]. Although the 30-amino-acid deletion is not associated with the virulence of the HP-PRRSV, it could be used as an epidemiological genetic marker for the classic HP-PRRSV [9], [32].

PRRSV is an enveloped, single-stranded positive RNA virus [2, 3]. With the 5′ UTR and 3′ UTR locating in the head and tail, the virus genome is about 15.5 kb in length, which contains 10 open reading frames (ORFs) [4]. The first two ORF1a and ORF1b, encode virus non-structural protein (Nsps), are designated as Nsp1α, Nsp1β, Nsp2-Nsp6, Nsp7α, Nsp7β and Nsp8 and Nsp9-Nsp12, respectively [12], [19], [20]. ORFs2-7 which are located in the 3'terminal region of the genome, encode virus structural protein, including four minor structural proteins (GP2a, GP2b, GP3, GP4 and GP5a) and three major structural proteins (GP5, M, N) [8], [23]. Among these proteins, Nsp2-coding region has been identified to be the most variable region in the whole genome with natural point mutations and deletions, and the ORF5 encoding the major envelope protein is highly variable among the structural proteins coding regions. Thus, Nsp2 and ORF5 have become the important target regions for the evolution and molecular epidemiology research on PRRSV [4], [15], [22].

According to different geographic origins, PRRSVs can be classified into two genotypes: European type (Genotype I) and North American type (Genotype II) [13], [16]. The similarity of nucleotide sequence is only about 60% between Genotype I strains and Genotype II strains [24]. One hallmark of PRRSV is its high genetic diversity. Due to lack of 3′-5′ correction activity in RNA polymerase, the genome is prone to mutation during replication. On this account, the mutation rate of PRRSV is the highest in all RNA viruses reported so far [7]. Outbreak of PRRS recently (2013–2014), which was proved to be caused by a novel HP-PRRSV, had been observed in multiple provinces in China. It had a distinct genetic background and showed high homology with NADC30, a moderately virulent strain originated in Canada and then transmitted to the United States. Sequence analysis revealed that this NADC30-like strain has a genetic exchange with the classic HP-PRRSVs in China [30], [31]. But, in Central China, only limited viral information could be available since 2010 [14]. The diversity of PRRSV strains prevailing now and their genetic relationship is unclear. Therefore, we collected clinical samples from different farms in Central China since 2014 and tested the PRRSV antibody level. The result showed that 82.3% (2242/2724) and 84.9% (3097/3648) were respectively positive for the antibody of PRRSV in 2014 and 2015. Finally, we investigated epidemiology and genetic diversity of outbreak PRRSV by analyzing 17 partial Nsp2 sequences and 30 full-length ORF5 sequences. We found that HP-PRRSV strains were relatively conserved, and NADC30-like PRRSV strains had been the dominating strains and undergone rapid evolution in late years characterized by the high mutation rate in the Nsp2 and GP5 in Central China. However, whether the existing vaccines in China could prevent the pigs from infecting the evolving strains still needs more study. This work would provide updated knowledge about the evolution and genetic diversity of PRRSVs and contribute to research of epidemiology surveillance of PRRSV in China.