Genetic diversity and phylogenetic analysis of the ORF5 gene of PRRSV from central China
Introduction
Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is an enveloped virus belonging to the family Arteriviridae in the order Nidovirales (Gorbalenya et al., 2006, Dokland, 2010) and can be divided into two Types based on pairwise genetic distances and phylogenetic analyses of European and North American isolates (Meng et al., 1995, Nelsen et al., 1999): Type 1, represented by the European prototype Lelystad (LV) (Wensvoort et al., 1991); and Type 2, exemplified by the North American prototype VR-2332 (Benfield et al., 1992, Collins et al., 1992).
PRRSV has a linear, single-stranded, positive-sense RNA genome of about 15 kb that consists of ten overlapping open reading frames (ORFs) including ORFs 1a/1b, 2a/2b, 3, 4, 5/5a, 6 and 7 (Stadejek et al., 2002, Firth et al., 2011, Johnson et al., 2011). The ORF5 encodes the viral envelope glycoprotein 5 (GP5) that is an important immunogenic protein and is associated with PRRSV neutralization (Wissink et al., 2005, Ansari et al., 2006, Ostrowski et al., 2002, Plagemann, 2004). The critical primary neutralizing epitope (PNE) within GP5 has been mapped to the protein's N-terminal ectodomain (Ostrowski et al., 2002, Plagemann, 2004). Nearby the PNE, a decoy epitope can induce non-neutralizing antibodies against GP5 and tends to postpone the production of neutralizing antibodies against PRRSV (Ostrowski et al., 2002, Lopez and Osorio, 2004). GP5 usually contains 2–4 potential N-linked glycosylation sites and the hyper-glycosylation of GP5 could be a major pathway via which PRRSV might evade vaccine induced immune responses (Gagnon et al., 2003, Ansari et al., 2006, Vu et al., 2011). Mutations, such as R13 → Q13 and R151 → G151, are considered to be involved in PRRSV attenuation (Allende et al., 2000, Wesley et al., 1999). Accordingly, in the attenuated vaccine isolate, RespPRRSV MLV, residues R13 and R151 of GP5 are respectively altered to Q13 and G151 (Allende et al., 2000). GP5 is also one of the most variable structural proteins of PRRSV (Pirzadeh et al., 1998). The ORF5 gene is therefore useful for investigating the phylogenetic relationships and genetic distances between PRRSV isolates (Zhou et al., 2009a, Zhou et al., 2009b, Thanawongnuwech et al., 2004, Mateu et al., 2006, Stadejek et al., 2006).
PRRS was first confirmed in mainland China in 1996 with the characterization of the CH-1a isolate (Guo et al., 1996a). Subsequently PRRS became endemic within China. Further, in 2006, highly pathogenic PRRSV (HP-PRRSV) represented by JXA1 broke out in mainland China and spread quickly to neighboring provinces, which brought great losses to Chinese pig production (Li et al., 2007, Tian et al., 2007, Tong et al., 2007), and for this reason, vaccination against PRRSV is likely to be the most effective way of mitigating losses to PRRS in China. Following the registration in China of the commercialized live vaccine for prevention and control of PRRS, Ingelvac® PRRS MLV (containing the attenuated vaccine isolate, RespPRRSV MLV), has been used extensively within China and has so-far only provided partial immune protection to Chinese pigs (Zhou and Yang, 2010).
Henan province is China's main swine husbandry hub. In this region, PRRS is a particularly important threat to porcine production. As the main candidate protein for development subunits vaccine, also due to the variation and immunological characteristics, GP5 has become the main target protein for analysis of genetic variation of PRRSV (An et al., 2007). So, in the present study, we focused on sequencing and analyzing the ORF5 genes of 112 PRRSV isolates circulating within Henan province between 2006 and 2015 to better understand the genetic diversity and molecular epidemiology of prevailing PRRSV in this region.
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Sample collection
A total of 198 clinical lung samples were collected from different swine herds that experienced outbreaks of severe reproductive failure in pregnant sows and respiratory problems in sucking and post-weaning piglets in Henan province. Porcine lung tissues were minced, diluted 1:10 in Dulbecco's modified Eagle medium, homogenized, and centrifuged at 1500g for 10 min to obtain supernatant for RNA extraction (Li et al., 2011) and the remaining samples were kept at − 70 °C.
RNA extraction and RT-PCR
Total RNA was extracted from
Phylogenetic analysis
To determine the genetic relationships of 112 PRRSV isolates sampled in the Henan province of China (Table 1) (30 isolates from this study and 82 isolates from GenBank) and 41 reference PRRSV isolates from elsewhere in the world (Table 2), we constructed a phylogenetic tree based on the full-length ORF5 gene sequences of these isolates. As shown in Fig. 1, the 153 PRRSV isolates could be divided into two major groups representing the Type 1 and Type 2 genotypes. All the Henan isolates belonged
Discussion
Since PRRS was first confirmed in China in 1996, nearly all described PRRSV isolates in China, including HP-PRRSV isolates (Tian et al., 2007), were of the Type 2 genotype (An et al., 2011). In the present study, sequence similarity revealed that all the Henan strains shared 84.2–99.5% nucleotide and 81.1–98.0% amino acid sequence identity with VR-2332, and 60.4–64.8% and 48.8–58.2% with LV, respectively. Additionally, phylogenetic analysis of the full-length ORF5 genes of all the Henan strains
Conflict of interest
The authors declare that they have no competing interests.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Acknowledgments
This work was supported by National Natural Science Foundation of China (31572520 and 31490600).
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These authors contributed equally to this work.