Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates

Abstract

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88–95% nucleotide and 88–96% amino acid sequence identities to each other, 87–97% nucleotide and 84–96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.

Introduction

Porcine reproductive and respiratory syndrome (PRRS), characterized by severe reproductive failure in sows and respiratory diseases in young pigs, was first recognized in the US in 1987 (Hill, 1990). Since its first appearance, PRRS has been causing immense economic losses in the swine industry (Polson et al., 1992). The causative agent of PRRS, PRRS virus (PRRSV), was first isolated by Wensvoort et al. (1991) in The Netherlands by using porcine alveolar macrophages and was designated as Lelystad virus (LV). In the US, PRRSV was isolated and characterized in a number of continuous cell lines (Benfield et al., 1992, Collins et al., 1992, Meng et al., 1994, Meng et al., 1996a). Meulenberg et al. (1993) first sequenced the complete genome of LV. Subsequently, partial sequences of another European isolate (Conzelmann et al., 1993) and two North American isolates of PRRSV (Mardassi et al., 1994, Meng et al., 1994) were reported. The genome of PRRSV is a positive strand RNA molecule of about 15 kb that encodes eight overlapping open reading frames (ORFs) (Meulenberg et al., 1993, Nelsen et al., 1999). The ORF1 is believed to encode the non-structural proteins. It has been shown that the ORF2–4 encode virion-associated proteins designated as GP2, GP3 and GP4, respectively (Meulenberg et al., 1995, Van Nieuwstadt et al., 1996). ORF5–7 encode the major envelope (GP5), membrane (M) and nucleocapsid (N) proteins, respectively (Mardassi et al., 1996, Meulenberg et al., 1995). It has been shown that monoclonal antibodies directed against GP4 and GP5 proteins are neutralizing (Pirzadeh and Dea, 1997). PRRSV, along with equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV), is now classified within a single genus Arterivirus, in the family Arteriviridae in the order Nidovirales (Cavanagh, 1997).

PRRS has now been recognized worldwide and is considered to be a global disease with important economic impact. Although PRRSV isolates identified from around the world cause similar diseases in pigs, increasing data indicates that PRRSV is antigenically, genetically and pathologically very heterogenic (Andreyev et al., 1997; Halbur et al., 1995, Halbur et al., 1996a, Halbur et al., 1996b; Kapur et al., 1996; Meng et al., 1994, Meng et al., 1995a, Meng et al., 1995b, Meng et al., 1996a, Meng et al., 1996b; Murtaugh et al., 1995). Despite the availability of several PRRSV vaccines, the disease remains difficult to control. Recently, a more virulent form of PRRSV has been causing high abortion and mortality rates in vaccinated swine populations in the US (Bell, 1998, Botner et al., 1997, Bush et al., 1999, Halbur and Bush, 1997, Lager et al., 1998, Mengeling et al., 1998). These severe outbreaks of PRRS have been referred to as acute PRRS, atypical PRRS, hot PRRS, abortion storm, or sow abortion and mortality syndrome. The term “acute PRRS” is used throughout this manuscript. Zimmerman et al. (1997) described the criteria for the diagnosis of acute PRRS which include acute onset, clinical signs occurring over a 2–4 weeks period, high mortality (>5%) in sows and boars, and a high rate of abortions (>10%). Many of the herds affected by acute PRRS were on a PRRS vaccination program with the available modified live-vaccines (MLVs), suggesting that the vaccine-induced antibodies failed to neutralize the acute PRRS virus.

There are no published data on the genetic characterization of acute PRRSV isolates, and the mechanism for the emergence of acute PRRS in vaccinated pigs is still not known. In pigs vaccinated with MLVs, both cell-mediated immunity and humoral immune responses against the GP5 and other structural proteins of PRRSV play important roles in protection against PRRS. The GP5 protein is exposed outside the virion (Meulenberg et al., 1995) and, thus, is constantly under positive or negative selection forces. It has been shown that the ORF5 gene sequence of PRRSV is very polymorphic (Andreyev et al., 1997, Kapur et al., 1996). Therefore, genetic comparison of the ORF5 gene sequences of the acute PRRSV isolates to those of MLVs and other PRRSV isolates identified prior to acute PRRS outbreaks may provide insight into the genetic evolution and origin of the acute PRRSV isolates.