Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing
© Lu et al.; licensee BioMed Central Ltd. 2014
Received: 3 December 2013
Accepted: 24 February 2014
Published: 4 March 2014
Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV.
We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5.
Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.
Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is the causative agent of a significant disease of the domestic pig (Sus scrofa) with global consequences. The severity of PRRSV infection ranges from subclinical to lethal and it affects pigs in both growing and reproductive stages. The virus has a positive-sense 15 kb RNA genome and its genetic diversity has been well characterised within and between European and North American strains . Extensive viral genetic heterogeneity may have contributed towards the observed variations between PRRSV isolates and clones in term of virulence, interactions with the immune system, and antigenic properties of viral proteins. Such a broad diversity indeed poses serious challenges to diagnostics and control measures.
Most previous studies of PRRSV genetic diversity have been restricted to the ORF5 and ORF7 sequences of type 2 “North American-like” viruses that also include the Asian variants. Only 14 of the 303 completed PRRSV genomes in Genbank belong to genotype 1. Furthermore, studies have shown that PRRSV mutates rapidly and multiple intra-strain variants can coexist in individually infected pigs . The extensive genetic diversity displayed by PRRSV and other RNA viruses such as HIV and influenza reflects the error prone nature of RNA polymerases, which lack a proofreading function [3, 4].
To identify PRRSV quasispecies, previous studies have employed conventional methodologies including reverse-transcription, PCR, cloning and Sanger sequencing of a subset of PRRSV structural and non-structural proteins [2, 5]. More recently next-generation sequencing (NGS) of fragments generated by long range RT-PCR has been used to characterise multiple PRRSV genomes . However, this approach relies upon prior knowledge of the target sequence and the assumption that the PCR primer binding sites are non-variable. Here we describe an approach which requires no prior knowledge of the target sequences and which should enable the detection of low-frequency nucleotide variants and hence provides a snapshot of the microevolution in the entire viral population.
We analysed the intra-strain sequence diversity of low passage PRRSV Olot/91 strain, passaged exclusively on primary porcine alveolar macrophages (PAM). First reported in Spain in 1991, Olot/91 is the parent strain of the commercial Suvaxyn PRRSV inactivated vaccine which is used in Spain and Portugal. Only a partial sequence of 3,383 nt [GenBank:X92942], that covers ORFs 2-7 and the 3'-UTR of this strain has previously been published .
While variants in the major Olot/91 strain do congregate on known highly hypervariable B- and T-cell epitopes of type I PRRSV (Figure 2 and Additional file 3: Figure S2) [19–23], most of the variants representative of the probable quasispecies population map to the nsp12 and gp2a genes of ORF1b and ORF2 respectively (Figure 2); suggesting a higher microevolutionary rate at this region under the investigated culture conditions. In addition, the predicted short signal peptide of GP2a was found to harbour a high concentration of non-synonymous variants and a potential N-glycosylation site was created from a residue change (S37N) on GP5. Further analysis, like molecular modelling, may be necessary to decipher the potential impacts of these evolving variants have on such functions as interactions between these proteins and other viral or host cellular proteins.
Using ultra-deep NGS we have constructed the complete sequence of the PRRSV Olot/91 genome cultured in their natural host - porcine alveolar macrophages - and identified single-nucleotide variants present in the associated viral population. Further investigation using this methodology will help to establish if a link exists between the microevolutionary dynamics and pathogenesis of PRRSV viral strains. However, it is also important to note that the exact nature of the pathogenesis cannot be truly identified until the viral haplotypes within the quasispecies population can be reconstructed with confidence .
We are indebted to Lise K. Kvisgaarda and Lars E. Larsen for kindly providing the sequence of the Marc 145-adapted Olot/91 strain. TAA, ALA and ZHL were supported by UK Biotechnology and Biological Sciences Research Council (BBRSC) Institute Strategic Programme Grants (BB/J004235/1). ADW was supported by EC FP7 PoRRScon Grant (Agreement number 245141). Edinburgh Genomics is funded by BBSRC National Capacity Grants (BB/J004243/1). We are also grateful for support from the COST Action FA0902.
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