In vitro evaluation of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 sequences in samples containing PRRSV modified-live vaccine and wild-type strains
Restricted accessBrief reportFirst published online July, 2025
In vitro evaluation of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 sequences in samples containing PRRSV modified-live vaccine and wild-type strains
Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses and is a major challenge to the swine industry. The PRRS virus (PRRSV) has high rates of mutation and evolution. We examined the influence on ORF5 Sanger sequencing outcomes of various concentrations of a wild-type (WT) PRRSV (lineage 1A RFLP 1-7-4) and a modified-live vaccine (MLV) virus (lineage 5 RFLP 2-5-2) in the same sample. Vaccine-like sequences were detected more frequently than the WT virus when the MLV virus was present at equal or higher concentrations than the WT virus. This result suggests that ORF5 Sanger sequencing may preferentially detect the dominant virus in samples containing more than one virus, potentially masking WT viral infections in vaccinated herds. Although our findings highlight a limitation in identifying co-circulation of strains, Sanger sequencing is still widely used as an accessible tool to characterize PRRSVs. Advanced sequencing techniques, such as NGS or CLAMP-based approaches, would complement Sanger sequencing results and allow improved detection of co-circulating variants by minimizing consensus sequence bias and selectively blocking vaccine-like sequences.
Al-ShuhaibMBSHashimHO.Mastering DNA chromatogram analysis in Sanger sequencing for reliable clinical analysis. J Genet Eng Biotechnol2023;21:115.
2.
BrarMS, et al. Evolutionary diversification of type 2 porcine reproductive and respiratory syndrome virus. J Gen Virol2015;96:1570–1580.
3.
BrazdilovaK, et al. TraceTrack, an open-source software for batch processing, alignment and visualization of sanger sequencing chromatograms. Bioinform Adv2023;3:vbad083.
4.
ChaeH, et al. Development of a one-step reverse transcription-quantitative polymerase chain reaction assay for the detection of porcine reproductive and respiratory syndrome virus. PLoS One2023;18:e0293042.
5.
ChaoK-H, et al. sangeranalyseR: simple and interactive processing of Sanger sequencing data in R. Genome Biol Evol2021;13:evab028.
6.
ChengT-Y, et al. Detection of multiple lineages of PRRSV in breeding and growing swine farms. Front Vet Sci2022;9:884733.
7.
CorteyM, et al. Next-generation sequencing as a tool for the study of porcine reproductive and respiratory syndrome virus (PRRSV) macro-and micro-molecular epidemiology. Vet Microbiol2017;209:5–12.
8.
CrossleyBM, et al. Guidelines for Sanger sequencing and molecular assay monitoring. J Vet Diagn Invest2020;32:767–775.
9.
GaugerPHarmonK.PRRS CLAMP: molecular diagnostic tools to distinguish wild-type and vaccine strains of PRRSV. In: Proc 52nd AASV Annual Meeting; Virtual; March 2021.
10.
HoltkampDJ, et al. Proposed modifications to porcine reproductive and respiratory syndrome virus herd classification. J Swine Health Prod2021;29:261–270.
11.
KatohKStandleyDM.MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol2013;30:772–780.
12.
KikutiM, et al. Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) genetic diversity and occurrence of wild type and vaccine-like strains in the United States swine industry. PLoS One2021;16:e0259531.
13.
LinharesDC, et al. Comparison of time to PRRSv-stability and production losses between two exposure programs to control PRRSv in sow herds. Prev Vet Med2014;116:111–119.
14.
LinharesDC, et al. Effect of modified-live porcine reproductive and respiratory syndrome virus (PRRSv) vaccine on the shedding of wild-type virus from an infected population of growing pigs. Vaccine2012;30:407–413.
15.
OleksiewiczMB, et al. Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes. Vet Microbiol1998;64:7–22.
PanJ, et al. Research progress on the detection methods of porcine reproductive and respiratory syndrome virus. Front Microbiol2023;14:1097905.
18.
PaploskiIA, et al. Phylogenetic structure and sequential dominance of sub-lineages of PRRSV type-2 lineage 1 in the United States. Vaccines2021;9:608.
19.
PaploskiIAD, et al. Temporal dynamics of co-circulating lineages of porcine reproductive and respiratory syndrome virus. Front Microbiol2019;10:2486.
20.
PecmanA, et al. Systematic comparison of nanopore and Illumina sequencing for the detection of plant viruses and viroids using total RNA sequencing approach. Front Microbiol2022;13:883921.
21.
RauschT, et al. Tracy: basecalling, alignment, assembly and deconvolution of sanger chromatogram trace files. BMC Genomics2020;21:230.
22.
SangerF, et al. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A1977;74:5463–5467.
23.
SanhuezaJM, et al. Spatial relative risk and factors associated with porcine reproductive and respiratory syndrome outbreaks in United States breeding herds. Prev Vet Med2020;183:105128.
24.
VanderWaalK, et al. Phylogenetic-based methods for fine-scale classification of PRRSV-2 ORF5 sequences: a comparison of their robustness and reproducibility. Front Virol2024;4:1433931.
25.
VilaltaC, et al. A review of quantitative tools used to assess the epidemiology of porcine reproductive and respiratory syndrome in US swine farms using Dr. Morrison’s Swine Health Monitoring Program data. Front Vet Sci2017;4:94.
26.
WernikeK, et al. Detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-PCR. PLoS One2012;7:e38251.
27.
WesleyRD, et al. Differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from North American field strains by restriction fragment length polymorphism analysis of ORF 5. J Vet Diagn Invest1998;10:140–144.
28.
ZhangJ, et al. High-throughput whole genome sequencing of porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next- generation sequencing technology. J Vet Diagn Invest2017;29:41–50.
