Today, we are sharing a publication from recent PhD graduate Joaquin Alvarez Norambuena in collaboration with the VDL and the MSHMP team in the Journal of Veterinary Diagnostic Investigation. In this paper, Dr. Alvarez Norambuena explores how Sanger sequencing performs when testing samples with multiple PRRS strains.
Methods
- Two PRRSV strains were used in this study: 1 vaccine based and 1 field based
- Nine different solutions were created with concentration of each virus ranging between 104 and 106 copies/mL.
- Each solution was tested by Rt-PCR and ORF5 Sanger sequencing.
- Each testing was done in triplicates to evaluate repeatability of the findings.
Results
- Sequences were retrieved from 22 out of the 27 samples.
- When the vaccine concentration was a 104 copies/mL and wild type concentration was less than 106 copies/mL, sequences were not found, despite a positive Rt-PCR.
- The vaccine sequence was found most often if the vaccine strain concentration was higher or equal to vaccine concentration. If the field virus concentration was higher in the sample, the sequence isolated was really similar to the wild one.
- 10 ambiguous nucleotides were found in 4 mixed samples.
- When 106. copies/mL of field virus were mixed with 105 copies/mL of vaccine strain, one test result showed a sequence with a RFLP pattern similar to the field, strain, one similar to the vaccine one and a last sequence with a new RFLP pattern.
Abstract
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.