Experimental in-vitro evaluation of PRRSv modified-live vaccine and wild-type virus sequence detection in co-infections

This is our Friday rubric: every week a new Science Page from the Bob Morrison’s Swine Health Monitoring Project. The previous editions of the science page are available on our website.

In this week’s science page UMN Veterinary Population Medicine researchers Joaquin Alvarez-Norambuena, Mariana Kikuti, Albert Rovira and Cesar Corzo investigate the detection of wild-type or modified-live vaccine sequences in in-vitro samples.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) virus is known to cause great economic losses in the swine industry (1). Modified-live vaccines (MLV) have been used in the field to mitigate the clinical impact and reduce wild-type virus shedding before wild-type (WT) exposure (2). Vaccines have also been used during an outbreak as a control strategy (3, 4). The main diagnostic test to confirm a new viral introduction is PCR together with ORF5 sequencing. However, interpretation of these results can be complex when two strains in the same sample are present since the sequencing of one strain may be favored (5). Therefore, the aim of this study is to assess the detection of wild-type or vaccine-like sequences in in-vitro samples spiked with both strains at different concentrations.

Materials and Methods

Two viruses were used for this study, a WT strain RFLP 1-7-4 lineage 1A and a MLV strain RFLP 2-5-2 lineage 5 from Ingelvac PRRS MLV (Boehringer Ingelheim Animal Health, Duluth, Georgia, USA). Each strain was subjected to quantitative PCR (qPCR) at the University of Minnesota Veterinary Diagnostic Laboratory (UMNVDL), and subsequently diluted to obtain three different concentrations of RNA copies per mL (i.e. 106, 105, 104). Samples comprised a mix of all possible concentration combinations of WT and MLV to create 9 different groups with 3 replicates each (Table 1). Samples were then RT-PCR tested and ORF5 sequenced.

Results

An MLV-like sequence (RFLP 2-5-2) was most frequently obtained when the vaccine had a concentration equal or higher to the WT strain (in 5 out of 9 groups, 55.6%). A WT sequence (RFLP 1-7-4) was most frequently obtained in 1 out of 9 (11.1%) groups, when the concentration of WT was 2 logs higher than the concentration of the MLV. In group 4, one sequence was MLV-like (RFLP 2-5-2) and 2 sequences were WT (RFLP 1-7-4 and 1-10-4). Only one sequence was generated in group 8 (vaccine at 104 and wild-type at 105), which was classified as WT (RFLP 1-7-4), and none of the replicated for group 9 yielded a sequence. One to three nucleotide differences from either the original MLV or WT sequences were observed in 8 samples, as shown in the percent nucleotide identity in Table 1. This resulted in changes in the RFLP pattern of the consensus sequence for two samples.

Conclusions

We observed a tendency to sequence the vaccine strain when the MLV sample concentration was at the same or higher than the wild-type strain in the same sample. The relevancy of this study is based on field scenarios in which a co-infection can occur. An example is regular diagnosis of reproductive or respiratory disease or when vaccinated herds are undergoing WT elimination. In both cases it is important to consider that the vaccine strain may be masking wild-type infections. 

Table 1. PRRS ORF5 sequencing results by group according to
RFLP classification and percent nucleotide identity to the
original vaccine and wild-type strain.

References

  1. Holtkamp DJ, Kliebenstein JB, Neumann EJ, et al. Assessment of the economic impact of porcine reproductive and respiratory syndrome virus on United States pork producers. J Swine Health
  2. Linhares, D. C., Cano, J. P., Wetzell, T., Nerem, J., Torremorell, M., & Dee, S. A. (2012). 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. Vaccine, 30(2), 407-413.
  3. Dee, S. A., Joo, H. S., Henry, S., Tokach, L., Park, B. K., Molitor, T., & Pijoan, C. (1996). Detecting subpopulations after PRRS virus infection in large breeding herds using multiple serologic tests. Swine Health and Production, 4(4), 181.
  4. Linhares, D. C. L., Cano, J. P., Torremorell, M., & Morrison, R. B. (2014). Comparison of time to PRRSv-stability and production losses between two exposure programs to control PRRSv in sow herds. Preventive veterinary medicine, 116(1-2), 111-119.
  5. Kikuti, M., Sanhueza, J., Vilalta, C., Paploski, I. A. D., VanderWaal, K., & Corzo, C. A. (2021). 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 one, 16(11), e0259531.

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