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 today’s Science Page the Veterinary Diagnostic Laboratory and the Production Animal Medicine team at Iowa State University look into whether cell culture isolates and viruses in the associated clinical samples are sequence equivalent or not.
Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in cell culture is a primary means of obtaining virus isolates for autogenous vaccine production and other applications. The objective of this study was to determine whether cell culture isolate and the virus in the clinical sample are sequence equivalent or not.
Materials and Methods
In total, 1024 PRRSV PCR-positive (995 PRRSV-2, 26 PRRSV-1, and 3 co-infected PRRSV-1 and PRRSV-2 PCR-positive) clinical samples and their isolates obtained in MARC-145 and/or ZMAC cells during 2010-2020 were included in this study. ORF5 sequences of 1024 clinical samples, 837 MARC-145 isolates (passage 0 or P1), and 270 ZMAC isolates (P0 or P1) were determined and compared for RFLP patterns, genetic lineages, and nucleotide identities. For those cases with non-matching PRRSV between clinical sample and cell culture isolate, next-generation sequencing (NGS) and vaccine-specific PCR were conducted to elucidate the differences.
Major findings and implications
- PRRSV virus isolation (VI) success rate was significantly higher in ZMAC than in MARC-145 cells for serum and lung samples containing PRRSV-1, PRRSV-2, or PRRSV-1 & PRRSV-2 co-infection.
- For clinical samples evaluated in this study (3 positive for both PRRSV-1 and PRRSV-2, 26 PRRSV-1, and 96.2% [957/995] of PRRSV-2), the predominant ORF5 sequences of PRRSV in the clinical samples and the respective cell culture isolates were matching in regards to RFLP patterns, genetic lineages, and nucleotide identities (Table 1).
- Small percentage of PRRSV-2 PCR-positive clinical samples (2.4%, Category 2) and their MARC-145 and/or ZMAC isolates had 98.6-99.8% ORF5 nucleotide identity and the same genetic lineages but different RFLP patterns due to point mutation(s) located at the HincII or SacII restriction site, but were considered as the same virus strains (Table 1).
- In the PRRSV-2 Category 3 (14/995; 1.4%), the predominant PRRSV-2 ORF5 sequences derived directly from clinical samples were different from those from their corresponding MARC-145 isolates but were similar to their corresponding ZMAC isolates (Table 1). In those cases, most isolates obtained in MARC-145 cells contained Ingelvac PRRS MLV vaccine-like virus while the predominant viral sequences detected in clinical samples and ZMAC isolates were wild-type strains. This is concerning because autogenous vaccines produced from MARC-145 isolates may not contain the desired wild-type virus strain found on the farm.
- Vaccine-specific PCR and NGS performed on selected cases in PRRSV-2 Category 3 confirmed presence of ≥2 PRRSV-2 strains (mixed infection) in such clinical samples. In co-infected samples, while Sanger sequencing determines the predominant strain ORF5 sequence from the clinical sample (i.e. wild-type A), if wild-type strain A has lower growth adaptability or kinetics compared to the other strain (i.e. vaccine-like strain B) in MARC-145 cells, vaccine-like strain B could be isolated in MARC-145 cells although wild-type strain A is isolated in ZMAC cells.
- Characterizing PRRSV sequences from clinical samples and cell culture isolates should be conducted before using isolates for producing autogenous vaccines or other applications.