Characterization of PRRSV in clinical samples and the corresponding cell culture isolates

Abstract

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. However, it has not been well characterized whether cell culture isolate and the virus in clinical sample are equivalent. This study compared PRRSV ORF5 sequences from 1024 clinical samples (995 PRRSV‐2, 26 PRRSV‐1, and three PRRSV‐1 and PRRSV‐2 PCR‐positive) and their isolates in MARC‐145 and/or ZMAC cells. For three PRRSV‐1 and PRRSV‐2 PCR‐positive clinical samples, both PRRSV‐1 and PRRSV‐2 were isolated in ZMAC cells, whereas either PRRSV‐1 or PRRSV‐2, but not both, was isolated in MARC‐145 cells, with isolate sequences matching the respective viruses in clinical samples. Twenty‐six PRRSV‐1 and most of 995 PRRSV‐2 PCR‐positive clinical samples had matching viral ORF5 sequences with their cell culture isolates. However, 14 out of 995 PRRSV‐2 cases (1.4%) had nonmatching viral sequences between clinical samples and MARC‐145 isolates, although viral sequences from clinical samples and ZMAC isolates matched. This is concerning because, if the MARC‐145 isolate is directly used for autogenous vaccine production without sequencing confirmation against the virus in the clinical sample, it is possible that the produced autogenous vaccine does not include the desired wild‐type virus strain found on the farm and instead contains vaccine‐like virus. Vaccine‐specific PCR and next‐generation sequencing performed on six selected cases indicated presence of ≥2 PRRSV‐2 strains (mixed infection) in such clinical samples. In summary, PRRSV ORF5 sequences from clinical samples and cell culture isolates matched each other for majority of the cases. However, PRRSV sequences between clinical sample and MARC‐145 cell culture isolate could occasionally be different when the clinical sample contains ≥2 PRRSV‐2 strains. Characterizing PRRSV sequences from clinical samples and cell culture isolates should be conducted before using isolates for producing autogenous vaccines or other applications.

Spatiotemporal Distribution of PRRSV-1 Clades in Hungary with a Focus on the Era of Disease Eradication

“…One crucial pillar of disease control is the genetic characterization of circulating virus strains in order to identify the origin of infection and determine the routes of transmission between pig farms, within a geographical region or an administrative area. Sequencing one of the most variable genomic regions, the ORF5, is still a gold-standard procedure for surveying these issues [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. The information obtained by means of molecular epidemiology further assists the improvement of external disease control measures of a given pig herd and provides the basis for reliable production of animal goods.…”

A recombinant porcine reproductive and respiratory syndrome virus type 2 field strain derived from two PRRSV-2-modified live virus vaccines

“…An indigenous clinical sample could potentially contain more than one PRRSV strain and could produce genetic sequences of either strain when submitted for Sanger or NGS sequencing. A virus isolation process favors the selection of one strain ( 50 ). Therefore, the NGS performed on a virus isolate made it conclusive for recovering and discerning the presence of a single recombinant strain in the sample.…”

Development of a one-step reverse transcription-quantitative polymerase chain reaction assay for the detection of porcine reproductive and respiratory syndrome virus

“…Therefore, it is often preceded by other molecular diagnostic tests. Porcine alveolar macrophages and MARC-145 are the most susceptible cells for PRRSV [ 54 – 57 ]. However, the susceptibility can vary between batches of cells and viruses.…”

“…However, the susceptibility can vary between batches of cells and viruses. For instance, field-isolated viruses, in particular, are generally present in low concentrations, and the susceptibility of the cell could be variable, leading to increased rates of culture failure [ 54 , 57 ]. Furthermore, confirmation of PRRSV growth often requires subsequent RT-PCR analysis, which introduces the risk of contamination.…”