Divergence in the molecular diagnosis of Porcine Reproductive and Respiratory Syndrome virus

The Porcine Reproductive and Respiratory Syndrome virus (PRRSv) is a disease endemic on most porcine herds causing significant economic impact in the pig sector. Two genotypes were identified nearly simultaneously in Europe and the USA with antigenic differences that lead to two distinct genotypes: the European type (genotype 1) and the North American type (genotype 2).

The PRRSv infection status has been followed in Flemish voluntarily participating herds in 2015 (More details in companion abstracts ‘Piglet Monitoring’ in Northern Belgium). Forty two pools of 3 sera samples previously tested positive for PRRSv by means of the vetMAX PRRSV EU/NA real-time PCR assay (LSI) were retested individually (n=126) for PRRSv by 3 different RT-PCR tests including a conventional RT-PCR targeting the ORF5 region; a Sybr green RT-PCR assay targeting the ORF7 region and the commercial VIROTYPE real-time RT-PCR (Qiagen). The ORF5 amplicons were sequenced in order to confirm the present viral genotype. The Sybr green and Virotype PCR assays allow discrimination between PRRSv genotypes on basis of melting temperature (Tm) or probes specificity respectively.

Among the samples only 44% were identified as positive by the 3 molecular assays. Considering the assays individually 84% of samples gave positive result with the conventional PCR, 68% with the Sybr green assay and 65% with the Virotype PCR assay.

Sequencing performed on partial ORF 5 amplicons (n=106) indicated the predominance of the genotype 1 (91%). The European type (n=96) was confirmed for 37% of the samples by the Sybr green assay and for 53% by the Virotype PCR assay whereas 13 and 12% of them were identified as genotype 2 by the same tests respectively and for 15 other % the Tm obtained in Sybr green assay did not allow a distinction between genotypes 1 and 2.

In parallel the Sybr green and the Virotype PCR assays have confirmed the North American genotype identification obtained by sequencing for 2 and 3 out of the 4 samples whereas one sample was identified as European strain by both tests

. Considering the results of pool testing by the fourth PCR assay (vetMAX, LSI) the accuracy of genotype identification was 83% for the conventional PCR assay; 58% for the Sybrgreen assay and 78% for the Virotype PCR assay.

The divergence observed between the different molecular assays is problematic as it demonstrated that none of the tested methods was efficient to ensure a confidant detection of virus presence in herds. Moreover, it appears that both the melting temperature range from the Sybr green assay and the specificity of the probes could no more be considered as a confident criteria for genotype identification.

Disclosure of Interest: None Declared.