Implementing a user‐friendly format to analyze PRRSV next‐generation sequencing results and associating breeding herd production performance with number of PRRSV strains and recombination events

Abstract

The open reading frames (ORF)5 represents approximately 4% of the porcine reproductive and respiratory syndrome virus (PRRSV)-2 genome (whole-PRRSV) and is often determined by the Sanger technique, which rarely detects >1 PRRSV strain if present in the sample. Next-generation sequencing (NGS) may provide a more appropriate method of detecting multiple PRRSV strains in one sample. This work assessed the effect of PRRSV genetic variability and recombination events, using NGS, on the time-to-low prevalence (TTLP) and total losses in breeding herds (n 20) that detected a PRRSV outbreak and adopted measures to eliminate PRRSV. Serum, lung or live virus inoculation material collected within 3-weeks of outbreak, and subsequently, processing fluids (PFs) were tested for PRRSV by RT-qPCR and NGS. Recovered whole-PRRSV or partial sequences were used to characterize within and between herd PRRSV genetic variability. Whole-PRRSV was recovered in five out of six (83.3%) lung, 16 out of 22 (72.73%) serum and in five out of 95 (5.26%) PF. Whole-PRRSV recovered from serum or lung were used as farm referent strains in 16 out of 20 (80%) farms.In four farms, only partial genome sequences were recovered and used as farm referent strains. At least two wild-type PRRSV strains (wt-PRRSV) were circulating simultaneously in 18 out of 20 (90%) and at least one vaccine-like strain co-circulating in eight out of 20 (40%) farms. PRRSV recombination events were detected in 12 farms (59%), been 10 out of 12 between wt-PRRSV and two out of 12 between wt-PRRSV and vaccine-like strains. Farms having ≥3 strains had a 12-week increase TTLP versus herds ≤2 strains detected. Farms with ≤2 strains (n 10) had 1837 and farms with no recombination events detected (n 8) had 1827 fewer piglet losses per 1000 sows versus farms with ≥3 PRRSV strains (n 8) or detected recombination (n 10), respectively. NGS outcomes and novel visualization methods provided more thorough insightThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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

“…Alignment of sample contigs to the specific reference sequences could identify PRRSV‐2 mixed infections, as demonstrated in the current study (e.g. ISU1014, ISU1015, ISU1017, and ISU1020 clinical samples) and a recently published study (Trevisan et al., 2022). However, if the sequences of two PRRSV strains in one sample are not diverged enough or if most of sequence reads are from one virus strain, different contigs covering the same genomic region will not be obtained and instead only consensus sequences are assembled.…”

“…One intriguing question is whether the sequence reads observed in the samples in this study under the IGV This study also provides insights on how to identify ≥2 PRRSV-2 strains in one sample using the NGS data. and a recently published study (Trevisan et al, 2022). However, if the sequences of two PRRSV strains in one sample are not diverged enough or if most of sequence reads are from one virus strain, different contigs covering the same genomic region will not be obtained and instead only consensus sequences are assembled.…”

“…In order to confirm that the PRRSV‐2 Category 3 samples contained ≥2 PRRSV‐2 strains, selected clinical samples and cell culture isolates in this category were further investigated by Ingelvac PRRS MLV vaccine‐specific PCR and NGS. When the standard procedures of mapping sequence reads to PRRSV reference sequence data set using the BWA mapping software and de novo assembly (Trevisan et al., 2022; Zhang et al., 2017) were conducted, sequence contigs or near full‐length genome sequences were obtained from the clinical samples and near full‐length PRRSV‐2 genome sequences were obtained from all of the MARC‐145 and ZMAC isolates. Alignment of the sequence contigs of clinical samples with the corresponding ZMAC isolate and MARC‐145 isolate sequences indicated that each of the clinical samples ISU1014, ISU1015, ISU1017, and ISU1020 includes ≥2 PRRSV‐2 strains (Figure 1).…”

“…For NGS data analysis, reads were mapped to PRRSV reference sequences using the Burrow–Wheeler Aligner (BWA) mapping software with the default threshold identity setting and then de novo assembly was performed following the previous studies (Trevisan et al., 2022; Zhang et al., 2017) to obtain a consensus whole‐genome sequence or contigs of the viral genome. Sequences of PRRSV from clinical samples were compared with the virus sequences in the corresponding MARC‐145 and ZMAC isolates.…”

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

“…Co-circulation of multiple PRRSV strains in a breeding herd is a reality ( 23 ). The emergence of new PRRSV strains can be due to low fidelity during RNA virus replication, recombination events, or random mutations ( 1 , 24 ).…”