Establishment of a nanoparticle-assisted RT-PCR assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus

Abstract

Porcine epidemic diarrhea virus (PEDV) can cause serious disease and even death in neonatal piglets, resulting in serious damage to the swine industry worldwide. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. Previous studies have indicated that PEDV vaccine strains have a partial deletion in ORF3. In this study, a nanoparticle-assisted polymerase chain reaction (nanoparticle-assisted RT-PCR) assay targeting the ORF3 of PEDV was developed to distinguish PEDV field strains from attenuated strains by using a specific pair of primers. The PCR products of field strains and attenuated strains were 264 bp and 215 bp in length, respectively. The sensitivity and specificity of this assay were also assessed. The nanoparticle-assisted RT-PCR assay was 10-100 times more sensitive than the conventional RT-PCR assay, with no cross-reactions when amplifying porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (RV), and porcine transmissible gastroenteritis virus (TGEV). The nanoparticle-assisted RT-PCR assay we describe here can be used to distinguish field strains from vaccine strains of PEDV, and it shows promise for reducing economic loss due to PEDV infection.

Development of a multiplex RT‐PCR for the detection of major diarrhoeal viruses in pig herds in China

“…Although several standard detection methods, for example virus isolation, virus neutralization tests, and indirect immunofluorescence assay, are available for the detection of viruses, these techniques are time‐consuming and not suitable for detecting large‐scale samples. Currently, polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA) methods for the detection of these viruses have been reported (Luo et al, ; Ma et al, ; Zhu, Wang, Cui, & Cui, ). Due to the high pathogenicity of these viruses to suckling piglets, which have immature immune systems and few antibodies, ELISA is inefficient for detecting these viruses compared to PCR.…”

Emergence and evolution of highly pathogenic porcine epidemic diarrhea virus by natural recombination of a low pathogenic vaccine isolate and a highly pathogenic strain in the spike gene

“…Since an attenuated live vaccine (CV777) was used for immunizations before the PEDV outbreak, we investigated whether PEDV was caused by a wild strain or a vaccine strain. The CV777 vaccine strain was characterized by a 49-bp deletion in the ORF3 gene, which is usually used in the differential diagnosis between wild and vaccine strains ( Zhu et al. 2016 ).…”

“…When obvious cytopathic effects were apparent, including the characteristic fused-syncytium cell formation, the viral supernatant was collected and used for RNA extraction for subsequent cloning and sequencing. The 49-bp deletion of the ORF3 gene was assessed for the differential diagnosis between vaccine and field strains for PEDV positive samples ( Zhu et al. 2016 ; He et al.…”

Differentiation of PEDV Classical Attenuated Vaccine Strains from Wild-type Strains using One-Step Real-Time Fluorescent Reverse Transcription PCR Assay Targeting ORF1 Nucleotides Deletion Region

“…There is a short period of carrying attenuated vaccine virus in the orally immunized pig population, and the possibility that the wild-type strains and the attenuated vaccine strains will exist simultaneously in the pig herds [12,13]. In recent study, multiple strains were also found in many immunized pig population in China [4,14,15], and phylogenetic analysis of the whole genome also showed that there are multiple variation sites in PEDV genome [16,17]. The antigenicity of vaccines derived from classical vaccine strains may be altered due to these genetic mutations, resulting in low vaccination e ciency and inability to protect pigs from variant PEDV strains [16].…”