Abstract:Multiple commercial porcine reproductive and respiratory syndrome (PRRS) modified live vaccines are currently utilized in Chinese swine herds due to the limited cross‐protection of vaccines and coexistence of different PRRS viruses. In this study, an infectious cDNA clone of the highly pathogenic PRRS (HP‐PRRS) vaccine JXA1‐R strain was generated. We successfully rescued the virus from direct in vitro DNA transfection of rJXA1‐R clone, which has similar growth kinetics to the parental JXA1‐R virus in Marc‐145 … Show more
“…The rJSTZ1712-12 and rJS-ORF2-6-CON full-length cDNA clones were generated using the reverse-genetic system as we previously described (Figure 2 ) [ 25 ]. After five time passages in Marc-145 cells, typical PRRSV-specific cytopathic effects (CPE) could be observed at about 3–4 dpi.…”
Section: Resultsmentioning
confidence: 99%
“… Figure 2 Strategy to construct the full-length cDNA clones of avirulent HP-PRRSV2 JSTZ1712-12 isolate and a chimeric virus containing ORF2-6-CON . The strategy was adopted from our previous study [ 25 ]. The pACYC177-CMV-Stuffer fragment including the unique restriction enzymes was shown in the upper part ( A ).…”
Section: Resultsmentioning
confidence: 99%
“…To generate an infectious clone carrying the ORF2-6-CON sequence, a full-genome cDNA clone of the avirulent HP-PRRSV2 isolate, JSTZ1712-12, was first constructed as we previously described [ 25 ]. Three overlapped fragments (F1, F2 and F3) spanning the full-length genome of JSTZ1712-12 were produced by PCR amplification using high fidelity PrimeSTAR MAX DNA Polymerase (TaKaRa, Shiga, Japan) with a panel of primers (Table 1 ).…”
Section: Methodsmentioning
confidence: 99%
“…Therefore, a consensus sequence of ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins of PRRSV was designed and synthesized. An infectious clone of avirulent HP-PRRSV2 isolate JSTZ1712-12 was first generated (named as rJSTZ1712-12) as we previously described [ 24 , 25 ], and then used as the backbone for the construction of a chimeric virus containing ORF2-6-CON (designated as rJS-ORF2-6-CON). The chimeric virus is fully infectious in vitro and in vivo.…”
Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.
“…The rJSTZ1712-12 and rJS-ORF2-6-CON full-length cDNA clones were generated using the reverse-genetic system as we previously described (Figure 2 ) [ 25 ]. After five time passages in Marc-145 cells, typical PRRSV-specific cytopathic effects (CPE) could be observed at about 3–4 dpi.…”
Section: Resultsmentioning
confidence: 99%
“… Figure 2 Strategy to construct the full-length cDNA clones of avirulent HP-PRRSV2 JSTZ1712-12 isolate and a chimeric virus containing ORF2-6-CON . The strategy was adopted from our previous study [ 25 ]. The pACYC177-CMV-Stuffer fragment including the unique restriction enzymes was shown in the upper part ( A ).…”
Section: Resultsmentioning
confidence: 99%
“…To generate an infectious clone carrying the ORF2-6-CON sequence, a full-genome cDNA clone of the avirulent HP-PRRSV2 isolate, JSTZ1712-12, was first constructed as we previously described [ 25 ]. Three overlapped fragments (F1, F2 and F3) spanning the full-length genome of JSTZ1712-12 were produced by PCR amplification using high fidelity PrimeSTAR MAX DNA Polymerase (TaKaRa, Shiga, Japan) with a panel of primers (Table 1 ).…”
Section: Methodsmentioning
confidence: 99%
“…Therefore, a consensus sequence of ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins of PRRSV was designed and synthesized. An infectious clone of avirulent HP-PRRSV2 isolate JSTZ1712-12 was first generated (named as rJSTZ1712-12) as we previously described [ 24 , 25 ], and then used as the backbone for the construction of a chimeric virus containing ORF2-6-CON (designated as rJS-ORF2-6-CON). The chimeric virus is fully infectious in vitro and in vivo.…”
Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.
“…Porcine epidemic diarrhea virus (PEDV) strain YC2014 was from Dr. Hongjie Fan in Nanjing Agricultural University China. Porcine reproductive and respiratory syndrome virus (PRRSV) strain JXA1-R and anti-N rabbit antibody were preserved in our laboratory ( 32 ).…”
The RLRs play critical roles in sensing and fighting viral infections especially RNA virus infections. Despite the extensive studies on RLRs in humans and mice, there is a lack of systemic investigation of livestock animal RLRs. In this study, we characterized the porcine RLR members RIG-I, MDA5 and LGP2. Compared with their human counterparts, porcine RIG-I and MDA5 exhibited similar signaling activity to distinct dsRNA and viruses, via similar and cooperative recognitions. Porcine LGP2, without signaling activity, was found to positively regulate porcine RIG-I and MDA5 in transfected porcine alveolar macrophages (PAMs), gene knockout PAMs and PK-15 cells. Mechanistically, LGP2 interacts with RIG-I and MDA5 upon cell activation, and promotes the binding of dsRNA ligand by MDA5 as well as RIG-I. Accordingly, porcine LGP2 exerted broad antiviral functions. Intriguingly, we found that porcine LGP2 mutants with defects in ATPase and/or dsRNA binding present constitutive activity which are likely through RIG-I and MDA5. Our work provided significant insights into porcine innate immunity, species specificity and immune biology.
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