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The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine. [ABSTRACT FROM AUTHOR]

Since we first reported porcine reproductive and respiratory syndrome virus 1 (PRRSV1) wild type strains in mainland China in 2011, PRRSV1 infection has been detected in more than 20 provinces in China. During the routine investigation of PRRSV1 epidemiology in 2022, we isolated a novel PRRSV1 strain (SD1291) from an adult slaughter pig in Linyi, Shandong Province. The SD1291 could only be isolated with primary alveolar macrophages (PAMs), not with Marc-145 cells. In addition, the 2022 SD1291 isolate has higher in vitro replication efficacy than the 2014 PRRSV1 HLJB1 isolate in PAMs. Due to high genetic variation, the complete genome of SD1291 was determined by metagenomic sequencing, which showed that SD1291 shares the highest genome similarity (88.12%) with the PRRSV1 HeB47 isolate. Sequence alignment results identified a four-amino-acid deletion in nsp2 and a five-amino-acid deletion in the GP3 and GP4 overlap region of SD1291. A complete-genome-based phylogenetic tree showed that SD1291 is grouped with BJEU06-1-like PRRSV1 isolates. A piglets' challenge study showed that SD1291 can cause high fever (the highest is 41°C), reduced weight gain, mild lung consolidation, and interstitial pneumonia indicating that SD1291 is a pathogenic PRRSV1 isolate. Overall, this study first identified a novel pathogenic PRRSV1 isolate from an adult slaughter pig in China. Our findings also suggested that new PRRSV1 variants could escape the current PRRSV vaccination system and circulate in adult swine herds, which deserve more attention. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 cells. To further evaluate the potential use of the cloned rJXA1‐R virus as a live vector for foreign gene expression, the enhanced green fluorescent protein (EGFP) was inserted between non‐structural and structural genes. Our results showed that the dynamic expression of EGFP can be visualized by live cell imaging system during the infection in Marc‐145 cells. The availability of our cloned JXA1‐R viruses provides a crucial platform to study the fundamental biology of HP‐PRRS virus vaccine and also serves as a potential effective vector for developing live vector vaccines against swine pathogens. [ABSTRACT FROM AUTHOR]

• Two chimeric PRRSV strains containing consensus sequences of ORF2-4 or ORF5-6 genes were generated. • These chimeric viruses had distinct in vitro and in vivo replication efficacy. • Pig inoculation of both chimeric viruses did not cause obvious clinical signs. • Both chimeric viruses could induce heterologous nAbs but only confer partial cross protection. • Minor and major envelope proteins probably have synergistic effects on inducing heterologous nAbs and conferring cross protection. High genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) isolates is a major obstacle for the development of effective PRRS vaccines. A chimeric highly pathogenic PRRSV2 (HP-PRRSV2) strain containing the consensus sequence of ORF2-6 genes was constructed in our previous study, which could induce broadly neutralizing antibodies (bnAbs) and confer satisfied cross protection against virulent NADC30-like isolate. To further elucidate the roles of minor and major envelope proteins encoded by ORF2-4 and ORF5-6 genes in conferring cross protection, two chimeric HP-PRRSV2 strains (rJS-ORF2-4-CON and rJS-ORF5-6-CON) containing consensus sequences of ORF2-4 or ORF5-6 were constructed and rescued in this study. The rJS-ORF5-6-CON strain has similar replication efficiency as the backbone HP-PRRSV2 rJSTZ1712-12 virus, while rJS-ORF2-4-CON has significantly lower in vitro and in vivo replication efficiency comparing to rJS-ORF5-6-CON. Animal inoculation indicated that both rJS-ORF2-4-CON and rJS-ORF5-6-CON did not cause obvious clinical signs in piglets and could induce heterologous nAbs after immunization. Challenge with a virulent heterologous NADC30-like SD17-38 isolate showed that even though both immunized groups presented lower viremia, faster virus elimination, less fever and alleviated lung gross lesions when compared with the only challenged pigs, rJS-ORF2-4-CON and rJS-ORF5-6-CON could not confer enough cross protection. Considering the bnAbs and satisfied cross protection induced by the chimeric virus containing ORF2-6 consensus sequence, our results support that minor and major envelope proteins play synergistic roles in inducing broader nAbs and conferring better cross protection. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates show high genetic and pathogenic diversity. The mechanisms underlying different virulence of PRRSV isolates are still not fully clarified. Two highly homologous PRRSV isolates (XJ17-5 and JSTZ1712-12) with distinct virulence were identified in our previous study. To evaluate the association between host responses and different virulence, here we investigated the transcriptomic profiles of porcine alveolar macrophages (PAMs) infected with these two isolates. RNA-Seq results showed that there are 1932 differential expression genes (DEGs) between two PRRSV infected groups containing 1067 upregulation and 865 downregulation genes. Compared with the avirulent JSTZ1712-12 infected group, GO analysis identified significant enrichment gene sets not only associated with virus infection but also innate immune response in the virulent XJ17-5 infected group. In addition, KEGG analysis indicated significantly enriched genes associated with NOD-like and RIG-I-like receptor signaling pathways in XJ17-5 vs JSTZ1712-12 group. Furthermore, XJ17-5 isolate induced significantly higher levels of innate immune response associated genes (IL-1β, CXCL2, S100A8, OAS2, MX1, IFITM3, ISG15 and IFI6) than JSTZ1712-12 isolate, which were further confirmed by real-time PCR. Given that these two isolates share similar replication efficiency in vivo and in vitro , our results indicated that distinct virulence of PRRSV isolates is associated with different host innate immune responses. • PRRSV virulent XJ and avirulent JS isolates had similar replication efficiency in PAMs. • RNA-seq analysis identified significantly different expression genes in XJ vs JS group. • GO analysis suggested that XJ and JS infection induced different innate immune responses. • KEGG analysis identified DEGs associated with NOD-like and RIG-I-like receptor signaling pathways in XJ vs JS group. • Real-time PCR results supported that XJ and JS infection induced different innate immune responses. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to the global swine industry. Even though several control strategies have been applied, PRRS is still not effectively controlled due to the continuous emergence of new variants and limited cross-protection by current vaccines. During the routine epidemiological investigation in 2017, two PRRSV variants were identified from a severe abortion farm and a clinically healthy farm, respectively. The viruses were isolated and denominated as XJ17-5 and JSTZ1712-12. Genomic sequencing indicated that their genomes are both 14,960 bp in length sharing 99.45% nucleotide identity. Sequence alignments identified a discontinuous 30-amino-acid deletion and a continuous 120-amino-acid deletion in nsp2 of both isolates. Genome-based phylogenetic analysis confirmed that XJ17-5 and JSTZ1712-12 belong to the HP-PRRSV subtype but form a new branch with other isolates containing the same 150-amino-acid deletion in nsp2. Pathogenic analysis showed that XJ17-5 is highly virulent causing 60% mortality, while JSTZ1712-12 is avirulent for piglets. Furthermore, fragment comparisons identified 34-amino-acid differences between XJ17-5 and JSTZ1712-12 that might be associated with the distinct virulence. The identification of highly homologous HP-PRRSV variants with new genetic feature and distinct virulence contributes to further analyze the pathogenesis and evolution of PRRSV in the field. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) has been prevalent in more than 20 provinces of China. However, no PRRSV-1-specific vaccine is commercially available in China. To evaluate the feasibility of using a low virulent PRRSV-1 isolate against potential outbreaks caused by virulent Chinese PRRSV-1 isolates, here we evaluated the efficacy of a low virulent PRRSV-1 HLJB1 strain isolated in 2014 as live vaccine against a virulent PRRSV-1 SD1291 strain isolated in 2022. Genome-based phylogenetic analysis showed that both HLJB1 and SD1291 were grouped within BJEU06–1-like isolates. However, they shared only 85.27% genomic similarity. Piglet inoculation and challenge study showed that HLJB1 inoculation could reduce viremia but did not significantly alleviate clinical signs and tissue lesions. Virus neutralization test indicated that HLJB1 inoculation could induce homologous neutralizing antibodies (NAbs) but no heterologous NAbs at 42 dpi. In addition, flow cytometric analyses showed that no memory T follicular helper (Tfh) cells against SD1291 and SD1291-specific IFN-γ secreting cells were induced by HLJB1 pre-inoculation. These results supported that HLJB1 inoculation only provides partial cross-protection against SD1291 infection even though they are clustered within the same PRRSV-1 subgroup, which is closely related to the failure in conferring cross-protective adaptive immune responses. • HLJB1 and SD1291 are both BJEU06-1-like PRRSV-1 isolates. • HLJB1 inoculation only confers partial cross-protection against SD1291 challenge. • HLJB1 inoculation cannot induce memory Tfh cells against SD1291 infection. • HLJB1 inoculation may induce homologous NAbs but no heterologous NAbs. • HLJB1 inoculation cannot generate SD1291-specific IFN-γ secreting cells. [ABSTRACT FROM AUTHOR]