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The 5' untranslated region (5'UTR) of many positive-stranded RNA viruses contain functional regulatory sequences. Here, we show that the porcine reproductive and respiratory syndrome virus (PRRSV), a member of arteriviruses, harbors small upstream open reading frames (uORFs) in its 5'UTR. Bioinformatics analysis shows that this feature is relatively well conserved among PRRSV strains and Arteriviridae. We also identified a uORF, namely uORF2, in the PRRSV strain JXwn06, that possesses translational activity and exerts a suppressive effect on the expression of the primary ORF evidenced by in vitro reporter assays. We tested its importance via reverse genetics by introducing a point mutation into the PRRSV infectious cDNA clone to inactivate the start codon of uORF2. The recovered mutant virus Mut2 surprisingly replicated to the same level as the wild-type virus (WT), but induced a higher level of inflammatory cytokines (e.g., TNF-α, IL-1ß, and IL-6) both in vitro and in animal experiments, correlating well with more severe lung injury and higher death rate. In line with this, over-expression of uORF2 in transfected cells significantly inhibited poly(I:C)-induced expression of inflammatory cytokines. Together, our data support the idea that uORF2 encodes a novel, functional regulator of PRRSV virulence despite of its short size. IMPORTANCE: PRRSV has remained a major challenge to the world swine industry, but we still do not know much about its biology and pathogenesis. Here, we provide evidence to show that the 5'UTR of PRRSV strain JXwn06 harbors a functional uORF that has the coding capacity and regulates induction of inflammation as demonstrated by in vitro assays and animal experiment. The findings reveal a novel viral factor that regulates cellular inflammation and provide insight into the understanding of PRRSV pathogenesis.
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Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.
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African swine fever virus (ASFV) is notoriously known for evolving strategies to modulate IFN signaling. Despite lots of efforts, the underlying mechanisms have remained incompletely understood. This study concerns the regulatory role of viral inner membrane protein p17. We found that the ASFV p17 shows a preferential interaction with cGAS-STING-IRF3 pathway, but not the RIG-I-MAVS-NF-κB signaling, and can inhibit both poly(I:C)- and poly(A:T)-induced activation of IRF3, leading to attenuation of IFN-ß induction. Mechanistically, p17 interacts with STING and IRF3 and recruits host scaffold protein PR65A, a subunit of cellular phosphatase PP2A, to down-regulate the level of p-IRF3. Also, p17 targets STING for partial degradation via induction of cellular apoptosis that consequently inhibits activation of both p-TBK1 and p-IRF3. Thus, our findings reveal novel regulatory mechanisms for p17 modulation of IFN signaling and shed light on the intricate interplay between ASFV proteins and host immunity.
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The porcine reproductive and respiratory syndrome virus (PRRSV) has significantly impacted the global pork industry for over three decades. Its high mutation rates and frequent recombination greatly intensifies its epidemic and threat. To explore the fidelity characterization of Chinese highly pathogenic PRRSV JXwn06 and the NADC30-like strain CHsx1401, self-recombination and mutation in PAMs, MARC-145 cells, and pigs were assessed. In vitro, CHsx1401 displayed a higher frequency of recombination junctions and a greater diversity of junction types than JXwn06. In vivo, CHsx1401 exhibited fewer junction types yet maintained a higher junction frequency. Notably, JXwn06 showed more accumulation of mutations. To pinpoint the genomic regions influencing their fidelity, chimeric viruses were constructed, with the exchanged nsp9-10 regions between JXwn06 and CHsx1401. The SJn9n10 strain, which incorporates JXwn06's nsp9-10 into the CHsx1401 genome, demonstrated reduced sensitivity to nucleotide analogs compared to CHsx1401. Conversely, compared with JXwn06, the JSn9n10 strain showed increased sensitivity to these inhibitors. The swapped nsp9-10 also influences the junction frequency and accumulated mutations as their donor strains. The results indicate a propensity for different types of genetic variations between these two strains and further highlight the nsp9-10 region as a critical determinant of their fidelity.
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Genoma Viral , Mutación , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/clasificación , Animales , Porcinos , Síndrome Respiratorio y de la Reproducción Porcina/virología , Línea Celular , Recombinación Genética , Replicación ViralRESUMEN
The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.
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Claudinas , Células Endoteliales , Pulmón , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Pulmón/metabolismo , Pulmón/virología , Pulmón/patología , Pulmón/irrigación sanguínea , Células Endoteliales/metabolismo , Células Endoteliales/virología , Claudinas/metabolismo , Claudinas/genética , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/patología , Claudina-4/metabolismo , Claudina-4/genética , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virología , Endotelio Vascular/metabolismo , Endotelio Vascular/virología , Endotelio Vascular/patología , Células Cultivadas , Permeabilidad Capilar , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/virología , Lesión Pulmonar Aguda/patología , Citocinas/metabolismoRESUMEN
Pyroptosis is a newly discovered type of pro-inflammatory programmed cell death that plays a vital role in various processes such as inflammations, immune responses, and pathogen infections. As one of the main executioners of pyroptosis, gasdermin D (GSDMD) is a membrane pore-forming protein that typically exists in a self-inhibitory state. Once activated, GSDMD will be cleaved into an N-terminal fragment with pore-forming activity, becoming the key indicator of pyroptosis activation, and a C-terminal fragment. Although commercial antibodies against human and murine GSDMD proteins are currently available, their reactivity with porcine GSDMD (pGSDMD) is poor, which limits research on the biological functions of pGSDMD and pyroptosis in pigs in vivo and in vitro. Here, five monoclonal antibodies (mAbs) were prepared by immunizing BALB/c mice with procaryotically expressed full-length pGSDMD, all of which did not cross react with human and murine GSDMD proteins. Epitope mapping demonstrated that 15H6 recognizes amino acids (aa) at positions 28-34 of pGSDMD (LQTSDRF), 19H3 recognizes 257-260aa (PPQF), 23H10 and 27A10 recognize 78-82aa (GPFYF), and 25E2 recognizes 429-435aa (PPTLLGS). The affinity constant and isotype of 15H6, 19H3, 23H10, 27A10, and 25E2 mAbs were determined to be 1.32 × 10-9, 3.66 × 10-9, 9.04 × 10-9, 1.83 × 10-9, and 8.00 × 10-8 mol/L and IgG1/κ, IgG2a/κ, IgG2a/κ, IgG1/κ, and IgG1/κ, respectively. Heavy- and light-chain variable regions sequencing showed that the heavy-chain complementarity-determining region (CDR) sequences of all five mAbs are completely different, while the light-chain CDR sequences of the four mAbs that recognize the N-terminus of pGSDMD are identical. Our prepared mAbs provide valuable materials for studying pGSDMD function and pyroptosis. KEY POINTS: ⢠A total of five mouse anti-pGSDMD mAbs were prepared, of which four recognize the N-terminus of pGSDMD and one recognize its C-terminus. ⢠The main performance parameters of the five mAbs, including epitope, antibody titer, affinity constant, isotype, and heavy- and light-chain CDR, were characterized. ⢠All five mAbs specifically recognize pGSDMD protein and do not cross react with human and murine GSDMD proteins.
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Anticuerpos Monoclonales , Gasderminas , Humanos , Porcinos , Animales , Ratones , Inmunosupresores , Porinas , Inmunoglobulina G , Ratones Endogámicos BALB CRESUMEN
It is a common sense that porcine reproductive and respiratory syndrome virus (PRRSV) infection could cause immune failure of classical swine fever (CSF) vaccine, and porcine alveolar macrophages (PAMs) are the target cells of both. To elucidate the role of macrophage polarization in PRRSV infection induced CSF vaccine failure, an immortal porcine alveolar macrophage line PAM39 cell line was used to investigate the effect of PRRSV or/and CSFV C-strain (CSFV-C) infection on macrophage polarization in vitro. Interestingly, PRRSV single infection or PRRSV co-infection with CSFV-C promoted PAM39 cells to M1, while CSFV-C single infection induced PAM39 cells to M2. After the construction of M1 and M2 PAM39 cells polarization models, M1 polarized PAM39 cells were found to inhibit the replication of CSFV-C, and Chinese medicine such as matrine, ginsenosides and astragalus polysaccharides could alleviate the polarization of PAM39 cells and the replication of CSFV-C. Furthermore, interferon (IFN)-γ and lipopolysaccharide (LPS) co-stimulation induced NF-κB activation while matrine treatment blocked M1 polarization-induced NF-κB pathway activation. These findings provided a theoretical basis for designing a new strategy to improve the immune effect of CSFV-C based on porcine alveolar macrophage polarization subtypes.
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Peste Porcina Clásica , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Vacunas , Porcinos , Animales , FN-kappa B/metabolismo , Matrinas , Peste Porcina Clásica/prevención & control , Macrófagos Alveolares , Replicación Viral , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Enfermedades de los Porcinos/metabolismoRESUMEN
Senecavirus A (SVA) is a newly emerging picornavirus associated with swine vesicular lesions and neonatal mortality, threatening the global pig industry. Despite sustained efforts, the molecular mechanisms of SVA pathogenesis have not yet been fully elucidated. Here, we demonstrate for the first time that SVA infection can induce complete mitophagy in host cells, which depends on SVA replication. Mitophagy has been subsequently proven to promote SVA replication in host cells. Genome-wide screening of SVA proteins involved in inducing mitophagy showed that although VP2, VP3, 2C, and 3A proteins can independently induce mitophagy, only the 2C protein mediates mitophagy through direct interaction with TUFM (Tu translation elongation factor, mitochondrial). The glutamic acids at positions 196 and 211 of TUFM were shown to be two key sites for its interaction with 2C protein. Moreover, TUFM was discovered to interact directly with BECN1 and indirectly with the ATG12-ATG5 conjugate. Further experiments revealed that TUFM needs to undergo ubiquitination modification before being recognized by the macroautophagy/autophagy receptor protein SQSTM1/p62, and E3 ubiquitin ligase RNF185 catalyzes K27-linked polyubiquitination of TUFM through the interaction between RNF185's transmembrane domain 1 and TUFM to initiate SVA-induced mitophagy. The ubiquitinated TUFM is recognized and bound by SQSTM1, which in turn interacts with MAP1LC3/LC3, thereby linking the 2C-anchored mitochondria to the phagophore for sequestration into mitophagosomes, which ultimately fuse with lysosomes to achieve complete mitophagy. Overall, our results elucidated the molecular mechanism by which SVA induces mitophagy to promote self-replication and provide new insights into SVA pathogenesis.Abbreviations: aa: amino acid; Baf A1: bafilomycin A1; BHK-21: baby hamster kidney-21; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GST: glutathione S-transferase; HA: hemagglutinin; hpi: hours post-infection; hpt: hours post-transfection; IPTG: isopropyl ß-D-1-thiogalactopyranoside; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor-1; MOI: multiplicity of infection; mRFP: monomeric red fluorescent protein; MS: mass spectrometry; ORF: open reading frame; PBS: phosphate-buffered saline; SD: standard deviation; SQSTM1/p62: sequestosome 1; ST: swine testis; SVA: Senecavirus A; TCID50: 50% tissue culture infectious dose; TIMM23: translocase of inner mitochondrial membrane 23; TM: transmembrane; TOMM20: translocase of outer mitochondrial membrane 20; TUFM: Tu translation elongation factor, mitochondrial; Ub: ubiquitin; UV: ultraviolet; VDAC1: voltage dependent anion channel 1; WT: wild-type; µg: microgram; µm: micrometer; µM: micromole.
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African swine fever virus (ASFV), a devastating pathogen to the worldwide swine industry, mainly targets macrophage/monocyte lineage, but how the virus enters host cells has remained unclear. Here, we report that ASFV utilizes apoptotic bodies (ApoBDs) for infection and cell-cell transmission. We show that ASFV induces cell apoptosis of primary porcine alveolar macrophages (PAMs) at the late stage of infection to productively shed ApoBDs that are subsequently swallowed by neighboring PAMs to initiate a secondary infection as evidenced by electron microscopy and live-cell imaging. Interestingly, the virions loaded within ApoBDs are exclusively single-enveloped particles that are devoid of the outer layer of membrane and represent a predominant form produced during late infection. The in vitro purified ApoBD vesicles are capable of mediating virus infection of naive PAMs, but the transmission can be significantly inhibited by blocking the "eat-me" signal phosphatidyserine on the surface of ApoBDs via Annexin V or the efferocytosis receptor TIM4 on the recipient PAMs via anti-TIM4 antibody, whereas overexpression of TIM4 enhances virus infection. The same treatment however did not affect the infection by intracellular viruses. Importantly, the swine sera to ASFV exert no effect on the ApoBD-mediated transmission but can partially act on the virions lacking the outer layer of membrane. Thus, ASFV has evolved to hijack a normal cellular pathway for cell-cell spread to evade host responses.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Vesículas Extracelulares , Porcinos , Animales , Virus de la Fiebre Porcina Africana/fisiología , Macrófagos/metabolismo , Monocitos/metabolismo , Vesículas Extracelulares/metabolismoRESUMEN
Porcine enteric alphacoronavirus (PEAV) is a newly emerging swine enteropathogen that poses a threat to the swine industry. To understand the PEAV genome evolution, we performed a comprehensive analysis of the codon usage patterns in fifty-nine PEAV strains currently available. Phylogenetic analysis showed that PEAV can be divided into six lineages. Effective number of codons analysis demonstrated that the PEAV genome exhibits a low codon usage bias (CUB). Nucleotide composition analysis indicated that the PEAV genome has the most abundant nucleotide U content, with GC content (39.37% ± 0.08%) much lower than AU content (60.63% ± 0.08%). Neutrality and effective number of codons plot analyses suggested that natural selection rather than mutation pressure dominates the CUB of PEAV. Host adaptation analysis revealed that PEAV fits the codon usage pattern of non-human primates, humans and mice better than that of pigs. Our data enriches information on PEAV evolution, host adaptability, and cross-species transmission.
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Alphacoronavirus , Uso de Codones , Animales , Porcinos , Ratones , Filogenia , Codón , Alphacoronavirus/genética , Selección Genética , Nucleótidos , Evolución MolecularRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen for the global pork industry. Although modified live virus (MLV) vaccines are commonly used for PRRSV prevention and control, they still carry a risk of infecting the host and replicating in target cells, thereby increasing the likehood of virus recombination and reversion to virulence. In this study, we inserted the target sequence of miR-142 into the nsp2 hypervariable region of PRRSV to inhibit viral replication in its host cells of pigs, with the aim of achieving virus attenuation. The chimeric virus RvJX-miR-142t was successfully rescued and retained its growth characteristics in MARC-145 cells. Furthermore, it did not replicate in MARC-145 cells transfected with miRNA-142 mimic. We also observed limited replication ability of RvJX-miR-142t in pulmonary alveolar macrophages, which are the main cell types that PRRSV infects. Our animal inoculation study showed that pigs infected with RvJX-miR-142t displayed less severe clinical symptoms, lower viremia titers, lighter lung lesions, and significantly lower mortality rates during the first 7 days post-inoculation, in comparison to pigs infected with the backbone virus RvJXwn. We detected a partially deletion of the miR-142 target sequence in the RvJX-miR-142t genome at 14 dpi. It is highly possible that the reversion of viral virulence observed in the later timepoints of our animal experiment was caused by that. Our study provided a new strategy for attenuating PRRSV and confirmed its effectiveness. However, further studies are necessary to increase the stability of this virus under host selection pressure.
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PRRSV and CSFV are both common infectious pathogens in porcine populations, posing significant threats to the healthy development of the porcine industry. Vaccine immunization is the main way to prevent and control these two diseases. Increasing studies have demonstrated that there is an interaction between PRRSV co-infection and CSFV vaccine immune failure. To investigate the effect of PRRSV infection on CSFV proliferation and its molecular mechanism, the proliferation dynamics of PRRSV/CSFV, the NLRP3 inflammasome components, and IL-1ß expression levels were detected in PRRSV/CSFV alone- or co-infection. Subsequently, the relationship between inflammasome activation, IL-1ß expression, and CSFV proliferation was analyzed through the construction of an inflammasome activation model, specific siRNA interference, and specific inhibitor treatment. The results showed that CSFV infection had a poor regulatory effect on NLRP3 inflammasome activation and IL-1ß maturation, but PRRSV and CSFV co-infection could significantly up-regulate the expression of NLRP3 and ASC, induce Caspase-1 activation, and promote IL-1ß maturation. It was further determined that NLRP3 inflammasome components played important roles in IL-1ß maturation and inhibiting CSFV proliferation by PRRSV. Additional experiments indicated that PRRSV replication is essential for NLRP3 inflammasome activation, IL-1ß maturation, and CSFV proliferation inhibition. More importantly, NLRP3 inflammasome activation is regulated by the TLR4-MyD88-NF-κB pathways. In conclusion, PRRSV infection induced IL-1ß maturation by activating the NLRP3 inflammasome through the TLR4-MyD88-NF-κB pathways and then inhibited the proliferation of CSFV. These data further improved the theoretical basis for PRRSV inducing inflammatory factors and leading to the failure of CSFV immunization.
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Coinfección , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Vacunas , Porcinos , Animales , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , FN-kappa B/metabolismo , Transducción de Señal , Factor 88 de Diferenciación Mieloide/genética , Receptor Toll-Like 4 , Coinfección/veterinaria , Proliferación Celular , Interleucina-1beta/genéticaRESUMEN
Fast evolution in the field of the replicase nsp2 represents a most prominent feature of porcine reproductive and respiratory syndrome virus (PRRSV). Here, we determined its biological significance in viral pathogenesis by constructing interlineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 (lineage 8) and the low-virulent NADC30-like strain CHsx1401 (lineage 1). Replacement with nsp2 from JXwn06 was surprisingly lethal to the backbone virus CHsx1401, but combined substitution with the structural protein-coding region (SP) gave rise to viable virus CHsx1401-SPnsp2JX. Meanwhile, a derivative carrying only the SP region (CHsx1401-SPJX) served as a control. Subsequent animal experiments revealed that acquisition of SP alone (CHsx1401-SPJX) did not allow CHsx1401 to gain much virulence, but additional swapping of HP-PRRSV nsp2 (CHsx1401-SPnsp2JX) enabled CHsx1401 to acquire some properties of HP-PRRSV, exemplified by prolonged high fever, microscopic lung hemorrhage, and a significant increase in proinflammatory cytokines in the acute stage. Consistent with this was the transcriptomic analysis of persistently infected secondary lymphoid tissues that revealed a much stronger induction of host cellular immune responses in this group and identified several core immune genes (e.g., TLR4, IL-1ß, MPO, etc.) regulated by HP-PRRSV nsp2. Interestingly, immune activation status in the individual groups correlated well with the rate of viremia clearance and viral tissue load reduction. Overall, the above results suggest that the Chinese HP-PRRSV nsp2 is a critical virulence regulator and highlight the importance of nsp2 genetic variation in modulating PRRSV virulence and persistence via immune modulation. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to the world swine industry. In the field, rapid genetic variations (e.g., deletion, mutation, recombination, etc.) within the nsp2 region present an intriguing conundrum to PRRSV biology and pathogenesis. By making chimeric mutants, here, we show that the Chinese highly pathogenic PRRSV (HP-PRRSV) nsp2 is a virulence factor and a much stronger inducer of host immune responses (e.g., inflammation) than its counterpart, currently epidemic, NADC30-like strains. Differences in the ability to modulate host immunity provide insight into the mechanisms of why NADC30-like strains and their derivatives are rising to be the dominant viruses, whereas the Chinese HP-PRRSV strains gradually give away center stage in the field. Our results have important implications in understanding PRRSV evolution, interlineage recombination, and persistence.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , China/epidemiología , Citocinas , Variación Genética , Genoma Viral , Filogenia , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos , Virulencia/genéticaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is a globally important disease threatening the pork industry, and modified live-virus (MLV) vaccines are widely used for its prevention. However, PRRS MLV shows high potential for reversion to virulence, leading to a major concern about its safety. Yet the revertant mechanism is still poorly understood. Here, attenuated virus JXwn06-P80, derived from the highly pathogenic PRRS virus (PRRSV) strain JXwn06 by serial passaging in MARC-145 cells, was reversely passaged in pigs through intranasal inoculation to mimic natural infection for 13 rounds, and the pathogenicity of viruses at the 3rd, 5th, 9th, 10th, and 11th passages was evaluated in pigs. From the 9th passage, the viruses caused mortality, which was related to their increased adaptability and replication efficiency (100 times higher than those of JXwn06-P80) in porcine alveolar macrophage (PAM) target cells. Similarly, JXwn06-P80 could also regain fatal virulence through reverse passage in PAMs for 25 or more passages, indicating that the increased adaptability in PAMs directly contributes to its regained fatal virulence. Next, the full-genome sequences were analyzed to explore the genetic evolutionary processes during adaptation both in vivo and in vitro. Finally, by a reverse genetic operation, four reverse mutation sites, NSP12-W121R, ORF2b (open reading frame 2b)-H9D, ORF5-H15L, and ORF5-V189L, were finally identified to partially contribute to the ability of the virus to adapt to PAMs, which may be related to virulence reversion during reverse passage. These findings provided direct scientific evidence for the virulence reversion of PRRS MLV and provided valuable clues for exploring its molecular mechanism. IMPORTANCE Reversion to virulence of a live attenuated vaccine is a public concern; however, direct scientific evidence is limited, and the mechanism is still poorly understood. Here, we present direct evidence for the reversion to virulence of PRRS MLV after serial passaging in pigs or target cells and found a correlation between virulence reversion and increased replication fitness in primary PAMs. The genetic evolutionary process during adaptation will provide valuable clues for exploring the molecular mechanism of PRRS MLV virulence reversion and offer important implications for understanding the reversion mechanisms of other vaccines.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Vacunas Virales , Porcinos , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virulencia/genética , Macrófagos Alveolares , Mutación , Vacunas Virales/genéticaRESUMEN
Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 °C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p < 0.001). Overall, the RT-RAA-LFD assay is a user-friendly diagnostic tool that can rapidly and visually detect PDCoV.
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Ácidos Nucleicos , Recombinasas , Animales , Deltacoronavirus , Humanos , Técnicas de Amplificación de Ácido Nucleico , Recombinasas/genética , Recombinasas/metabolismo , Reproducibilidad de los Resultados , Transcripción Reversa , Sensibilidad y Especificidad , PorcinosRESUMEN
PRRSV and CSFV are both the main pathogens of pigs and pose great threats to the pig industry. Previous studies have shown that PRRSV infection or attenuated virus vaccination can reduce the antibody level of attenuated CSFV vaccine and even cause immune failure. The higher pro-inflammatory cytokines induced by PRRSV might play a significant role in inhibiting the proliferation of CSFV-C. However, the molecular mechanism has not been elucidated yet. Here, the effect of IL-1ß, a central mediator of immune-regulating inflammatory responses, on CSFV-C proliferation was investigated, as well as the mechanisms responsible for the production of IL-1ß in the PRRSV and CSFV-C co-infection systems. The results showed that co-infection could significantly increase IL-1ß production both at mRNA and protein levels with the infection progressing, and the IL-1ß upregulation was mainly triggered by PRRSV infection. Additional experiments indicated that IL-1ß inhibited the proliferation of CSFV-C in a cell-type independent manner at the replication and release stages. Furthermore, the IL-1ß production induced via the TLR4/MyD88 pathway and the downstream signaling pathways NF-κB, ERK1/2, P38, and JNK were involved by treatment with specific inhibitors or siRNA knockdown assays. Finally, we clarified that the NLRP3 inflammasome played a meaningful role in the maturation and release of IL-1ß. Together, the accumulated results provided a deeper understanding of the vaccination failure of CSFV caused by PRRSV co-infection as well as targets for the development of novel approaches for the vaccination and control of CSF.
Asunto(s)
Coinfección , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Animales , Proliferación Celular , Coinfección/veterinaria , Inflamasomas/genética , Interleucina-1beta/genética , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Porcinos , Receptor Toll-Like 4/genéticaRESUMEN
It is a common phenomenon that PRRSV infection can interfere with the protective efficacy of the CSFV vaccine in clinical settings, and no effective treatment is available. In our previous study, we found that PRRSV infection could inhibit the replication of CSFV-C by promoting the high expression of inflammatory cytokines. In order to further investigate whether Chinese medicine could alleviate the inhibition effect, the PAM39 cells model, which was co-infected with PRRSV and CSFV-C, was established. The effects of Chinese medicine on this co-infection model, as well as the effect of astragalus polysaccharide on the TLRs/NF-κB/TNF-α pathways, were investigated. Our results demonstrated that PAM39 cells inoculated with different pathogenic PRRSV significantly inhibited the replication of CSFV-C and up-regulated the major inflammatory mediators, including TNF-α. For the following studies, 50 µM of astragalus polysaccharide was selected from six kinds of representative Chinese medicine based on their cytotoxicity, viral titers, and inflammatory mediators. Further experiments indicated that astragalus polysaccharide could alleviate the inhibition of CSFV-C replication in the co-infection group with no influence on cell viability. In addition, astragalus polysaccharide treatment clearly reduced P65 phosphorylation and down-regulated the expression of TLR7, TLR9, and TNF-α in co-infection group, implying that the TLRs/NF-κB/TNF-α pathways may play an important role in astragalus polysaccharide's anti-inflammatory response. In conclusion, astragalus polysaccharide treatment alleviated PRRSV-mediated inhibition of CSFV-C replication via the TLRs/NF-κB/TNF-α pathways, and the molecular mechanism of PRRSV co-infection leading to the failure of CSFV vaccine immunization was partially elucidated, providing a scientific basis for effective CSF prevention and control in pig farms.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Coinfección , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Mediadores de Inflamación , FN-kappa B/metabolismo , Polisacáridos/farmacología , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Porcinos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), an economically devastating pathogen to the world swine industry. Here, we report that this event is to suppress cellular inflammation and is mediated by the viral replicase protein nsp1ß. We show that nsp1ß is a stress-responsive protein, enters virus-induced stress granules (SGs) during infection, and repurposes SGs into a proviral platform, where it co-opts the SG core component G3BP1 to interact with PKR in a regulated manner. RNA interference silencing of G3BP1 or mutation of specific nsp1ß residues (VS19GG) can abolish the antagonization of PKR activation. The viral mutant carrying the corresponding mutations induces elevated level of PKR phosphorylation and pronounced production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-6, and IL-8), whereas small-interfering RNA knockdown of PKR or treatment with C16, a PKR inhibitor, blocks this effect. Thus, PRRSV has evolved a unique strategy to evade PKR restriction to suppress host inflammatory responses.
Asunto(s)
Factores de Restricción Antivirales , ADN Helicasas , Evasión Inmune , Proteínas de Unión a Poli-ADP-Ribosa , Virus del Síndrome Respiratorio y Reproductivo Porcino , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Proteínas no Estructurales Virales , eIF-2 Quinasa , Animales , Factores de Restricción Antivirales/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Gránulos de Estrés/virología , Porcinos , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , eIF-2 Quinasa/metabolismoRESUMEN
Porcine circovirus 3 (PCV3) is an emerging virus, causing substantial economic losses in pig populations, that was first detected in 2016. Furthermore, the virus has already been reported in Europe, the Americas, and Asia, including China, indicating that the virus has spread worldwide. However, the molecular epidemiology of PCV3 still needs further study. To investigate PCV3 epidemiological characteristics in China, 2707 serum samples of pigs were randomly collected from 17 provinces in China between September 2018 and March 2022 and analyzed via PCR assays. The study showed that PCV3 infection was prevalent in the overall population with 31.07% (841/2707) and 100.0% (17/17) at sample and province levels, respectively, though the positivity rate of PCV3 varied from 7.41 to 70.0% in different provinces, suggesting that PCV3 infection has a widespread distribution in China. We selected 22 serum samples from different regions that had high levels of viral DNA for amplification and sequenced their ORF2 (Cap) gene. According to the phylogenetic analysis, all isolates in the current study could be grouped into two separate subclades, with 15 strains belonging to clade 3a and 7 strains belonging to clade 3b, indicating that PCV3a and PCV3b were the predominant subtypes in the regions of China studied. Meanwhile, additional analysis revealed that the capsid gene sequences identified in this study displayed 97.46~99.8% nucleotide (nt) and 97.06~100% amino acid (aa) sequence similarity with other PCV3 available reference strains, respectively. In general, our studies provide important insights for understanding the prevalence and evolution of PCV3 in China and will guide future efforts to develop measures for preventing and controlling the disease.
RESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for the global pork industry, characterized for its genetic variation and unsatisfied heterological protection from vaccines. A high-throughput screening platform for developing anti-PRRSV therapies is urgently needed. Here, an 11-amino-acid subunit HiBiT derived from NanoLuc luciferase was inserted into the PRRSV genome at four loci of the Nsp2 coding region or as an additional TRS2 driven open reading frame (ORF) between the ORF7 and 3'-untranscribed region (3'-UTR), respectively, and five recombinant viruses with luciferase activity were successfully rescued. The virological characteristics of the representative virus RvJX-Nsp2325-HiBiT were investigated. In vitro, it displayed similar growth kinetics as the parental virus and keeps the luciferase activity and genetic stability after eight rounds of serial passages. The concept-proof test confirmed that RvJX-Nsp2325-HiBiT can be easily used to evaluate the efficacy of antiviral reagents by detecting the reduction of luciferase activity, showing a consistent trend with infectious titers, as well as to set a novel convenient virus neutralization assay based on the intensity of luciferase activity. Last, the viral proliferation, virulence, validity, and HiBiT stability were further investigated in pig inoculation study, showing that the luciferase activity can be directly detected in the tissue samples or indirectly from the MARC-145 cells inoculated with sera from RvJX-Nsp2325-HiBiT-inoculated pigs. Taken together, the results indicate that the HiBiT-tagged virus is a convenient and stable tool for evaluating viral propagation both in vitro and in vivo, which can provide a high-efficient platform for screening and evaluating anti-PRRSV therapies. IMPORTANCE Luciferase reporter tagged virus is crucial to viral quantification in the study of viral replication, pathogenesis and exploring antiviral reagents. It is urgently needed for PRRSV academia to construct a stable, fast, and high-throughput reporting system, which can be used both in vitro and in vivo. Here, an 11-amino-acid luciferase subunit was successfully inserted into the PRRSV genome; the feasibility, genetic stability, and efficiency for viral quantification both in vitro and in vivo were characterized; and the results demonstrated it has greatly improved the convenience and efficiency for screening the anti-PRRSV reagents. Furthermore, a novel luciferase-based virus neutralization assay was successfully set, which can eliminate the step of sample gradient dilution and greatly improve the convenience and throughput of neutralizing antibody testing. Predictably, it will greatly facilitate the screening and evaluating anti-PRRSV therapies, as well as the mechanistic study of its replication and pathogenesis in the future.