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Introduction: Muscovy duck parvovirus (MDPV), Goose parvovirus (GPV), Duck circovirus, (DuCV) and Duck adenovirus 3 (DAdV-3) are important pathogens that cause high morbidity and mortality in ducks, causing huge economic loss for the duck industry. Methods: The present study, a quadruplex one-step real time quantitative PCR method for the detection of MDPV, GPV, DuCV, and DAdV-3 was developed. Results: The results showed that assay had no cross-reactivity with other poultry pathogens [Duck plague virus (DPV), Duck tembusu virus (DTMUV), H6 avian influenza virus (H6 AIV), New duck reovirus (NDRV), Newcastle disease virus (NDV), H4 avian influenza virus (H4 AIV), Escherichia coli (E. coli), Muscovy duck reovirus (MDRV), Egg drop syndrome virus (EDSV), Pasteurella multocida (P. multocida)]. The sensitivity result showed that the limits of detection for MDPV, GPV, DuCV, and DAdV-3 were 10, 10, 1 and 10 copies/µl, respectively; The coefficients of variation intra- and inter-method was 1-2%; The range of linear (109 to 103 copies/µL) demonstrated the R2 values for MDPV, GPV, DuCV, and DAdV-3 as 0.9975, 0.998, 0.9964, and 0.996, respectively. The quadruplex real time quantitative PCR method efficiency was 90.30%, 101.10%, 90.72%, and 90.57% for MDPV, GPV, DuCV, and DAdV-3, respectively. 396 clinical specimens collected in some duck sausages from June 2022 to July 2023 were simultaneously detected using the established quadruplex real time quantitative PCR method and the reported assays. The detection rates for MDPV, GPV, DuCV, and DAdV-3 were 8.33% (33/396), 17.93% (71/396), 33.58% (133/396), and 29.04% (115/396), respectively. The agreement between these assays was greater than 99.56%. Discussion: The developed quadruplex real-time quantitative PCR assay can accurately detect these four viruses infecting ducks, providing a rapid, sensitive, specific and accurate technique for clinical testing.
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Patos , Enfermedades de las Aves de Corral , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , Patos/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/diagnóstico , Sensibilidad y Especificidad , Parvovirinae/genética , Parvovirinae/aislamiento & purificación , Parvovirinae/clasificación , Aviadenovirus/genética , Aviadenovirus/aislamiento & purificación , Aviadenovirus/clasificación , Circovirus/genética , Circovirus/aislamiento & purificación , Circovirus/clasificación , Infecciones por Parvoviridae/veterinaria , Infecciones por Parvoviridae/diagnóstico , Infecciones por Parvoviridae/virologíaRESUMEN
Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin ß1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-ß signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.
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Circovirus , Fibronectinas , Haemophilus parasuis , Enfermedades de los Porcinos , Tráquea , Animales , Porcinos , Fibronectinas/metabolismo , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/metabolismo , Haemophilus parasuis/metabolismo , Circovirus/metabolismo , Circovirus/patogenicidad , Tráquea/virología , Tráquea/microbiología , Tráquea/metabolismo , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/virología , Infecciones por Haemophilus/metabolismo , Adhesión Bacteriana , Serogrupo , Coinfección/virología , Coinfección/microbiología , Infecciones por Pasteurellaceae/veterinaria , Infecciones por Pasteurellaceae/virología , Infecciones por Pasteurellaceae/microbiología , Infecciones por Pasteurellaceae/metabolismoRESUMEN
INTRODUCTION: The novel Porcine circovirus 3 (PCV-3) has been associated in the past years to different porcine diseases, including reproductive failure. The potential occurrence of PCV-3 in abortions from Swiss pig herds has not been investigated so far. Thus, we conducted a retrospective study on pig aborted cases submitted to our laboratory in the University of Bern during the last 10 years with the main aim of investigating the possible presence of PCV-3 in foetal and/or placental tissue. Twelve out of the 53 studied cases showed mild histopathological changes as previously described in PCV-3 positive cases. However, in none of the cases, PCV-3 genetic material could be detected in the examined formalin-fixed, paraffin-embedded tissues. In only one third of the cases, a cause for the abortion was found, which is similar to other studies. Our survey suggests that PCV-3 was not involved in the porcine abortion cases submitted over the last decade at our institution in Switzerland.
INTRODUCTION: Le nouveau Circovirus porcin 3 (PCV-3) a été associé ces dernières années à différentes maladies porcines, y compris des troubles de la reproduction. La présence potentielle du PCV-3 dans les avortements de porcs en Suisse n'a pas été étudiée jusqu'à présent. Nous avons donc mené une étude rétrospective sur les cas d'avortements de porcs soumis à notre laboratoire de l'Université de Berne au cours des 10 dernières années, dans le but principal d'étudier la présence éventuelle du PCV-3 dans les tissus fÅtaux et/ou placentaires. Douze des 53 cas étudiés présentaient des changements histopathologiques légers, tels que décrits précédemment dans les cas positifs au PCV-3. Cependant, dans aucun des cas, le matériel génétique du PCV-3 n'a pu être détecté dans les tissus examinés fixés au formol et inclus en paraffine. Dans un tiers des cas seulement, une cause d'avortement a été trouvée, ce qui est similaire à d'autres études. Notre étude suggère que le PCV-3 n'a pas été impliqué dans les cas d'avortements porcins soumis au cours de la dernière décennie dans notre institution en Suisse.
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Aborto Veterinario , Infecciones por Circoviridae , Circovirus , Enfermedades de los Porcinos , Animales , Femenino , Embarazo , Aborto Veterinario/virología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/virología , Circovirus/aislamiento & purificación , Circovirus/genética , Formaldehído , Adhesión en Parafina/veterinaria , Placenta/virología , Placenta/patología , Estudios Retrospectivos , Porcinos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/patología , Suiza/epidemiologíaRESUMEN
Porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome in piglets. Differences in the infectivity and horizontal transmissibility of different isolates of PCV2a, PCV2b, and PCV2d in pigs were evaluated by HE and IHC staining, PCR, virus titration, and IPMA to determine their clinical symptoms, pathological changes, levels of virus and antibody, and cohabitation infectivity. In the cohabitation infection experiment, weak viremia and low levels of antibodies were detected in the pigs challenged with PCV2a-CL, whereas no viremia or antibodies were detected in the corresponding cohabiting pigs. Furthermore, no PCV2 was isolated from any organ of pigs that were challenged with PCV2a-CL, as well as from those of their cohabiting pigs. In contrast, persistent viremia and pathological changes, including swollen inguinal lymph nodes, were detected in both the challenged and cohabiting pigs after PCV2b-BY or PCV2d-LNHC infection. Alive PCV2 was detected in the tonsils, inguinal lymph nodes, spleen, and kidneys of the experimental pigs by virus titration, and the highest viral titer was detected in the tonsils, followed by the inguinal lymph nodes. In a comparative analysis of the challenged and cohabiting pigs, a 1-week delay in viremia and specific antibodies was observed in the cohabiting pigs. Moreover, the number of viruses isolated from the tonsils and inguinal lymph nodes of the pigs cohabiting with PCV2d-LNHC-challenged pigs was significantly greater than those in the pigs that were directly challenged with PCV2d-LNHC in cohabitation infection experiment (P<0.05). Together, these results indicated that the infectivity and horizontal transmissibility of the strains PCV2b-BY and PCV2d-LNHC were much greater than those of the strain PCV2a-CL and provided some insights into PCV2 pathogenicity.
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Anticuerpos Antivirales , Infecciones por Circoviridae , Circovirus , Animales , Circovirus/patogenicidad , Circovirus/clasificación , Circovirus/aislamiento & purificación , Porcinos , Infecciones por Circoviridae/virología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/transmisión , Anticuerpos Antivirales/sangre , Viremia/transmisión , Viremia/virología , Viremia/veterinaria , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/transmisión , Síndrome Multisistémico de Emaciación Posdestete Porcino/virología , Síndrome Multisistémico de Emaciación Posdestete Porcino/transmisión , Carga ViralRESUMEN
As one genotype of porcine circovirus (PCV) identified in 2016, PCV3 has brought huge hidden dangers to the global swine industry together with PCV2. Virus-like particles (VLPs) of capsid protein (Cap) of PCV2 serve as an alternative nano-antigen delivery strategy to efficiently induce antiviral immune response against PCV2 and/or other covalently displayed swine pathogens. However, the current understanding is limited on the capability of PCV3 as a nano-vaccine vehicle. Here we systematically compared the characteristics and the immunogenic efficacy of PCV3 Cap (Cap3) and PCV2 Cap (Cap2) in a VLP form. Cap3 VLPs presented higher internalization efficiency into cells and cytokines production compared to those of Cap2. Meanwhile, cross-reactive immunity between Cap3 VLPs and Cap2 VLPs was detected. Furthermore, to evaluate the function of Cap3 VLPs and Cap2 VLPs as vaccine vehicles carrying foreign proteins, the non-structural protein 6 of porcine reproductive and respiratory syndrome virus (PRRSV) was fused to C-terminus of Cap. Cap3-based chimeric particles induced a higher level of nsp6-specific immune response and PRRSV inhibition. Collectively, these self-assembling, Cap-based VLPs offer a compelling platform for enhancing the effectiveness of subunit vaccinations against newly emerging diseases and hold great promise for the development of Cap3-based chimeric subunit vaccines.
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Proteínas de la Cápside , Circovirus , Vacunas de Partículas Similares a Virus , Vacunas Virales , Animales , Circovirus/inmunología , Circovirus/genética , Porcinos , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Anticuerpos Antivirales/inmunología , Infecciones por Circoviridae/prevención & control , Infecciones por Circoviridae/inmunologíaRESUMEN
BACKGROUND: This field efficacy study was designed to determine the efficacy of a new bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae at three independent pig farms. METHODS: Three pig farms were selected based on their history of subclinical PCV2 infection and enzootic pneumonia. Each farm housed a total of 40, 18-day-old pigs that were randomly allocated to 1 of 2 treatment groups. Pigs were administered a 2.0 mL dose of the bivalent vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate-buffered saline at the same age. RESULTS: Clinically, the average daily weight gain of vaccinated groups was significantly higher (p < 0.05) than those of unvaccinated animals during the growing (70-112 days of age), finishing (112-175 days of age) and overall (3-175 days of age) stages of production. Vaccinated animals elicited neutralizing anti-PCV2 antibodies and PCV2d-specific interferon-γ secreting cells (IFN-γ-SC), which reduced the amount of PCV2d genomic copies in blood and reduced lymphoid lesions severity when compared with unvaccinated animals. Similarly, vaccinated animals elicited M. hyopneumoniae-specific IFN-γ-SC, which reduced the amount of M. hyopneumoniae in the larynx and reduced lung lesions severity. CONCLUSIONS: The result of the field trial demonstrated that the bivalent vaccine was efficacious in the protection of swine herds suffering from subclinical PCV2d infection and enzootic pneumonia.
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Vacunas Bacterianas , Infecciones por Circoviridae , Circovirus , Mycoplasma hyopneumoniae , Neumonía Porcina por Mycoplasma , Vacunas Virales , Animales , Circovirus/inmunología , Mycoplasma hyopneumoniae/inmunología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/prevención & control , Porcinos , Neumonía Porcina por Mycoplasma/prevención & control , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Combinadas/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/microbiología , Distribución Aleatoria , Sus scrofa , Infecciones AsintomáticasRESUMEN
Introduction: Since their identification in 1974, circoviruses have caused clinicopathological diseases in various animal species, including humans. However, their origin, transmission, and genetic evolution remain poorly understood. Methods: In this study, the genome sequences of circovirus were obtained from GenBank, and the Bayesian stochastic search variable selection algorithm was employed to analyzed the evolution and origin of circovirus. Results: Here, the evolutionary origin, mode of transmission, and genetic recombination of the circovirus were determined based on the available circovirus genome sequences. The origin of circoviruses can be traced back to fish circovirus, which might derive from fish genome, and human contributes to transmission of fish circovirus to other species. Furthermore, mosquitos, ticks, bats, and/or rodents might play a role as intermediate hosts in circovirus intra- and inter-species transmission. Two major lineages (A and B) of circoviruses are identified, and frequent recombination events accelerate their variation and spread. The time to the most recent common ancestor of circoviruses can be traced back to around A.D. 600 and has been evolving at a rate of 10-4 substitutions site-1 year-1 for a long time. Discussion: These comprehensive findings shed light on the evolutionary origin, population dynamics, transmission model, and genetic recombination of the circovirus providing valuable insights for the development of prevention and control strategies against circovirus infections.
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Infecciones por Circoviridae , Circovirus , Evolución Molecular , Filogenia , Recombinación Genética , Animales , Humanos , Circovirus/genética , Infecciones por Circoviridae/transmisión , Infecciones por Circoviridae/virología , Infecciones por Circoviridae/veterinaria , Genoma Viral , Teorema de BayesRESUMEN
Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6-10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3-4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 1010 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity.
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Células de la Médula Ósea , Circovirus , Animales , Porcinos , Circovirus/fisiología , Circovirus/aislamiento & purificación , Circovirus/genética , Células de la Médula Ósea/virología , Células Cultivadas , Infecciones por Circoviridae/virología , Infecciones por Circoviridae/veterinaria , Enfermedades de los Porcinos/virología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cultivo de Virus/métodosRESUMEN
Porcine circoviruses (PCVs) contain four types: PCV1, PCV2, PCV3, and PCV4, all of which can infect pigs. Among them, PCV1 is non-pathogenic, and PCV2 can cause porcine circovirus diseases (PCVD) or porcine circovirus-associated diseases (PCVAD). Although the pathogenicity of PCV3 and PCV4 is still controversial, increasing evidence shows that PCV3 and PCV4 can cause PCV-related disease. However, mixed infection of PCV2, PCV3, and PCV4 with other pathogens often occurs in large-scale pig breeding, bringing severe economic losses to the global pig industry. In this study, the soluble recombinant proteins of PCV2, PCV3, and PCV4 Cap were expressed by the prokaryotic expression system and biotinylated to combine with the Streptavidin magnetic beads, followed by immunogenicity evaluation of the recombinant proteins. Furthermore, we also assessed the efficacy and immunogenicity of trivalent recombinant proteins conjugated with different adjuvants in mice. The results showed that the highly effective anti-PCV serum was successfully prepared, and the recombinant proteins conjugated with different adjuvants produced various degrees of humoral and cellular immunity in mice. Three recombinant proteins are effective immunogens, and the trivalent proteins coupled with the aluminum adjuvant or GM-CSF-CpG for two-dose immunization can stimulate prominent humoral and cellular immunity against PCVs in vivo. The soluble recombinant proteins are the most promising candidate for developing a trivalent vaccine against PCVs (PCV2, PCV3, and PCV4) infection simultaneously.
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Proteínas de la Cápside , Infecciones por Circoviridae , Circovirus , Circovirus/inmunología , Circovirus/genética , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/prevención & control , Infecciones por Circoviridae/virología , Infecciones por Circoviridae/inmunología , Porcinos , Ratones , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Proteínas Recombinantes/inmunología , Femenino , Vacunas Virales/inmunología , Ratones Endogámicos BALB C , Anticuerpos Antivirales/sangreRESUMEN
Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.
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Proteínas de la Cápside , Circovirus , Ubiquitina-Proteína Ligasas , Ubiquitinación , Replicación Viral , Circovirus/genética , Circovirus/metabolismo , Circovirus/fisiología , Animales , Porcinos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Humanos , Células HEK293 , Proteolisis , Línea Celular , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/metabolismo , Infecciones por Circoviridae/virología , Infecciones por Circoviridae/metabolismo , Unión ProteicaRESUMEN
Current evidence suggests that porcine circovirus type 2 (PCV2) infection induces immunosuppression in piglets. Sophora subprostrate polysaccharide (SSP) exhibits various pharmacological activities, including immunoregulatory, anti-inflammatory, antiviral, and antioxidant properties. However, the acts of lncRNAs in regulating the therapeutic effects of SSP on PCV2-infected RAW264.7 cells remains poorly understood. This study aimed to investigate the molecular mechanisms by which lncRNAs regulate PCV2-induced immunosuppression during SSP treatment. Our findings revealed that 1699 mRNAs, 373 lncRNAs, and 129 miRNAs were differentially expressed in PCV2-infected RAW264.7 cells. Additionally, 359 mRNAs, 271 lncRNAs, and 79 miRNAs exhibited differential expression in SSP-treated PCV2-infected RAW264.7 cells. GO and KEGG analyses indicated that the candidate genes were enriched in the TNF/NF-κB signaling pathway. Furthermore, based on GO and KEGG pathway analysis, a ceRNA network involving chemokine (C-X-C motif) ligand 2 (CXCL2), miR-217-x, and MSTRG.5823.1 was constructed. We demonstrated that lncRNA MSTRG.5823.1 localized to the cytoplasm. Moreover, we found that silencing or overexpressing lncRNA MSTRG.5823.1 significantly modulated PCV2-induced immunosuppression by regulating the activation of the TNF/NF-κB signaling pathway. Specifically, lncRNA MSTRG.5823.1 overexpression increased the expression of TNF/NF-κB signaling pathway-related genes and proteins in PCV2-infected RAW264.7 cells. Conversely, silencing lncRNA MSTRG.5823.1 decreased their expression. Rescue assays further revealed that the suppressive effects of miR-217-x overexpression on TNF/NF-κB signaling pathway-related genes and proteins could be reversed by MSTRG.5823.1 overexpression. These findings highlight the critical role of lncRNA MSTRG.5823.1 in PCV2 infection progression and suggest a new strategy for the prevention and treatment of PCV2 infection.
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Infecciones por Circoviridae , Circovirus , FN-kappa B , Polisacáridos , ARN Largo no Codificante , Transducción de Señal , Sophora , Animales , Ratones , Circovirus/inmunología , FN-kappa B/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Infecciones por Circoviridae/inmunología , Polisacáridos/farmacología , Porcinos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , MicroARNs/genética , MicroARNs/metabolismo , Tolerancia Inmunológica/efectos de los fármacosRESUMEN
The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.
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Circovirus , ADN Viral , Circovirus/genética , Porcinos , Animales , ADN Viral/genética , Clonación Molecular , Genoma Viral , Genética Inversa/métodos , Infecciones por Circoviridae/virología , Línea CelularRESUMEN
BACKGROUND: Canine circovirus (CanineCV), a non-enveloped virus with a circular DNA genome, has been identified in various avian and mammalian species, including domestic and wild canids. This study aimed to comprehensively analyze the prevalence of CanineCV across diverse animal species in 11 provinces of China. RESULTS: A total of 1,666 serum samples were collected, revealing a 5.82% prevalence of CanineCV in dogs, with the highest rates being observed in southern and eastern China. Phylogenetic analysis of 266 global CanineCV genomes sourced from the NCBI identified six distinct genotypes, elucidating the complex dynamics of their evolution. Evidence suggested a potential bat origin for CanineCV, with positive selection and high rates of evolution being observed. Recombination analysis revealed dynamic genetic exchange, highlighting the intricate nature of CanineCV evolution. Mutational analysis identified key amino acid substitutions likely to influence the virus's adaptation. Additionally, glycosylation, palmitoylation, and SUMOylation sites were predicted, shedding light on crucial functional properties of the virus. CONCLUSIONS: This study provides a global perspective on the origin, genetic diversity, and evolutionary dynamics of CanineCV. Understanding these factors is crucial for elucidating its epidemiology and potential health risks.
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Infecciones por Circoviridae , Circovirus , Enfermedades de los Perros , Filogenia , Animales , Circovirus/genética , Circovirus/clasificación , Perros , Enfermedades de los Perros/virología , Enfermedades de los Perros/epidemiología , China/epidemiología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/epidemiología , Infecciones por Circoviridae/virología , Evolución Molecular , Genoma Viral , Variación Genética , Prevalencia , GenotipoRESUMEN
Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.
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Infecciones por Circoviridae , Circovirus , Herpesvirus Suido 1 , Animales , Circovirus/inmunología , Circovirus/genética , Ratones , Herpesvirus Suido 1/inmunología , Herpesvirus Suido 1/genética , Infecciones por Circoviridae/inmunología , Infecciones por Circoviridae/prevención & control , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Vacunas Virales/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Porcinos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Vacunas Sintéticas/inmunología , Seudorrabia/inmunología , Seudorrabia/prevención & control , Femenino , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Canine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene (Cap). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach. RESULTS: According to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution. CONCLUSIONS: Our study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response.
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Proteínas de la Cápside , Circovirus , Filogenia , Tailandia , Circovirus/genética , Proteínas de la Cápside/genética , Animales , Perros , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/virología , Variación Genética , Enfermedades de los Perros/virología , Secuencia de AminoácidosRESUMEN
Since PCV4 was first described in 2019, the virus has been identified in several countries in Southeast Asia and Europe. Most studies have been limited to detecting PCV4 by PCR. Thus, PCV4 has an unclear association with clinical disease. This study utilized 512 porcine clinical lung, feces, spleen, serum, lymphoid tissue, and fetus samples submitted to the ISU-VDL from June-September 2023. PCV4 was detected in 8.6% of samples with an average Ct value of 33. While detection rates among sample types were variable, lymphoid tissue had the highest detection rate (18.7%). Two ORF2 sequences were obtained from lymphoid tissue samples and had 96.36-98.98% nucleotide identity with reference sequences. Direct detection of PCV4 by RNAscope revealed viral replication in B lymphocytes and macrophages in lymph node germinal centers and histiocytic and T lymphocyte infiltration in the lamina propria of the small intestine. PCV4 detection was most commonly observed in nursery to finishing aged pigs displaying respiratory and enteric disease. Coinfection with PCV2, PCV3, and other endemic pathogens was frequently observed, highlighting the complex interplay between different PCVs and their potential roles in disease pathogenesis. This study provides insights into the frequency of detection, tissue distribution, and genetic characteristics of PCV4 in the US.
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Infecciones por Circoviridae , Circovirus , Enfermedades de los Porcinos , Animales , Circovirus/genética , Circovirus/aislamiento & purificación , Porcinos , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/virología , Enfermedades de los Porcinos/virología , Estados Unidos/epidemiología , Tejido Linfoide/virología , Coinfección/virología , Coinfección/veterinaria , Pulmón/virologíaRESUMEN
This study was aimed to investigate the frequency of PiCV recombination, the kinetics of PiCV viremia and shedding and the correlation between viral replication and host immune response in young pigeons subclinically infected with various PiCV variants and kept under conditions mimicking the OLR system. Fifteen racing pigeons originating from five breeding facilities were housed together for six weeks. Blood and cloacal swab samples were collected from birds every seven days to recover complete PiCV genomes and determine PiCV genetic diversity and recombination dynamics, as well as to assess virus shedding rate, level of viremia, expression of selected genes and level of anti-PiCV antibodies. Three hundred and eighty-eight complete PiCV genomes were obtained and thirteen genotypes were distinguished. Twenty-five recombination events were detected. Recombinants emerged during the first three weeks of the experiment which was consistent with the peak level of viremia and viral shedding. A further decrease in viremia and shedding partially corresponded with IFN-γ and MX1 gene expression and antibody dynamics. Considering the role of OLR pigeon rearing system in spreading infectious agents and allowing their recombination, it would be reasonable to reflect on the relevance of pigeon racing from both an animal welfare and epidemiological perspective.
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Enfermedades de las Aves , Infecciones por Circoviridae , Circovirus , Columbidae , Esparcimiento de Virus , Animales , Columbidae/virología , Circovirus/genética , Circovirus/inmunología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/virología , Infecciones por Circoviridae/epidemiología , Infecciones por Circoviridae/inmunología , Enfermedades de las Aves/virología , Enfermedades de las Aves/epidemiología , Enfermedades de las Aves/inmunología , Viremia/epidemiología , Viremia/virología , Viremia/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Genoma Viral , Recombinación Genética , Genotipo , Replicación Viral , FilogeniaRESUMEN
This study was designed to investigate the different sequential order of infection for porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV). Thirty-six pigs were randomly assigned to six different treatment groups. The first (hereafter referred to as PRRSV-PCV2) group was inoculated with PRRSV first followed by PCV2d. The second (hereafter referred to as PCV2+PRRSV) group was co-infected with both viruses at the same timepoint (42 days of age). The third (hereafter referred to as PCV2-PRRSV) group was inoculated with PCV2d first followed by PRRSV. A fourth group was only inoculated with PCV2d at 42 days of age, while a fifth group was only inoculated with PRRSV at the same timepoint. The sixth group served as a negative control group. The most important observation discovered that PRRSV only had a potentiation effect on PCV2 in both PRRS-PCV2 and PCV2+PRRSV groups. Both PRRSV-PCV2 and PCV2+PRRSV groups experienced a significant reduction in growth performance compared with control pigs. In addition, PRRSV-PCV2 and PCV2+PRRSV groups exhibited a greater severity in their clinical signs, and/or had higher PCV2 blood and lymphoid viral loads that resulted in a stronger severity of lymphoid lesions compared with PCV2-PRRSV group. Serum TNF-α levels were significantly higher in both PRRS-PCV2 and PCV2+PRRSV groups compared with those in PCV2-PRRS, PCV2, and PRRSV groups. The results of this study demonstrated that divergent clinical outcomes are dependent on the sequential infection order of PCV2 and PRRSV.
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Infecciones por Circoviridae , Circovirus , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Circovirus/fisiología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Infecciones por Circoviridae/virología , Infecciones por Circoviridae/veterinaria , Coinfección/virología , Coinfección/veterinaria , Carga Viral , Enfermedades de los Porcinos/virologíaRESUMEN
Since Porcine Circovirus 3 (PCV3) was first identified in 2016, our understanding of the humoral response is still relatively scarce. Current knowledge of the PCV3 humoral response is primarily based on field studies identifying the seroprevalence of PCV3 Cap-induced antibodies. Studies on the humoral response following experimental PCV3 infection have conflicting results where one study reports the development of the Cap IgG response 7 days postinfection with no concurrent Cap IgM response, while a second study shows a Cap IgM response at the same time point with no detection of Cap IgG. The dynamics of the PCV3 Cap and Rep IgG following maternal antibody transfer and experimental infection have not been well characterized. Additionally, the cross-reactivity of convalescent serum from PCV2 and PCV3 experimentally infected animals to serologic methods of the alternate PCV has limited evaluation. Here, we show that maternally derived antibodies were detectable in piglet serum 7-9 weeks postfarrowing for the Cap IgG and 5-weeks-post farrowing for the Rep IgG using Cap- and Rep-specific enzyme linked immunosorbent assays (ELISA) and immunofluorescent assays (IFA) methods. Following experimental inoculation, Cap IgG was detected at 2-weeks-post inoculation and Rep IgG detection was delayed until 4-weeks-post inoculation. Furthermore, convalescent serum from either PCV2 or PCV3 methods displayed no cross-reactivity by serological methods against the other PCV. The information gained in this study highlights the development of both the Cap- and Rep-specific antibodies following experimental infection and through the transfer of maternal antibodies. The increased understanding of the dynamics of maternal antibody transfer and development of the humoral response following infection gained in the present study may aid in the establishment of husbandry practices and potential application of prophylactics to control PCV3 clinical disease. IMPORTANCE: Research on Porcine Circovirus 3 (PCV3) immunology is vital for understanding and controlling this virus. Previous studies primarily relied on field observations, but they have shown conflicting results about the immunological response against PCV3. This study helps fill those gaps by looking at how antibodies develop in pigs, especially those maternal-derived, and their impact in neonatal pigs preventing PCV3-associated disease in piglets. In addition, we look at the dynamics of antibodies in experimental infections mimicking infection in pigs in the grower-phase condition. Understanding this process can help to develop better strategies to prevent PCV3 infection. Also, this research found that PCV2 and PCV3 do not cross-react, which is crucial for serological test development and results interpretation. Overall, this work is essential for improving swine health and farming practices in the face of PCV3 infections.
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Anticuerpos Antivirales , Infecciones por Circoviridae , Circovirus , Inmunidad Humoral , Inmunidad Materno-Adquirida , Inmunoglobulina G , Enfermedades de los Porcinos , Animales , Circovirus/inmunología , Porcinos , Infecciones por Circoviridae/inmunología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/virología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Femenino , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Ensayo de Inmunoadsorción Enzimática , Reacciones Cruzadas/inmunologíaRESUMEN
In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured populations of turbot (Scophthalmus maximus L.). Histopathological examination using light microscopy revealed extensive necrosis within the cardiac, splenic, and renal tissues of the afflicted fish. Utilizing transmission electron microscopy (TEM), we detected the presence of circovirus particles within the cytoplasm of these cells, with the virions consistently exhibiting a spherical morphology of 20-40 nm in diameter. TEM inspections confirmed the predominance of these virions in the heart, spleen, and kidney. Subsequent molecular characterization through polymerase chain reaction (PCR) analysis corroborated the TEM findings, with positive signals in the aforementioned tissues, in stark contrast to the lack of detection in gill, fin, liver, and intestinal tissues. The TEM observations, supported by PCR electrophoresis data, strongly suggest that the spleen and kidney are the primary targets of the viral infection. Further characterization using biophysical, biochemical assays, and genomic sequencing confirmed the viral classification within the genus Circovirus, resulting in the nomenclature of turbot circovirus (TurCV). The current research endeavors to shed light on the pathogenesis of this pathogen, offering insights into the infection mechanisms of TurCV in this novel piscine host, thereby contributing to the broader understanding of its impact on turbot health and aquaculture.