RESUMO
African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Suínos , Animais , Vírus da Febre Suína Africana/fisiologia , Baço/patologia , Replicação Viral , Macrófagos/patologiaRESUMO
African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Sistemas CRISPR-Cas , Endossomos , Proteínas de Membrana , Internalização do Vírus , Replicação Viral , Animais , Suínos , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Febre Suína Africana/genética , Endossomos/metabolismo , Endossomos/virologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Técnicas de Inativação de GenesRESUMO
African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly infectious disease afflicting domestic pigs and wild boars. It exhibits an alarming acute infection fatality rate of up to 100%. Regrettably, no commercial vaccines or specific drugs for combating this disease are currently available. This study evaluated the anti-ASFV activities in porcine alveolar macrophages, 3D4/21 cells, and PK-15 cells of four bis-benzylisoquinoline alkaloids (BBAs): cepharanthine (CEP), tetrandrine, fangchinoline, and iso-tetrandrine. Furthermore, we demonstrated that CEP, which exhibited the highest selectivity index (SI = 81.31), alkalized late endosomes/lysosomes, hindered ASFV endosomal transport, disrupted virus uncoating signals, and thereby inhibited ASFV internalization. Additionally, CEP disrupted ASFV DNA synthesis, leading to the inhibition of viral replication. Moreover, berbamine was labeled with NBD to synthesize a fluorescent probe to study the cellular location of these BBAs. By co-staining with Lyso-Tracker and lysosome-associated membrane protein 1, we demonstrated that BBAs target the endolysosomal compartments for the first time. Our data together indicated that BBAs are a class of natural products with significant inhibitory effects against ASFV infection. These findings suggest their potential efficacy as agents for the prevention and control of ASF, offering valuable references for the identification of potential drug targets.IMPORTANCEThe urgency and severity of African swine fever (ASF) underscore the critical need for effective interventions against this highly infectious disease, which poses a grave threat to domestic pigs and wild boars. Our study reveals the potent anti-African swine fever virus (ASFV) efficacy of bis-benzylisoquinoline alkaloids (BBAs), particularly evident in the absence of progeny virus production under a 5 µM concentration treatment. The structural similarity among cepharanthine, tetrandrine, fangchinoline, and iso-tetrandrine, coupled with their analogous inhibitory stages and comparable selectivity indexes, strongly suggests a shared antiviral mechanism within this drug category. Further investigation revealed that BBAs localize to lysosomes and inhibit the internalization and replication of ASFV by disrupting the endosomal/lysosomal function. These collective results have profound implications for ASF prevention and control, suggesting the potential of the investigated agents as prophylactic and therapeutic measures. Furthermore, our study offers crucial insights into identifying drug targets and laying the groundwork for innovative interventions.
Assuntos
Vírus da Febre Suína Africana , Antivirais , Benzilisoquinolinas , Endossomos , Lisossomos , Internalização do Vírus , Replicação Viral , Animais , Vírus da Febre Suína Africana/efeitos dos fármacos , Vírus da Febre Suína Africana/fisiologia , Internalização do Vírus/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Replicação Viral/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/virologia , Suínos , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/virologia , Antivirais/farmacologia , Linhagem Celular , Febre Suína Africana/virologia , Febre Suína Africana/tratamento farmacológico , Febre Suína Africana/metabolismo , Guanina/análogos & derivados , Guanina/farmacologia , Alcaloides/farmacologia , Macrófagos Alveolares/virologia , Macrófagos Alveolares/efeitos dos fármacos , BenzodioxóisRESUMO
The spread of the African swine fever virus (ASF virus) genotype ii in the Eurasian region has been very successful and often inexplicable. The virus spreads rapidly and persists in areas with wild boar populations, but areas without feral pig populations are also affected. The virus has shown the ability to survive for a long time in the environment without a population of susceptible hosts, both pigs and Ornithodoros soft ticks. Published data indicated that ASF viruses persist significantly longer in an environment with some freshwater snails (especially Pomacea bridgesii, Tarebia granifera, Asolene spixii, Melanoides tuberculate, and Physa fontinalis), compared to freshwater without snails. Data obtained in this study suggest that gastropods theoretically can be the hosts of the ASF virus. Also, we have proven the possibility of long-term existence of an infectious virus when infected in vitro.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Animais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Vírus da Febre Suína Africana/isolamento & purificação , Suínos , Febre Suína Africana/virologia , Gastrópodes/virologia , Ornithodoros/virologiaRESUMO
BACKGROUND: African swine fever virus (ASFV) is a major threat to pig production and the lack of effective vaccines underscores the need to develop robust antiviral countermeasures. Pathologically, a significant elevation in pro-inflammatory cytokine production is associated with ASFV infection in pigs and there is high interest in identifying dual-acting natural compounds that exhibit antiviral and anti-inflammatory activities. METHODS: Using the laboratory-adapted ASFV BA71V strain, we screened a library of 297 natural, anti-inflammatory compounds to identify promising candidates that protected Vero cells against virus-induced cytopathic effect (CPE). Virus yield reduction, virucidal, and cell cytotoxicity experiments were performed on positive hits and two lead compounds were further characterized in dose-dependent assays along with time-of-addition, time-of-removal, virus entry, and viral protein synthesis assays. The antiviral effects of the two lead compounds on mitigating virulent ASFV infection in porcine macrophages (PAMs) were also tested using similar methods, and the ability to inhibit pro-inflammatory cytokine production during virulent ASFV infection was assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The screen identified five compounds that inhibited ASFV-induced CPE by greater than 50% and virus yield reduction experiments showed that two of these compounds, tetrandrine and berbamine, exhibited particularly high levels of anti-ASFV activity. Mechanistic analysis confirmed that both compounds potently inhibited early stages of ASFV infection and that the compounds also inhibited infection of PAMs by the virulent ASFV Arm/07 isolate. Importantly, during ASFV infection in PAM cells, both compounds markedly reduced the production of pro-inflammatory cytokines involved in disease pathogenesis while tetrandrine had a greater and more sustained anti-inflammatory effect than berbamine. CONCLUSIONS: Together, these findings support that dual-acting natural compounds with antiviral and anti-inflammatory properties hold promise as preventative and therapeutic agents to combat ASFV infection by simultaneously inhibiting viral replication and reducing virus-induced cytokine production.
Assuntos
Vírus da Febre Suína Africana , Anti-Inflamatórios , Antivirais , Animais , Vírus da Febre Suína Africana/efeitos dos fármacos , Vírus da Febre Suína Africana/fisiologia , Antivirais/farmacologia , Suínos , Anti-Inflamatórios/farmacologia , Chlorocebus aethiops , Células Vero , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Macrófagos/imunologia , Febre Suína Africana/virologia , Replicação Viral/efeitos dos fármacos , Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Efeito Citopatogênico Viral/efeitos dos fármacos , Citocinas/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015-January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Genótipo , Sus scrofa , Animais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Suínos , Itália , Febre Suína Africana/virologia , Genoma Viral , Fenótipo , Deleção de Sequência , Macrófagos/virologiaRESUMO
Since the reintroduction of African swine fever virus (ASFV) in Europe in 2007 and its subsequent spread to Asia, wild boar has played a crucial role in maintaining and disseminating the virus. There are significant gaps in the knowledge regarding infection dynamics and disease pathogenesis in domestic pigs and wild boar, particularly at the early infection stage. We aimed to compare domestic pigs and wild boar infected intranasally to mimic natural infection with one of the original highly virulent genotype II ASFV isolates (Armenia 2007). The study involved euthanising three domestic pigs and three wild boar on days 1, 2, 3, and 5 post-infection, while four domestic pigs and four wild boar were monitored until they reached a humane endpoint. The parameters assessed included clinical signs, macroscopic lesions, viremia levels, tissue viral load, and virus shedding in nasal and rectal swabs from day 1 post-infection. Compared with domestic pigs, wild boar were more susceptible to ASFV, with a shorter incubation period and earlier onset of clinical signs. While wild boar reached a humane endpoint earlier than domestic pigs did, the macroscopic lesions were comparatively less severe. In addition, wild boar had earlier viremia, and the virus was also detected earlier in tissues. The medial retropharyngeal lymph nodes were identified as key portals for ASFV infection in both subspecies. No viral genome was detected in nasal or rectal swabs until shortly before reaching the humane endpoint in both domestic pigs and wild boar, suggesting limited virus shedding in acute infections.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Genótipo , Sus scrofa , Animais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/virologia , Suínos , Eliminação de Partículas Virais , Viremia/veterinária , Viremia/virologia , Carga Viral/veterinária , VirulênciaRESUMO
African swine fever (ASF), caused by the African swine fever virus (ASFV), is one of the most important infectious diseases that cause high morbidity and mortality in pigs and substantial economic losses to the pork industry of affected countries due to the lack of effective vaccines. The need to develop alternative robust antiviral countermeasures, especially anti-ASFV agents, is of the utmost urgency. This study shows that fangchinoline (FAN), a bisbenzylisoquinoline alkaloid found in the roots of Stephania tetrandra of the family Menispermaceae, significantly inhibits ASFV replication in porcine alveolar macrophages (PAMs) at micromolar concentrations (IC50 = 1.66 µM). Mechanistically, the infection of ASFV triggers the AKT/mTOR/NF-κB signaling pathway. FAN significantly inhibits ASFV-induced activation of such pathways, thereby suppressing viral replication. Such a mechanism was confirmed using an AKT inhibitor MK2206 as it inhibited AKT phosphorylation and ASFV replication in PAMs. Altogether, the results suggest that the AKT/mTOR pathway could potentially serve as a treatment strategy for combating ASFV infection and that FAN could potentially emerge as an effective novel antiviral agent against ASFV infections and deserves further in vivo antiviral evaluations.
Assuntos
Vírus da Febre Suína Africana , Antivirais , Benzilisoquinolinas , Macrófagos Alveolares , NF-kappa B , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Replicação Viral , Animais , Macrófagos Alveolares/virologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Replicação Viral/efeitos dos fármacos , Vírus da Febre Suína Africana/efeitos dos fármacos , Vírus da Febre Suína Africana/fisiologia , Suínos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais/efeitos dos fármacos , NF-kappa B/metabolismo , Benzilisoquinolinas/farmacologia , Antivirais/farmacologia , Febre Suína Africana/virologia , Febre Suína Africana/tratamento farmacológico , Febre Suína Africana/metabolismoRESUMO
African Swine Fever (ASF) disease transmission parameters are crucial for making response and control decisions when faced with an outbreak, yet they are poorly quantified for smallholder and village contexts within Southeast Asia. Whilst disease-specific factors - such as latent and infectious periods - should remain reasonably consistent, host, environmental and management factors are likely to affect the rate of disease spread. These differences are investigated using Approximate Bayesian Computation with Sequential Monte-Carlo methods to provide disease parameter estimates in four naïve pig populations in villages of Lao People's Democratic Republic. The villages represent smallholder pig farmers of the Northern province of Oudomxay and the Southern province of Savannakhet, and the model utilised field mortality data to validate the transmission parameter estimates over the course of multiple model generations. The basic reproductive number between-pigs was estimated to range from 3.08 to 7.80, whilst the latent and infectious periods were consistent with those published in the literature for similar genotypes in the region (4.72 to 6.19 days and 2.63 to 5.50 days, respectively). These findings demonstrate that smallholder village pigs interact similarly to commercial pigs, however the spread of disease may occur slightly slower than in commercial study groups. Furthermore, the findings demonstrated that despite diversity across the study groups, the disease behaved in a consistent manner. This data can be used in disease control programs or for future modelling of ASF in smallholder contexts.
Assuntos
Febre Suína Africana , Teorema de Bayes , Animais , Febre Suína Africana/transmissão , Febre Suína Africana/epidemiologia , Suínos , Laos/epidemiologia , Número Básico de Reprodução , Criação de Animais Domésticos/métodos , Método de Monte Carlo , Sus scrofa , Vírus da Febre Suína Africana/fisiologia , Surtos de Doenças/veterináriaRESUMO
Despite the great interest in the development of a vaccine against African swine fever (ASF) in wild boar, the immunological mechanisms that induce animal protection are still unknown. For this purpose, tertiary lymphoid organs (TLOs) of wild boar were characterised and compared with mucosa-associated lymphoid tissues (MALTs) by histopathology, histomorphometry and immunohistochemistry (CD3, CD79, PAX5, LYVE1, fibronectin). In addition, real-time polymerase chain reaction (qPCR) and immunohistochemistry (p72) were used to evaluate the presence of ASF virus (ASFV) in blood and tissues samples, respectively. TLOs were observed in animals infected with a low-virulent ASFV isolate (LVI), animals co-infected with low and high-virulent ASFV isolates (LVI-HVI) and animals infected only with the high virulence isolate (HVI). TLOs in LVI and LVI-HVI groups were located adjacent to the mucosa and presented a similar structure to MALT. Immunoexpresion of p72 observed in the inflammatory cells adjacent to TLOs/MALTs confirmed its development and reactivity generated by ASF attenuated isolates. Immunohistochemical evaluation, based on cellular composition (T and B lymphocytes), and histomorphometrical study revealed a more pronounced maturation of TLOs/MALTs in the LVI-HVI group. It is currently unclear whether these formations play a protective role by contributing to local immunity in chronic inflammatory diseases. However, the structural similarities between TLOs and MALTs and the location of TLOs close to the mucosa suggest that they may perform a similar function, facilitating a local protective response. Nevertheless, further investigations are warranted to assess the cellular and humoral dynamics of these lymphoid organs induced by attenuated isolates.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Suínos , Animais , Sus scrofa , Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/prevenção & controle , VirulênciaRESUMO
African swine fever (ASF) is a disease that is a growing threat to the global swine industry. Regulations and restrictions are placed on swine movement to limit the spread of the virus. However, these are costly and time-consuming. Therefore, this study aimed to determine if high-pressure processing (HPP) sanitization techniques would be effective against the ASF virus. Here, it was hypothesized that HPP could inactivate or reduce ASF virus infectivity in tissue homogenates. To test this hypothesis, 30 aliquots of each homogenate (spleen, kidney, loin) were challenge-infected with the Turin/83 strain of ASF, at a 10 7.20 median hemadsorption dose (HAD)50/mL. Subsequently, eight aliquots of each homogenate were treated with 600 millipascal (600 MPa) HPP for 3, 5, and 7 min. Six untreated aliquots were used as the controls. Virological results showed a reduction in the viral titer of more than 7-log. These results support the validity of the study hypothesis since HPP treatment was effective in inactivating ASFV in artificially prepared samples. Overall, this study suggests the need for further investigation of other ASFV-contaminated meat products.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Animais , Vírus da Febre Suína Africana/fisiologia , Suínos , Febre Suína Africana/virologia , Pressão , Rim/virologia , Carga Viral , Inativação de Vírus , Baço/virologiaRESUMO
African swine fever (ASF) is a devastating disease with a high impact on the pork industry worldwide. ASF virus (ASFV) is a very complex pathogen, the sole member of the family Asfaviridae, which induces a state of immune suppression in the host through infection of myeloid cells and apoptosis of lymphocytes. Moreover, haemorrhages are the other main pathogenic effect of ASFV infection in pigs, related to the infection of endothelial cells, as well as the activation and structural changes of this cell population by proinflammatory cytokine upregulation within bystander monocytes and macrophages. There are still many gaps in the knowledge of the role of proteins produced by the ASFV, which is related to the difficulty in producing a safe and effective vaccine to combat the disease, although few candidates have been approved for use in Southeast Asia in the past couple of years.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Vírus da Febre Suína Africana/patogenicidade , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/fisiologia , Animais , Febre Suína Africana/virologia , Febre Suína Africana/imunologia , Suínos , VirulênciaRESUMO
African swine fever, caused by African swine fever virus (ASFV), is a highly contagious and fatal disease that poses a significant threat to the global pig industry. The limited information on ASFV pathogenesis and ASFV-host interactions has recently prompted numerous transcriptomic studies. However, most of these studies have focused on elucidating the transcriptome profiles of ASFV-infected porcine alveolar macrophages in vitro. Here, we analyzed dynamic transcriptional patterns in vivo in nine organ tissues (spleen, submandibular lymph node, mesenteric lymph node, inguinal lymph node, tonsils, lungs, liver, kidneys, and heart) obtained from pigs in the early stages of ASFV infection (1 and 3 d after viremia). We observed rapid spread of ASFV to the spleen after viremia, followed by broad transmission to the liver and lungs and subsequently, the submandibular and inguinal lymph nodes. Profound variations in gene expression patterns were observed across all organs and at all time-points, providing an understanding of the distinct defence strategies employed by each organ against ASFV infection. All ASFV-infected organs exhibited a collaborative response, activating immune-associated genes such as S100A8, thereby triggering a pro-inflammatory cytokine storm and interferon activation. Functional analysis suggested that ASFV exploits the PI3K-Akt signalling pathway to evade the host immune system. Overall, our findings provide leads into the mechanisms underlying pathogenesis and host immune responses in different organs during the early stages of infection, which can guide further explorations, aid the development of efficacious antiviral strategies against ASFV, and identify valuable candidate gene targets for vaccine development.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Transcriptoma , Animais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Suínos , Febre Suína Africana/virologia , Perfilação da Expressão Gênica , Linfonodos/virologia , Baço/virologia , Baço/metabolismo , Viremia , Pulmão/virologia , Fígado/virologia , Fígado/metabolismoRESUMO
Virus replication relies on complex interactions between viral proteins. In the case of African swine fever virus (ASFV), only a few such interactions have been identified so far. In this study, we demonstrate that ASFV protein p72 interacts with p11.5 using co-immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS). It was found that protein p72 interacts specifically with p11.5 âat sites amino acids (aa) 1-216 of p72 and aa 1-68 of p11.5. To assess the importance of p11.5 in ASFV infection, we developed a recombinant virus (ASFVGZΔA137R) by deleting the A137R gene from the ASFVGZ genome. Compared with ASFVGZ, the infectious progeny virus titers of ASFVGZΔA137R were reduced by approximately 1.0 logs. In addition, we demonstrated that the growth defect was partially attributable to a higher genome copies-to-infectious virus titer ratios produced in ASFVGZΔA137R-infected MA104 âcells than in those infected with ASFVGZ. This finding suggests that MA104 âcells infected with ASFVGZΔA137R may generate larger quantities of noninfectious particles. Importantly, we found that p11.5 did not affect virus-cell binding or endocytosis. Collectively, we show for the first time the interaction between ASFV p72 and p11.5. Our results effectively provide the relevant information of the p11.5 protein. These results extend our understanding of complex interactions between viral proteins, paving the way for further studies of the potential mechanisms and pathogenesis of ASFV infection.
Assuntos
Vírus da Febre Suína Africana , Proteínas Virais , Replicação Viral , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Animais , Suínos , Proteínas Virais/metabolismo , Proteínas Virais/genética , Chlorocebus aethiops , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Linhagem Celular , Ligação Proteica , Cromatografia Líquida , Células Vero , Espectrometria de MassasRESUMO
African swine fever (ASF) is a highly contagious, often fatal viral disease caused by African swine fever virus (ASFV), which imposes a substantial economic burden on the global pig industry. When screening for the virus replication-regulating genes in the left variable region of the ASFV genome, we observed a notable reduction in ASFV replication following the deletion of the MGF300-4L gene. However, the role of MGF300-4L in ASFV infection remains unexplored. In this study, we found that MGF300-4L could effectively inhibit the production of proinflammatory cytokines IL-1ß and TNF-α, which are regulated by the NF-κB signaling pathway. Mechanistically, we demonstrated that MGF300-4L interacts with IKKß and promotes its lysosomal degradation via the chaperone-mediated autophagy. Meanwhile, the interaction between MGF300-4L and IκBα competitively inhibits the binding of the E3 ligase ß-TrCP to IκBα, thereby inhibiting the ubiquitination-dependent degradation of IκBα. Remarkably, although ASFV encodes other inhibitors of NF-κB, the MGF300-4L gene-deleted ASFV (Del4L) showed reduced virulence in pigs, indicating that MGF300-4L plays a critical role in ASFV pathogenicity. Importantly, the attenuation of Del4L was associated with a significant increase in the production of IL-1ß and TNF-α early in the infection of pigs. Our findings provide insights into the functions of MGF300-4L in ASFV pathogenicity, suggesting that MGF300-4L could be a promising target for developing novel strategies and live attenuated vaccines against ASF.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Quinase I-kappa B , Inibidor de NF-kappaB alfa , Animais , Vírus da Febre Suína Africana/fisiologia , Quinase I-kappa B/genética , Quinase I-kappa B/farmacologia , NF-kappa B/genética , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/farmacologia , Suínos , Fator de Necrose Tumoral alfa/genética , VirulênciaRESUMO
BACKGROUND: The African swine fever (ASF) virus (ASFV) and ASF-like viral sequences were identified in human samples and sewage as well as in different water environments. Pigs regularly experience infections by the ASFV. The considerable stability of the virus in the environment suggests that there is ongoing and long-term contact between humans and the ASFV. However, humans exhibit resistance to the ASFV, and the decisive factor in developing infection in the body is most likely the reaction of target macrophages to the virus. Therefore, this study aimed to characterize the responses of human macrophages to the virus and explore the distinct features of the viral replication cycle within human macrophages. METHODS: The ASFV Armenia/07 strain was used in all experiments. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the ASFV gene expression; flow cytometry analysis was performed to evaluate the effects of the inactive and active ASFV (inASFV and aASFV) treatments on the phenotype of THP-1-derived macrophages (Mφ0) and inflammatory markers. Moreover, other methods such as cell viability and apoptosis assays, staining techniques, phagocytosis assay, lysosome-associated membrane protein (LAMP-1) cytometry, and cytokine detection were used during experiments. RESULTS: Our findings showed that the virus initiated replication by entering human macrophages. Subsequently, the virus shed its capsid and initiated the transcription of numerous viral genes, and at least some of these genes executed their functions. In THP-1-derived macrophages (Mφ0), the ASFV implemented several functions to suppress cell activity, although the timing of their implementation was slower compared with virus-sensitive porcine alveolar macrophages (PAMs). Additionally, the virus could not complete the entire replication cycle in human Mφ0, as indicated by the absence of viral factories and a decrease in infectious titers of the virus with each subsequent passage. Overall, the infection of Mφ0 with the ASFV caused significant alterations in their phenotype and functions, such as increased TLR2, TLR3, CD80, CD36, CD163, CXCR2, and surface LAMP-1 expression. Increased production of the tumor necrosis factor (TNF) and interleukin (IL)-10 and decreased production of interferon (IFN)-α were also observed. Taken together, the virus enters human THP-1-derived macrophages, starts transcription, and causes immunological responses by target cells but cannot complete the replicative cycle. CONCLUSION: These findings suggest that there may be molecular limitations within human macrophages that at least partially restrict the complete replication of the ASFV. Understanding the factors that hinder viral replication in Mφ0 can provide valuable insights into the host-virus interactions and the mechanisms underlying the resistance of human macrophages to the ASFV.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Macrófagos , Replicação Viral , Vírus da Febre Suína Africana/fisiologia , Vírus da Febre Suína Africana/genética , Humanos , Macrófagos/virologia , Macrófagos/metabolismo , Animais , Febre Suína Africana/virologia , Febre Suína Africana/imunologia , Febre Suína Africana/metabolismo , Apoptose , Suínos , Fagocitose , Células THP-1 , Sobrevivência Celular , Citocinas/metabolismo , Citocinas/genéticaRESUMO
African swine fever virus (ASFV) infection causes African swine fever (ASF), a highly contagious and fatal disease that poses severe threat to swine production. To gain insights into the host responses to ASFV, we generated recombinant adenovirus Ad5 expressing viral membrane proteins p54, p17, and pB117L individually and infected an alveolar cell line, 3D4/21, with these recombinant viruses. Then, the cell lysates were analyzed using label-free quantification proteomic analysis method. A total of 2158 differentially expressed proteins (DEPs) were identified, of which 817, 466, and 875 proteins were from Ad5-p54-, Ad5-p17-, Ad5-pB117L-infected 3D4/21 cells, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed distinct yet interconnecting patterns of protein interaction networks. Specifically, the Ad5-p54 virus infection enriched the DEPs primarily involved in the metabolic pathways, endocytosis, adherens junction, and SNARE interactions in vesicular transport. The Ad5-p17 virus infection enriched the DEPs in endocytosis, ubiquitin-mediated proteolysis, N-Glycan biosynthesis, and apoptosis, while the Ad5-pB117L virus infection enriched the DEPs in metabolic pathways, endocytosis, oxidative phosphorylation, and focal adhesion. In summary, these results provide a comprehensive proteinomics analysis of the cellular responses to three ASFV membrane proteins, thus facilitating our understanding of ASFV pathogenesis.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Proteômica , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Vírus da Febre Suína Africana/metabolismo , Animais , Suínos , Proteômica/métodos , Linhagem Celular , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Interações Hospedeiro-Patógeno , Mapas de Interação de Proteínas , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/genéticaRESUMO
African Swine Fever Virus (ASFV) infection causes an acute and highly contagious disease in swine, resulting in significant economic losses and societal harm worldwide. Currently, there are no effective vaccines or antiviral drugs available for ASFV. Tetrandrine (TET) is extracted from the traditional Chinese herb Stephania tetrandrae, possesses diverse biological functions such as anti-inflammatory, anti-tumor, and antiviral activities. The study comprehensively evaluated the anti-ASFV effect of TET and validated it through biological assays. The dose-dependent inhibition of TET against ASFV was confirmed and a novel mechanism of TET's anti-ASFV activity was elucidated. TET effectively inhibits ASFV during internalization by blocking macropinocytosis through the inhibition of the PI3K/Akt pathway. The specific inhibitor LY294002, targeting the PI3K/Akt pathway, exhibits similar antiviral activity against ASFV as TET. Furthermore, the inhibitory effect of TET against other viruses such as Lumpy Skin Disease Virus (LSDV) and Porcine Epidemic Diarrhea Virus (PEDV) was also identified. Our findings suggest that TET effectively inhibits ASFV and reveal the potential for broad-spectrum antiviral drugs targeting the PI3K/Akt pathway.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Benzilisoquinolinas , Internalização do Vírus , Animais , Vírus da Febre Suína Africana/efeitos dos fármacos , Vírus da Febre Suína Africana/fisiologia , Antivirais/farmacologia , Antivirais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Suínos , Benzilisoquinolinas/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Human health is dependent on food safety and, therefore, on the health of farm animals. One of the most significant threats in regard to swine diseases is African swine fever (ASF). Infections caused by porcine circoviruses (PCVs) represent another important swine disease. Due to the ubiquitous nature of PCV2, it is not surprising that this virus has been detected in ASFV-affected pigs. However, recent data indicate that coinfection of PCV3 and ASFV also occurs. It is still unclear whether PCV infection plays a role in ASFV infection, and that subject requires further analysis. The aim of this study was to assess whether PCV3 and PCV4 are present in the wild boar population in Poland (real-time PCR). The analysis was performed on wild boar samples collected for routine ASF surveillance in Poland, between 2018 and 2021. By extension, the obtained data were compared in regard to ASFV presence in these samples, thus investigating the odds of ASFV infection on the grounds of the PCV carrier state in free-ranging Suidae in Poland. In addition, sequencing of PCV3 and phylogenetic analysis were performed, based on a full genome and a capsid gene. In the current study, we demonstrated the high prevalence of PCV3 in the wild boar population in Poland; meanwhile, PCV4 was not detected. The odds of ASFV infection on the grounds of the PCV3 carrier state in free-ranging Suidae in Poland was more than twice as high. Ten full genome sequences of PCV3 were obtained, all of them belonging to clade 3a. The similarity between them was in the range of 98.78-99.80%.