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1.
Talanta ; 246: 123467, 2022 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-35489097

RESUMO

Occupational health problems, such as asthma, in specific work environments arise from the presence of airborne fungi. Rapid detection of pathogenic airborne fungi is therefore important to reduce or avoid any adverse effects on staff health. Herein, we established a new integrated rapid Lyticase-Motor-Chemical reagent nucleic acid releasing (LMC) method for the release of fungal DNA. Aspergillus fumigatus, Aspergillus flavus, and Cryptococcus neoformans were chosen to evaluate the LMC method. The results of Loop-Mediated Isothermal Amplification (LAMP) analyses showed that this method could release the nucleic acid of 4 × 104 fungal spores, equaling to 400 copies per microliter. This rapid multiplex nucleic acid detection system of airborne fungi included an integrated DNA release device and a portable microfluidic chip. The integrated DNA release device combined mechanical lysing and biochemical reagent treatment to automate DNA release. The microfluidic chip was capable of multiplex nucleic acid detection. The detection limit of this system was 4 × 104 spores per test, meeting the requirement of early warnings. The whole analysis from the sample input to readout could be completed within 90 min, including 30 min for fungal DNA release and 45 min for LAMP analysis. The integrated DNA release device and microfluidic chip were portable, showing tremendous potential in point-of-care tests of airborne fungi.

2.
Cell Mol Immunol ; 2022 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-35332300

RESUMO

Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand ("trans" genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.

3.
Dis Markers ; 2022: 8241405, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35299867

RESUMO

Objective: This study is aimed at investigating the early diagnosis and efficacy of emergency treatments of nine patients with severe multiple injuries accompanied by traumatic aortic dissection (TAD). Methods: Patients who sustained severe multiple injuries accompanied by TAD following a car accident (n = 6) and falls from a height (n = 3) were treated in the emergency department of our hospital from October 2017 to July 2021. Data of these patients, including seven men and two women (average age, 53 ± 15.2 years; range, 18-83 years) were analysed retrospectively. Upon hospital arrival, the multidisciplinary treatment (MDT) trauma team, composed of doctors and nurses, immediately performed resuscitation following the Green Channel Consultation and Treatment Process for Severe Multiple Injuries. Life-threatening injuries were managed urgently. Blood tests and blood preparation and bedside B-scan ultrasonography and CT were performed. Aortic computed tomography angiography (CTA) was conducted decisively in patients suspected of TAD so that endovascular graft exclusion (EVGE) with the aortic covered stent can be performed promptly, followed by emergency management, second-stage surgery, and intensive care according to the injury control strategy. Results: This study included nine patients suffering from severe multiple injuries accompanied by Stanford type B TAD, with injury severity scores ranging from 35 to 43 points. Six patients underwent EVGE while receiving emergency treatment, whereas two patients who also had intracranial haemorrhage underwent selective EVGE. One case of TAD missed in the emergency department was detected 13 days after hospitalisation; therefore, the patient promptly underwent EVGE. Emergency procedures performed included exploratory laparotomy and splenectomy (n = 2), thoracic closed drainage (n = 5), haemothoracotomy (n = 3), second-stage fracture surgery (n = 4), and tracheotomy (n = 1). Postinjury complications included haemorrhagic shock, coagulation disorders, hyoxaemia, pulmonary infection, renal insufficiency, and hypoproteinaemia; however, all patients recovered after intensive care treatment. Aortic CTA after EVGE revealed the disappearance of the dissection and the resorption of the intermural haematoma. However, varying degrees of stenosis or occlusion were observed in the left subclavian artery. Nine patients with severe multiple injuries were treated satisfactorily by the MDT, without fatalities, and all patients were discharged for rehabilitation. Conclusion: In this study, procedures including resuscitation, urgent aortic CTA for definitive diagnosis, prompt EVGE, emergency injury control surgery, second-stage definitive surgery, intensive care treatment, and rehabilitation were rationally performed by the emergency MDT trauma team. Overall, this continuous and seamless process is a key factor for the successful treatment of patients with severe multiple injuries accompanied by TAD.


Assuntos
Aneurisma Dissecante/cirurgia , Aorta Torácica/cirurgia , Diagnóstico Precoce , Tratamento de Emergência , Traumatismo Múltiplo/terapia , Stents , Angiografia por Tomografia Computadorizada , Cuidados Críticos , Procedimentos Endovasculares , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Tomografia Computadorizada por Raios X , Transplantes , Resultado do Tratamento
4.
Viruses ; 14(2)2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35215764

RESUMO

Porcine parvovirus (PPV) is the main pathogen of reproductive disorders. In recent years, a new type of porcine parvovirus has been discovered and named porcine parvovirus 2 to 7 (PPV2-PPV7), and it is associated with porcine circovirus type 2 in pigs. Codon usage patterns and their effects on the evolution and host adaptation of different PPV sub-types are still largely unknown. Here, we define six main sub-types based on the Bayesian method of structural proteins of each sub-type of PPV, including PPV2, PPV3, PPV4, PPV5, PPV6, and PPV7, which show different degrees of codon usage preferences. The effective number of codons (ENC) indicates that all PPV sub-types have low codon bias. According to the codon adaptation index (CAI), PPV3 and PPV7 have the highest similarity with the host, which is related to the main popular tendency of the host in the field; according to the frequency of optimal codons (FOP), PPV7 has the highest frequency of optimal codons, indicating the most frequently used codons in its genes; and according to the relative codon deoptimization index (RCDI), PPV3 has a higher degree. Therefore, it is determined that mutational stress has a certain impact on the codon usage preference of PPV genes, and natural selection plays a very decisive and dominant role in the codon usage pattern. Our research provides a new perspective on the evolution of porcine parvovirus (PPV) and may help provide a new method for future research on the origin, evolutionary model, and host adaptation of PPV.


Assuntos
Uso do Códon , Variação Genética , Adaptação ao Hospedeiro , Infecções por Parvoviridae/virologia , Parvovirus Suíno/genética , Doenças dos Suínos/virologia , Animais , Teorema de Bayes , Evolução Molecular , Genótipo , Mutação , Filogenia , Seleção Genética , Suínos
5.
Viruses ; 14(2)2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35215787

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Circovirus/patogenicidade , Coinfecção/virologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Animais , China , Infecções por Circoviridae/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Circovirus/genética , Coinfecção/genética , Coinfecção/imunologia , Coinfecção/mortalidade , Feminino , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Pulmão/imunologia , Pulmão/virologia , Masculino , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/mortalidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Suínos , Virulência
6.
J Virol ; 96(1): e0169521, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-34643429

RESUMO

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrate that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2α phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2α pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2α also promoted the formation of stress granules (SGs), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR, and eIF2α) for antiviral drugs. IMPORTANCE Coronavirus diseases are caused by different coronaviruses of importance in humans and animals, and specific treatments are extremely limited. ERS, which can activate the UPR to modulate viral replication and the host innate response, is a frequent occurrence in coronavirus-infected cells. PHEV, a neurotropic betacoronavirus, causes nerve cell damage, which accounts for the high mortality rates in suckling piglets. However, it remains incompletely understood whether the highly developed ER in nerve cells plays an antiviral role in ERS and how ERS regulates viral proliferation. In this study, we found that PHEV infection induced ERS and activated the UPR both in vitro and in vivo and that the activated PERK/PKR-eIF2α axis inhibited PHEV replication through attenuating global protein translation and promoting SG formation. A better understanding of coronavirus-induced ERS and UPR activation may reveal the pathogenic mechanism of coronavirus and facilitate the development of new treatment strategies for these diseases.


Assuntos
Betacoronavirus 1/fisiologia , Infecções por Coronavirus/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Replicação Viral/fisiologia , eIF-2 Quinase/metabolismo , Animais , Betacoronavirus 1/metabolismo , Linhagem Celular , Infecções por Coronavirus/virologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático , Camundongos , Fosforilação , Biossíntese de Proteínas , Transdução de Sinais , Resposta a Proteínas não Dobradas
7.
Talanta ; 236: 122827, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635217

RESUMO

Cryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on microfluidic detection for CM diagnosis is still limited. We developed a multifunctional microfluidic module that integrated the pathogen enrichment and on-chip nucleic acid extraction. The module adopted a simple filtration membrane to effectively capture Cryptococcus cells and simplify the process, and combined lyticase digestion, alkaline lysis and heating methods to optimize the strategy to achieve nucleic acid extraction. The entire process was operated in the module, which reduced the exposure risk of directly processing cryptococcal samples. A portable one-pot lyophilized LAMP reagent bead with no temperature limit was developed, which improved the flexibility of operation. This module did not require any additional instrument, and is promising to develop a simple, rapid, and efficient approach to realize the "sample in and answer out" detection of real CSF samples. This microfluidic module had practical prospects and is expected to replace LFA for efficacy evaluation and follow-up in the middle and late stages of CM treatment, and could be used as an auxiliary method to confirm cases with questionable LFA results in the early diagnosis of CM.


Assuntos
Meningite Criptocócica , Ácidos Nucleicos , Humanos , Meningite Criptocócica/diagnóstico , Microfluídica , Análise de Sequência com Séries de Oligonucleotídeos
8.
Vet Microbiol ; 265: 109315, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34972029

RESUMO

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a neurotropic coronavirus and highly pathogenic in veterinary clinic. Spike (S) protein of PHEV interplays with host components to cross the plasma membrane of target cells, but characterization of its functional receptors is limited. Here, we discovered that cell-surface glycans, i.e., sialic acid (SA) and heparan sulfate (HS), act as critical interacting factors of PHEV, involving in viral attachment. As shown in glycans depletion assay, removing SA or HS from N2a cells inhibits PHEV infection. Soluble sugar monomers were utilized for competitive binding tests, and we found that both SA and HS could specifically bind to PHEV and affect the viral infectivity. Furthermore, the expression of heparan sulfate proteoglycans (HSPGs), including syndecans and glypicans, and endoglycosidase heparinase which cleaves HS were regulated by PHEV RNA replication. Together, we newly identified specificity recognition of cellular glycans and PHEV during infection, providing novel cellular targets for antiviral therapies and better understanding of pathogenesis.


Assuntos
Betacoronavirus 1 , Membrana Celular , Polissacarídeos , Ligação Viral , Animais , Linhagem Celular , Suínos
9.
J Pharm Biomed Anal ; 209: 114464, 2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-34915322

RESUMO

Staphylococcus aureus (SA) is one of the most common pathogenic bacteria, and methicillin-resistant SA (MRSA) is an equally common drug-resistant bacteria. MRSA detection is of great significance for clinical diagnosis, medication guidance, and prevention of antibiotic abuse. Traditional MRSA detection using the culture method is time-consuming, laborious, and difficult to conduct rapid on-site detection. In this research, we developed a device for rapid MRSA detection, which can detect the nuc gene in SA and mecA gene in MRSA simultaneously for 30-40 min. After simple sample processing, the mixture can be directly loaded onto the chip device. The detection results can be directly determined by a color change, with a limitation of approximately 102 copies. This isothermal amplification chip device can be widely applied in many fields, with simple operation and low contamination.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Proteínas de Bactérias/genética , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Técnicas de Amplificação de Ácido Nucleico , Testes Imediatos , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética
10.
Transbound Emerg Dis ; 2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34812572

RESUMO

As a member of the Alphavirus, Getah virus (GETV) was becoming more serious and posing a serious threat to animal safety and public health. However, the circulation, distribution and evolution of GETV is not well understood. Hence, we integrated a variety of bioinformatic methodologies, from genomic alterations to systematic analysis, phylogeography, selection, adaptive analysis, prediction of protein modification, structural biology and molecular dynamics simulations to understand the characteristics of GETV. The results of phylogeography and molecular evolution show that due to the lack of vaccine, GETV is rapidly expanding its host range and geographical distribution at a high evolutionary rate. We also predicted the important modification sites, and identified the adaptive and active selection sites. Finally, the analysis of spatial structure and function showed that six adaptive sites may be related to the structural stability, receptor binding ability, immunogenicity and immune evasion of the virus, respectively. The data from this study have important implications for the understanding of ongoing GETV outbreaks worldwide and will guide future efforts to develop effective preventive and control measures against GETV. In particular, biosafety measures should be strengthened immediately to prevent GETV from becoming a pandemic, especially in China, South Korea and Japan.

11.
Virol J ; 18(1): 209, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34674719

RESUMO

BACKGROUND: Porcine vesicular disease is caused by the Seneca Valley virus (SVV), it is a novel Picornaviridae, which is prevalent in several countries. However, the pathogenicity of SVV on 5-6 week old pigs and the transmission routes of SVV remain unknown. METHODS: This research mainly focuses on the pathogenicity of the CH-GX-01-2019 strain and the possible vector of SVV. In this study, 5-6 week old pigs infected with SVV (CH-GX-01-2019) and its clinical symptoms (including rectal temperatures and other clinical symptoms) were monitored, qRT-PCR were used to detect the viremia and virus distribution. Neutralization antibody assay was set up during this research. Mosquitoes and Culicoides were collected from pigsties after pigs challenge with SVV, and SVV detection within mosquitoes and Culicoides was done via RT-PCR. RESULTS: The challenged pigs presented with low fevers and mild lethargy on 5-8 days post infection. The viremia lasted more than 14 days. SVV was detected in almost all tissues on the 14th day following the challenge, and it was significantly higher in the hoofs (vesicles) and lymph nodes in comparison with other tissues. Neutralizing antibodies were also detected and could persist for more than 28 days, in addition neutralizing antibody titers ranged from 1:128 to 1:512. Mosquitoes and Culicoides were collected from the pigsty environments following SVV infection. Although SVV was not detected in the mosquitoes, it was present in the Culicoides, however SVV could not be isolated from the positive Culicoides. CONCLUSIONS: Our work has enriched the knowledge relating to SVV pathogenicity and possible transmission routes, which may lay the foundation for further research into the prevention and control of this virus.


Assuntos
Ceratopogonidae , Infecções por Picornaviridae , Picornaviridae , Doenças dos Suínos , Animais , Fazendas , Mosquitos Vetores , Infecções por Picornaviridae/veterinária , Suínos , Virulência
12.
J Virol ; 95(19): e0015321, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287041

RESUMO

Orf virus (ORFV) is a highly epitheliotropic parapoxvirus with zoonotic significance that induces proliferative lesions in the skin of sheep, goats, and humans. Several viral proteins carried by ORFV, including nuclear factor-κB (NF-κB) inhibitors, play important roles in hijacking host-associated proteins for viral evasion of the host innate immune response. However, the roles of proteins with unknown functions in viral replication and latent infection remain to be explored. Here, we present data demonstrating that the ORF120, an early-late ORFV-encoded protein, activates the NF-κB pathway in the early phase of infection, which implies that ORFV may regulate NF-κB through a biphasic mechanism. A DUAL membrane yeast two-hybrid system and coimmunoprecipitation experiments revealed that the ORF120 protein interacts with Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1). The overexpression of the ORF120 protein can efficiently increase the expression of G3BP1 and nuclear translocation of NF-κB-p65 in primary ovine fetal turbinate (OFTu) and HeLa cells. The knockdown of G3BP1 significantly decreased ORF120-induced NF-κB activation, indicating that G3BP1 is involved in ORF120-induced NF-κB pathway activation. A dual-luciferase reporter assay revealed that ORF120 could positively regulate the NF-κB pathway through the full-length G3BP1 or the domain of G3BP1RRM+RGG. In conclusion, we demonstrate, for the first time, that the ORF120 protein is capable of positively regulating NF-κB signaling by interacting with G3BP1, providing new insights into ORFV pathogenesis and a theoretical basis for antiviral drug design. IMPORTANCE As part of the host innate response, the nuclear factor-κB (NF-κB) pathway plays a partial antiviral role in nature by regulating the innate immune response. Thus, the NF-κB pathway is probably the most frequently targeted intracellular pathway for subversion by anti-immune modulators that are carried by a wide range of pathogens. Various viruses, including poxviruses, carry several proteins that prepare the host cell for viral replication by inhibiting cytoplasmic events, leading to the initiation of NF-κB transcriptional activity. However, NF-κB activity is hypothesized to facilitate viral replication to a great extent. The significance of our research is in the exploration of the activation mechanism of NF-κB induced by the Orf virus (ORFV) ORF120 protein interacting with G3BP1, which helps not only to explain the ability of ORFV to modulate the immune response through the positive regulation of NF-κB but also to show the mechanism by which the virus evades the host innate immune response.


Assuntos
DNA Helicases/metabolismo , Ectima Contagioso/virologia , NF-kappa B/metabolismo , Vírus do Orf/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , DNA Helicases/química , Células HeLa , Humanos , Vírus do Orf/genética , Vírus do Orf/crescimento & desenvolvimento , Vírus do Orf/patogenicidade , Proteínas de Ligação a Poli-ADP-Ribose/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ovinos , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Transcrição Genética , Ativação Transcricional , Proteínas Virais/genética , Virulência
13.
J Virol ; 95(19): e0085121, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287052

RESUMO

Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (ß-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing ULK1-knockout neuroblastoma cells, we have identified that ULK1 is not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of noncanonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic ß-CoV and the host. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic alongside the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) pose Betacoronavirus (ß-CoV) as a global public health challenge. Coronaviruses subvert, hijack, or utilize autophagy to promote proliferation, and thus, exploring the cross talk between ß-CoV and autophagy is of great significance in confronting future ß-CoV outbreaks. Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic ß-CoV that invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction is yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypasses the multifaceted regulation of ULK1 kinase. The PHEV-triggered noncanonical autophagy underscores the complex interactions of virus and host and will help in the development of therapeutic strategies targeting noncanonical autophagy to treat ß-CoV disease.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Betacoronavirus 1/metabolismo , Animais , Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , COVID-19 , Linhagem Celular , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/metabolismo , Fosforilação , SARS-CoV-2
14.
Virus Genes ; 57(3): 284-288, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33970402

RESUMO

Lyon IARC polyomavirus (LIPyV), a newly discovered polyomavirus (PyV), was first identified in 2017 in human skin samples in the USA. Later, it was detected in several other countries in samples of human and feline origin. Our aim was to find out if the virus occurs in China. To this end, 100 fecal samples were collected from cats with diarrhea in Guangxi Province during 2016 and 2018 and tested with polymerase chain reaction (PCR). Only 2 samples that originated from two related individuals were found to be positive. Based on the sequence identity of the 240-bp PCR products, the two positive samples supposedly contained identical viruses. Therefore, only one of them, which was designated as LIPyV-GXNN01, was selected for full genome amplification, cloning, sequencing and analysis. LIPyV-GXNN01, which comprises 5,263 nucleotides, has an early region that consists of small T antigen (ST-Ag) and large T antigen (LT-Ag) and a late region coding for the VP1, VP2, and VP3 structural proteins. Moreover, the LIPyV-GXNN01 strain structural proteins share 95.9-99.4%, 97.6-99.2%, and 97.1-99.2% nucleic acid identity with the VP1, VP2, and VP3of other LIPyV reference strains, respectively. A phylogenetic analysis revealed that GXNN01 clustered together with previously reported LIPyV strain. This present study is the first report of LIPyV in China.


Assuntos
Antígenos Virais de Tumores/genética , Diarreia/genética , Genoma Viral/genética , Polyomavirus/genética , Animais , Gatos , Diarreia/virologia , Humanos , Anotação de Sequência Molecular , Polyomavirus/isolamento & purificação , Polyomavirus/patogenicidade , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologia , Proteínas Estruturais Virais/genética , Sequenciamento Completo do Genoma
15.
Oncogene ; 40(23): 3974-3988, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33990700

RESUMO

5-Fluorouracil (5-FU)-based chemotherapy is the first-line treatment for colorectal cancer (CRC) but is hampered by chemoresistance. Despite its impact on patient survival, the mechanism underlying chemoresistance against 5-FU remains poorly understood. Here, we identified serine hydroxymethyltransferase-2 (SHMT2) as a critical regulator of 5-FU chemoresistance in CRC. SHMT2 inhibits autophagy by binding cytosolic p53 instead of metabolism. SHMT2 prevents cytosolic p53 degradation by inhibiting the binding of p53 and HDM2. Under 5-FU treatment, SHMT2 depletion promotes autophagy and inhibits apoptosis. Autophagy inhibitors decrease low SHMT2-induced 5-FU resistance in vitro and in vivo. Finally, the lethality of 5-FU treatment to CRC cells was enhanced by treatment with the autophagy inhibitor chloroquine in patient-derived and CRC cell xenograft models. Taken together, our findings indicate that autophagy induced by low SHMT2 levels mediates 5-FU resistance in CRC. These results reveal the SHMT2-p53 interaction as a novel therapeutic target and provide a potential opportunity to reduce chemoresistance.


Assuntos
Cloroquina/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Glicina Hidroximetiltransferase/metabolismo , Animais , Antimaláricos/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Glicina Hidroximetiltransferase/deficiência , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Arch Virol ; 166(7): 1951-1959, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33987752

RESUMO

A novel circovirus designated "porcine circovirus type 4" (PCV4) was recently reported in pigs with severe clinical disease in Hunan Province, China. Relatively little is known about the molecular epidemiology of this recently discovered virus. In order to assess the prevalence of PCV4 infection in pigs and to analyze its genomic characteristics, 1683 clinical samples were collected in Inner Mongolia, China, from 2016 to 2018. The overall infection rate of PCV4 was 1.6% (27/1683) at the sample level and 21.6% (11/51) at the farm level, with rates ranging from 3.2% (1/31) to 20.0% (6/30) on different PCV4-positive pig farms. In addition, the PCV4 infection rates at both the sample and farm level increased from 2016 to 2018. This also showed that PCV4 was present in pigs in 2016 in China and therefore did not arrive later than this date. Additionally, our findings showed that PCV4 infections had no association with PCV2 or PCV3 infections. We sequenced the complete genomes of three PCV4 strains and found that the PCV4 strains had a high degree of genetic stability but shared less than 80% sequence identity with other circoviruses. We identified six amino acid mutations in the Rep protein and seven in the Cap protein. Phylogenetic analysis based on Cap and Rep sequences confirmed that the PCV4 strains grouped in an independent branch. Our findings provide important information about the prevalence and genetic characteristics of PCV4 strains.


Assuntos
Infecções por Circoviridae/epidemiologia , Circovirus/genética , Doenças dos Suínos/epidemiologia , Animais , China/epidemiologia , Infecções por Circoviridae/virologia , Fazendas , Genoma Viral/genética , Genômica/métodos , Epidemiologia Molecular/métodos , Filogenia , Prevalência , Estudos Retrospectivos , Suínos , Doenças dos Suínos/virologia
17.
Vet Microbiol ; 258: 109099, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33984791

RESUMO

Poxviruses have evolved multiple strategies to modulate host-derived factors to create an optimal environment for viral efficient replication. Our previous study indicated that cyclophilin B (CypB) is a critical factor for ORFV replication in MDBK cells. However, the precise molecular mechanism by which CypB facilitates ORFV replication remains less understood. In the present study, the function of CypB in ORFV replication is further evaluated. The overexpression of CypB was observed to facilitate ORFV replication in OFTu cells and HeLa cells, however, RNA interference (RNAi)-mediated reduction of endogenous CypB decreased the levels of ORFV replication. Coimmunoprecipitation experiments revealed that the CypB interacted with ORFV ORF058 protein, a late protein involved in virus entry. The interaction of host factor CypB and ORF058 protein was further confirmed by confocal microscopy analysis and GST-pull down. In addition, the 52-55 aa was identified as the critical binding sites for CypB on ORF058 protein by GST-pull down with OFTu cells overexpressing CypB and purified GST-tagged truncated ORF058. In conclusion, we demonstrate that CypB is a critical host factor for ORFV replication in vitro by interacting with ORF058 protein, providing new insights into ORFV pathogenesis.


Assuntos
Ciclofilinas/metabolismo , Regulação da Expressão Gênica/imunologia , Vírus do Orf/efeitos dos fármacos , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Ovinos , Regulação para Cima
18.
Parasitol Res ; 120(6): 2165-2174, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33893549

RESUMO

Neospora caninum is an important pathogen commonly causing spontaneous abortion in livestock. The parasite is known to remain in cysts in an inactive state; or it can undergo expansive development within an intermediate host. However, the mechanisms that trigger the proliferation of N. caninum have not been thoroughly elucidated. For various organisms, it has been demonstrated that microRNAs (miRNAs) can act as important endogenous regulatory factors in gene regulation during cell differentiation and development. However, miRNAs and their function have not been studied in N. caninum. In this study, small RNA libraries from N. caninum tachyzoites (NC-1 strain) were analyzed using a high-throughput RNA sequencing technology combined with systematic bioinformatics analysis. A considerable number of novel miRNAs from N. caninum NC-1 strain tachyzoites were identified. Of the 300 miRNAs found by bioinformatics analysis, 10 were conserved miRNAs belonging to 10 metazoan miRNA families, while 290 were novel miRNAs. The expression of 13 novel miRNAs was verified by real-time quantitative PCR (qRT-PCR). Data from this study provided and identified authentic miRNAs for the first time in N. caninum. The study also introduces a framework for further investigations of RNAi-dependent regulatory mechanisms of the parasite and provides data for further understanding of N. caninum development.


Assuntos
MicroRNAs/metabolismo , Neospora/genética , RNA de Protozoário/metabolismo , Animais , Chlorocebus aethiops , Coccidiose , Regulação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Neospora/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Células Vero
19.
Transbound Emerg Dis ; 68(2): 289-295, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32657534

RESUMO

In this study, we detected a circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus (named Po-Circo-like (PCL) virus) in intestinal tissue samples of pigs, and the complete genome sequences of three strains (named PCL viruses GX14, GX15 and GX19) were obtained. Unlike PCL virus strains 21 and 22, whose genome sequences have 3,912 and 3,923 nucleotides (nt), respectively, the strains revealed in this study have a circular genome with 3,944 nt and five major open reading frames (ORFs). Among these ORFs, ORF1 encodes the Rep and not the typical capsid protein encoded in PCV. Furthermore, the strains in this study share 79.2%-96.0% nucleic acid identity and 83.0%-98.1% amino acid identity with ORF1 of the reference strains. Moreover, the Rep of the PCL virus in this study shared 19.9%-22.2% (<30%) identity of its amino acid sequence with PCV but shared 34.9%-94.8% (>30%) identity of its amino acid sequence with sequences of five proteins that are expressed by the family Kirkoviridae. [Correction added on 24 December 2020 after first online publication: The preceding sentence has been corrected in this version.] Interestingly, the stem loop of the PCL virus has one nucleotide substitution, T1328G. The Bo-Circo-like CH strain shares high nucleic acid and amino acid similarity (>80%) with the PCL virus. Moreover, Bo-Circo-like CH and GX-19 had similar stem-loop sequences. The PCL virus might therefore be transmitted to non-porcine hosts by cross-species transmission routes. Phylogenetic analysis classified the PCL virus into the new family Kirkoviridae and indicated its close relationship with the Bo-Circo-like virus. A phylogenetic divergence analysis based on the rep gene classified all PCL virus strains into two genotypes (PCLa and PCLb). In conclusion, the present study is the first detailed report of the PCL virus, which is a potential new virus in pigs that might be involved in cross-species transmission. Further investigation is needed to determine the pathogenesis of this virus and its epidemiologic impact.


Assuntos
Infecções por Vírus de DNA/veterinária , Vírus de DNA/classificação , Vírus de DNA/genética , Diarreia/veterinária , Genoma Viral , Doenças dos Suínos/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas do Capsídeo/genética , China , Circovirus/genética , Infecções por Vírus de DNA/virologia , Vírus de DNA/isolamento & purificação , DNA Viral/genética , Diarreia/virologia , Fases de Leitura Aberta , Filogenia , Suínos , Proteínas Virais/genética
20.
Virus Res ; 291: 198177, 2021 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-33038460

RESUMO

Seneca Valley virus (SVV) is a novel Picornaviridae that is closely associated with porcine idiopathic vesicular disease (PIVD). Here, a novel SVV strain (CH-GX-01-2019) was detected and isolated from swine in Guangxi Province, China. The complete genomic sequence of CH-GX-01-2019 exhibited 93.3-98.9 % identify with other SVV isolates at the nucleotide level. CH-GX-01-2019 showed the highest level of similarity (98.9 %) with Vietnamese strains. And CH-GX-01-2019 exhibited two consecutive amino acid mutations in VP1 gene. Phylogenetic analysis based on the complete genome and the VP1 gene showed that Chinese SVV isolates can be divided into three clusters. We analyzed the geographical distributions of SVV strains in China and found that the epidemiology of SVV in China is complicated; most strains are distributed predominantly in south and central China. Between 2015 and 2019, the dominant epidemic SVV isolates in China have changed from clusters 1 and 3 to cluster 2. CH-GX-01-2019 (cluster 3) is a recombinant strain from Colombia-2016 (cluster 2) and HB-CH-2016 (cluster 1). Our findings will enhance our understanding of the prevalence and genetic variation of SVV in the swine herds of China and provide important insights into the molecular epidemiology of SVV.


Assuntos
Evolução Molecular , Filogenia , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/veterinária , Picornaviridae/genética , Doenças dos Suínos/epidemiologia , Animais , Linhagem Celular , China/epidemiologia , Cricetinae , Fazendas , Genoma Viral , Gado/virologia , Picornaviridae/classificação , Picornaviridae/isolamento & purificação , Infecções por Picornaviridae/virologia , Prevalência , Recombinação Genética , Suínos , Doenças dos Suínos/virologia , Sequenciamento Completo do Genoma
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