RESUMO
Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.
Assuntos
Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Proteínas não Estruturais Virais , Replicação Viral , Animais , Camundongos , Linhagem Celular , Cisteína/metabolismo , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Lipoilação , Serina/metabolismo , Proteínas não Estruturais Virais/metabolismo , VirulênciaRESUMO
As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.
Assuntos
Vírus da Febre Suína Clássica/metabolismo , Peste Suína Clássica/virologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Vírus da Febre Suína Clássica/genética , Clatrina/metabolismo , Retículo Endoplasmático/metabolismo , Interações entre Hospedeiro e Microrganismos , Suínos , Vesículas Transportadoras , Internalização do Vírus , Replicação ViralRESUMO
Broad-spectrum antibiotics are frequently used to treat bacteria-induced infections, but the overuse of antibiotics may induce the gut microbiota dysbiosis and disrupt gastrointestinal tract function. Probiotics can be applied to restore disturbed gut microbiota and repair abnormal intestinal metabolism. In the present study, two strains of Enterococcus faecium (named DC-K7 and DC-K9) were isolated and characterized from the fecal samples of infant dogs. The genomic features of E. faecium DC-K7 and DC-K9 were analyzed, the carbohydrate-active enzyme (CAZyme)-encoding genes were predicted, and their abilities to produce short-chain fatty acids (SCFAs) were investigated. The bacteriocin-encoding genes in the genome sequences of E. faecium DC-K7 and DC-K9 were analyzed, and the gene cluster of Enterolysin-A, which encoded a 401-amino-acid peptide, was predicted. Moreover, the modulating effects of E. faecium DC-K7 and DC-K9 on the gut microbiota dysbiosis induced by antibiotics were analyzed. The current results demonstrated that oral administrations of E. faecium DC-K7 and DC-K9 could enhance the relative abundances of beneficial microbes and decrease the relative abundances of harmful microbes. Therefore, the isolated E. faecium DC-K7 and DC-K9 were proven to be able to alter the gut microbiota dysbiosis induced by antibiotic treatment.
Assuntos
Antibacterianos , Disbiose , Enterococcus faecium , Microbioma Gastrointestinal , Animais , Disbiose/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Antibacterianos/farmacologia , Camundongos , Fezes/microbiologia , Ácidos Graxos Voláteis/metabolismo , Probióticos/farmacologia , Cães , Bacteriocinas/farmacologiaRESUMO
OBJECTIVES: In this study, the distribution of the oxazolidinone/phenicol resistance gene optrA and the mobile genetic elements involved in its dissemination were analysed among enterococcal isolates from a farrow-to-finish swine farm. METHODS: Enterococcus faecium and Enterococcus faecalis isolates were obtained from all pig production stages in the farm. The optrA-carrying E. faecium and E. faecalis isolates were subjected to PFGE and antimicrobial susceptibility testing. Complete sequences of the genetically unrelated optrA-carrying E. faecium and E. faecalis isolates were determined using Illumina HiSeq and MinION platforms. RESULTS: The optrA gene was present in 12.2% (23/188) of the E. faecium and E. faecalis isolates, most of which originated from nursery and finishing stages. The 23 optrA-positive Enterococcus isolates represented 15 PFGE types. WGS of representative isolates of the 15 PFGE types showed that optrA was carried by diverse genetic elements either located in the chromosomal DNA or on plasmids. A novel optrA-bearing genetic element was identified on two distinct multi-resistance plasmids from E. faecium. Two new hybrid plasmids carrying several resistance genes were found in two E. faecalis isolates. pC25-1-like plasmids and chromosomally integrated Tn6674 and Tn6823-like transposons were prevalent in the remaining Enterococcus isolates. CONCLUSIONS: The gene optrA was found in genetically unrelated E. faecium and E. faecalis isolates from the same farm. Analysis of the genetic contexts of optrA suggested that horizontal transfer including different plasmids and transposons played a key role in the dissemination of optrA in this farm.
Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Animais , Suínos , Enterococcus faecalis , Antibacterianos/farmacologia , Fazendas , Genes Bacterianos , Farmacorresistência Bacteriana/genética , Enterococcus , Sequências Repetitivas Dispersas , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/veterinária , Testes de Sensibilidade MicrobianaRESUMO
The tumor suppressor p53 as an innate antiviral regulator contributes to restricting Japanese encephalitis virus (JEV) replication, but the mechanism is still unclear. The interferon-induced transmembrane protein 3 (IFITM3) is an intrinsic barrier to a range of virus infection, whether IFITM3 is responsible for the p53-mediated anti-JEV response remains elusive. Here, we found that IFITM3 significantly inhibited JEV replication in a protein-palmitoylation-dependent manner and incorporated into JEV virions to diminish the infectivity of progeny viruses. Palmitoylation was also indispensible for keeping IFITM3 from lysosomal degradation to maintain its protein stability. p53 up-regulated IFITM3 expression at the protein level via enhancing IFITM3 palmitoylation. Screening of palmitoyltransferases revealed that zinc finger DHHC domain-containing protein 1 (ZDHHC1) was transcriptionally up-regulated by p53, and consequently ZDHHC1 interacted with IFITM3 to promote its palmitoylation and stability. Knockdown of IFITM3 significantly impaired the inhibitory role of ZDHHC1 on JEV replication. Meanwhile, knockdown of either ZDHHC1 or IFITM3 expression also compromised the p53-mediated anti-JEV effect. Interestingly, JEV reduced p53 expression to impair ZDHHC1 mediated IFITM3 palmitoylation for viral evasion. Our data suggest the existence of a previously unrecognized p53-ZDHHC1-IFITM3 regulatory pathway with an essential role in restricting JEV infection and provide a novel insight into JEV-host interaction.
Assuntos
Aciltransferases/metabolismo , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Replicação Viral/fisiologia , Células A549 , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/metabolismo , Encefalite Japonesa/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Interferons/metabolismo , Lipoilação , Células VeroRESUMO
Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and ß production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and ß induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and ß production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and ß induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and ß production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and ß production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Mutação , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Animais , Antivirais/farmacologia , Patos , Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/tratamento farmacológico , Encefalite Japonesa/virologia , Interações Hospedeiro-Patógeno , Camundongos , Ligação Proteica , Suínos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The lungs possess an effective antimicrobial system and a strong ability to eliminate microorganisms in healthy organisms, and were once considered sterile. With the development of culture-independent sequencing technology, the richness and diversity of porcine lung microbiota have been gaining attention. In order to study the relationship between lung microbiota and porcine respiratory disease complex (PRDC), the lung microbiota in healthy and diseased swine bronchoalveolar lavage fluids were analyzed and compared using the Illumina MiSeq sequencing platform. The predominant microbial communities of healthy and diseased swine were similar at the phylum level, mainly composed of Proteobacteria, Firmicutes, Tenericutes, and Bacteroidetes. However, the bacterial taxonomic communities of healthy and diseased swine differed at the genus level. The higher relative abundances of Lactococcus, Enterococcus, Staphylococcus, and Lactobacillus genera in healthy swine might provide more benefits for lung health, while the enhanced richness of Streptococcus, Haemophilus, Pasteurella, and Bordetella genera in diseased swine might be closely related to pathogen invasion and the occurrence of respiratory disease. In conclusion, the observed differences in the richness and diversity of lung microbiota can provide novel insights into their relationship with PRDC. Analyses of swine lung microbiota communities might produce an effective strategy for the control and prevention of respiratory tract infections.
Assuntos
DNA Bacteriano/genética , Pulmão/microbiologia , Microbiota/genética , Infecções Respiratórias/microbiologia , Suínos/microbiologia , Animais , Bordetella/classificação , Bordetella/genética , Bordetella/isolamento & purificação , Bordetella/patogenicidade , Líquido da Lavagem Broncoalveolar/microbiologia , Enterococcus/classificação , Enterococcus/genética , Enterococcus/isolamento & purificação , Haemophilus/classificação , Haemophilus/genética , Haemophilus/isolamento & purificação , Haemophilus/patogenicidade , Sequenciamento de Nucleotídeos em Larga Escala , Lactobacillus/classificação , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Lactococcus/classificação , Lactococcus/genética , Lactococcus/isolamento & purificação , Pasteurella/classificação , Pasteurella/genética , Pasteurella/isolamento & purificação , Pasteurella/patogenicidade , Filogenia , RNA Ribossômico 16S/genética , Staphylococcus/classificação , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Streptococcus/classificação , Streptococcus/genética , Streptococcus/isolamento & purificação , Streptococcus/patogenicidadeRESUMO
Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death, and disability. Although the Culex mosquito is the primary vector of JEV, little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via the defensin/lipoprotein receptor-related protein 2 (LRP2) axis. Mosquito defensin bound the ED III domain of the viral envelope (E) protein and directly mediated efficient virus adsorption on the target cell surface; the receptor LRP2, which is expressed on the cell surface, affected defensin-dependent adsorption. As a result, mosquito defensin enhanced JEV infection in the salivary gland, increasing the possibility of viral transmission by mosquitoes. These findings demonstrate the novel role of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission.IMPORTANCE In this study, we observed the complex roles of mosquito defensin in JEV infection; mosquito defensin exhibited a weak antiviral effect but strongly enhanced binding. In the latter, defensin directly binds the ED III domain of the viral E protein and promotes the adsorption of JEV to target cells by interacting with lipoprotein receptor-related protein 2 (LRP2), thus accelerating virus entry. Together, our results indicate that mosquito defensin plays an important role in facilitating JEV infection and potential transmission.
Assuntos
Culex/genética , Defensinas/genética , Vírus da Encefalite Japonesa (Espécie)/genética , Proteínas de Insetos/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mosquitos Vetores/genética , Proteínas do Envelope Viral/genética , Adsorção , Animais , Culex/virologia , Defensinas/metabolismo , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/transmissão , Encefalite Japonesa/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas de Insetos/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mosquitos Vetores/virologia , Ligação Proteica , Glândulas Salivares/metabolismo , Glândulas Salivares/virologia , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV, species Betaarterivirus suid 1 or 2) is a major pathogen affecting pigs on farms throughout the world. miR-296-3p is a multifunctional microRNA involved in the regulation of the inflammatory response in mice and humans. However, little is known about the biological functions of miR-296-3p in pigs. In this study, we used a highly pathogenic PRRSV-2 (species Betaarterivirus suid 2) strain to show that PRRSV infection robustly downregulates the expression of miR-296-3p in porcine alveolar macrophages (PAMs). Furthermore, we demonstrated that overexpression of miR-296-3p increases the replication of highly pathogenic (HP)-PRRSV in PAMs. Notably, the overexpression of miR-296-3p inhibited the induction of TNF-α, even with increased viral replication, compared with that in the HP-PRRSV-infected control group. We also demonstrated that miR-296-3p targets IRF1-facilitated viral infection and modulates the expression of TNF-α in PAMs during HP-PRRSV infection and that IRF1 regulates the expression of TNF-α by activating the TNF promoter via IRF1 response elements. In summary, these findings show that HP-PRRSV infection activates the IRF1/TNF-α signaling axis in PAMs by downregulating host miR-296-3p. This extends our understanding of the inflammatory response induced by HP-PRRSV infection.
Assuntos
Regulação para Baixo/genética , Fator Regulador 1 de Interferon/genética , Macrófagos Alveolares/virologia , MicroRNAs/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Suínos/virologia , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Perfilação da Expressão Gênica/métodos , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Síndrome Respiratória e Reprodutiva Suína/virologia , Transdução de Sinais/genética , Suínos/genética , Transcriptoma/genética , Replicação Viral/genéticaRESUMO
The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.
Assuntos
Vírus da Febre Suína Africana/genética , Febre Suína Africana/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Suínos/sangue , Febre Suína Africana/sangue , Febre Suína Africana/virologia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Proteínas Associadas a CRISPR/biossíntese , Proteínas Associadas a CRISPR/isolamento & purificação , Sistemas CRISPR-Cas , China , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Viral/genética , Desoxirribonuclease I/genética , Endodesoxirribonucleases/biossíntese , Endodesoxirribonucleases/isolamento & purificação , Fluorescência , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real , Recombinases/metabolismo , Sensibilidade e EspecificidadeRESUMO
Neuroinflammation has been acknowledged as a primary factor contributing to the pathogenesis of neurodegenerative disease. However, the molecular mechanism underlying inflammation stress-mediated neuronal dysfunction is not fully understood. The aim of our study was to explore the influence of mammalian STE20-like kinase 1 (Mst1) in neuroinflammation using TNFα and CATH.a cells in vitro. The results of our study demonstrated that the expression of Mst1 was dose-dependently increased after TNFα treatment. Interestingly, knockdown of Mst1 using siRNA transfection significantly repressed TNFα-induced neuronal death. We also found that TNFα treatment was associated with mitochondrial stress, including mitochondrial ROS overloading, mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential reduction, and mitochondrial pro-apoptotic factor release. Interestingly, loss of Mst1 attenuated TNFα-triggered mitochondrial stress and sustained mitochondrial function in CATH.a cells. We found that Mst1 modulated mitochondrial homeostasis and cell viability via the JNK pathway in a TNFα-induced inflammatory environment. Inhibition of the JNK pathway abolished TNFα-mediated CATH.a cell death and mitochondrial malfunction, similar to the results obtained via silencing of Mst1. Taken together, our results indicate that inflammation-mediated neuronal dysfunction is implicated in Mst1 upregulation, which promotes mitochondrial stress and neuronal death by activating the JNK pathway. Accordingly, our study identifies the Mst1-JNK-mitochondria axis as a novel signaling pathway involved in neuroinflammation.
Assuntos
Inflamação/fisiopatologia , Sistema de Sinalização das MAP Quinases/genética , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antracenos/farmacologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Inflamação/induzido quimicamente , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfaRESUMO
Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.
Assuntos
Aedes/química , Aedes/virologia , Culex/química , Culex/virologia , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Animais , Linhagem Celular , Encefalite Japonesa/fisiopatologia , Encefalite Japonesa/transmissão , Encefalite Japonesa/virologia , Interações Hospedeiro-Patógeno , Lectinas Tipo C/química , RNA de Cadeia Dupla/farmacologia , Proteínas do Envelope Viral/metabolismo , Carga Viral , Internalização do VírusRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the ß2-microglobulin (ß2M) gene (B2M) and reduced cellular levels of ß2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of ß2M expression. Exogenous expression of Nsp4 downregulated ß2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4.
Assuntos
Antígenos de Histocompatibilidade Classe II/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Microglobulina beta-2/genética , Animais , Linhagem Celular , Regulação para Baixo , Expressão Gênica , Inativação Gênica/imunologia , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II/metabolismo , Interações Hospedeiro-Patógeno , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Sus scrofa , Suínos , Proteínas não Estruturais Virais , Microglobulina beta-2/metabolismoRESUMO
Japanese encephalitis (JE) is a viral encephalitis disease caused by infection with the Japanese encephalitis virus (JEV). The virus can cross the blood-brain barrier and cause death or long-term sequela in infected humans or animals. In this study, we first investigated the distribution of JEV infection in brain and further analyzed the dynamic change in inflammation related genes, chemokines, as well as pathological characteristics. Results demonstrated that CCR2 and CCR5 antagonist could significantly inhibit the inflammation. The mice treated with CCR2 and CCR5 antagonists had a higher survival rate between 60% and 70%, respectively. In summary, our study thoroughly illustrated the characteristics of the dynamic change in inflammation related genes and chemokines induced by JEV infection. We further indicated that CCR5 and CCR2 are potential targets for treatment of JE.
Assuntos
Antagonistas dos Receptores CCR5/farmacologia , Quimiocinas/metabolismo , Encefalite Japonesa/tratamento farmacológico , Encefalite Japonesa/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Receptores de Quimiocinas/antagonistas & inibidores , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Chlorocebus aethiops , Citocinas/metabolismo , Modelos Animais de Doenças , Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR2/antagonistas & inibidores , Receptores CCR5 , Células VeroRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is characterized by reproductive failure and respiratory disorders. The secretome of PRRSV-infected porcine alveolar macrophages (PAMs), which are the primary target cells of PRRSV, was analyzed by label-free quantitative proteomics to gain a profile of proteins secreted during PRRSV infection. A total of 95 secreted proteins with differentially expressed levels between PRRSV- and mock-infected PAMs was screened. Among these, the expression levels of 49 and 46 proteins were up-regulated and down-regulated, respectively, in PRRSV-infected cell supernatants, as compared with mock-infected cell supernatants. Bioinformatic analysis revealed that the differentially expressed proteins were enriched in several signaling pathways related to the immune and inflammatory responses, such as the Toll-like receptor signaling pathway and NF-kappa B signaling pathway, and involved in a great diversity of biological processes, such as protein binding and localization, as well as immune effector processes. In addition, PRRSV-infected cell supernatants induced significant expression of inflammatory cytokines in vascular endothelial cells. These findings suggest that the secreted proteins play potential roles in the host immune and inflammatory responses as well as PRRSV replication, thereby providing new insights into cell-to-cell communication during PRRSV infection.
Assuntos
Macrófagos Alveolares/metabolismo , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteoma/análise , Proteômica/métodos , Animais , Células Cultivadas , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Síndrome Respiratória e Reprodutiva Suína/virologia , Transdução de Sinais , SuínosRESUMO
This study was conducted to determine the prevalence of antimicrobial resistance in Campylobacter spp. isolates from broilers in live bird markets (LBMs). A total of 209 Campylobacter spp. isolates (84 Campylobacter jejuni; 125 Campylobacter coli) were recovered from 364 broiler cecum samples collected from five LBMs in Shanghai, China. Minimum inhibitory concentrations of 13 antimicrobials were determined using agar dilution method. More than 96% of the Campylobacter spp. isolates were resistant to quinolones and tetracyclines. A high prevalence of macrolide resistance (erythromycin, 84.0%; azithromycin, 80.8%) was observed in C. coli, but not in C. jejuni (erythromycin, 6.0%; azithromycin, 2.4%). C. coli also showed significantly higher resistance than C. jejuni to clindamycin, gentamicin, and kanamycin. In contrast, C. coli isolates had lower resistance to florfenicol than the C. jejuni isolates. The majority of the C. jejuni (88.1%) and C. coli (97.6%) isolates exhibited multidrug resistance (MDR) to three or more classes of antimicrobials. All of the 208 ciprofloxacin-resistant Campylobacter spp. isolates were positive for the C257T mutation of the gyrA gene. In addition, the tet(O) gene was identified in all of the 202 doxycycline-resistant Campylobacter spp. isolates. Furthermore, 75.7% and 20.4% of the 103 azithromycin-resistant Campylobacter spp. isolates were positive for the A2075G mutation of the 23S rRNA gene and the presence of the erm(B) gene, respectively. Moreover, the cat gene was found in 14.3% (8/56) and 76.8% (73/95) of the chloramphenicol-resistant C. jejuni and C. coli isolates, respectively. To the best of our knowledge, this is the first report of the prevalence of antimicrobial resistance among Campylobacter spp. isolates originating from LBMs. The high prevalence of MDR Campylobacter spp. isolates in LBMs highlights the need to implement efficient intervention measures to control not only Campylobacter contamination in LBMs but also dissemination of antimicrobial resistance among Campylobacter spp. in poultry production.
Assuntos
Proteínas de Bactérias/genética , Campylobacter coli/efeitos dos fármacos , Campylobacter jejuni/efeitos dos fármacos , Galinhas/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Animais , Antibacterianos/farmacologia , Campylobacter coli/genética , Campylobacter coli/isolamento & purificação , Campylobacter jejuni/genética , Campylobacter jejuni/isolamento & purificação , Proteínas de Transporte/genética , China , Ciprofloxacina/farmacologia , Clindamicina/farmacologia , DNA Girase/genética , Eritromicina/farmacologia , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Microbiologia de Alimentos , Gentamicinas/farmacologia , Canamicina/farmacologia , Metiltransferases/genética , Testes de Sensibilidade Microbiana , Aves Domésticas/microbiologia , Quinolonas/farmacologia , RNA Ribossômico 23S/isolamento & purificação , Tetraciclinas/farmacologiaRESUMO
Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2'-5'-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.
Assuntos
Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Interferon Tipo I/imunologia , Proteína Supressora de Tumor p53/deficiência , Animais , Antivirais/farmacologia , Linhagem Celular , Embrião de Galinha , Cães , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes p53 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A/fisiologia , Interferon Tipo I/genética , Interferon alfa-2 , Interferon-alfa/farmacologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Replicação ViralRESUMO
BACKGROUND: Japanese encephalitis virus (JEV) has a significant impact on public health. An estimated three billion people in 'at-risk' regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing. Consequently, there is an urgent need for the development of novel therapeutic agents against Japanese encephalitis. Nitazoxanide (NTZ) is a thiazolide anti-infective licensed for the treatment of parasitic gastroenteritis. Recently, NTZ has been demonstrated to have antiviral properties. In this study, the anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model. METHODS: JEV-infected cells were treated with NTZ at different concentrations. The replication of JEV in the mock- and NTZ-treated cells was examined by virus titration. NTZ was administered at different time points of JEV infection to determine the stage at which NTZ affected JEV replication. Mice were infected with a lethal dose of JEV and intragastrically administered with NTZ from 1 day post-infection. The protective effect of NTZ on the JEV-infected mice was evaluated. FINDINGS: NTZ significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 ± 0.04 µg/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 ± 0.31 µg/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated by daily intragastric administration of 100 mg/kg/day of NTZ were protected from a lethal challenge dose of JEV. CONCLUSIONS: Both in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of Japanese encephalitis.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Encefalite Japonesa/tratamento farmacológico , Encefalite Japonesa/virologia , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Animais , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nitrocompostos , Análise de Sobrevida , Resultado do Tratamento , Carga Viral , Replicação Viral/efeitos dos fármacosRESUMO
OBJECTIVE: To elucidate the extent of food contamination by enterohemorrhagic Escherichia coli (EHEC) O157 in Eastern China. METHODS: A total of 1100 food and animal fecal samples were screened for EHEC O157. Then, molecular characterization of each isolate was determined. RESULTS: EHEC O157 was isolated as follows: pig feces, 4% (20/500); cattle feces, 3.3% (2/60); chicken feces, 1.43% (2/140); pork, 2.14% (3/140), milk, 1.67% (1/60); and chicken meat, 1.67% (1/60). The stx1, stx2, eae, and hlyA genes were present in 26.7% (8/30), 40% (12/30), 63.3% (19/30), and 50% (15/30) of the O157 isolates, respectively. Molecular typing showed that strains from fecal and food samples were clustered into the same molecular typing group. Furthermore, the isolates from pork and pig feces possessed the same characterization as the clinical strains ATCC35150 and ATCC43889. Biofilm formation assays showed that 53.3% of the EHEC O157 isolates could produce biofilm. However, composite analyses showed that biofilm formation of EHEC O157 was independent of genetic background. CONCLUSIONS: Animal feces, especially from pigs, serve as reservoirs for food contamination by EHEC O157. Thus, it is important to control contamination by EHEC O157 on farms and in abattoirs to reduce the incidence of foodborne infections in humans.
Assuntos
Escherichia coli O157/isolamento & purificação , Fezes/microbiologia , Microbiologia de Alimentos , Animais , Biofilmes , Bovinos , Galinhas , Escherichia coli O157/fisiologia , Carne/microbiologia , SuínosRESUMO
Japanese encephalitis virus (JEV) causes acute encephalitis in humans and is of major public health concern in most Asian regions. Dogs are suitable sentinels for assessing the risk of JEV infection in humans. A neutralization test (NT) or an enzyme-linked immunosorbent assay (ELISA) is used for the serological detection of JEV in dogs; however, these tests have several limitations, and, thus, a more convenient and reliable alternative test is needed. In this study, a colloidal gold immunochromatographic strip (ICS), using a purified recombinant EDIII protein, was established for the serological survey of JEV infection in dogs. The results show that the ICSs could specifically detect JEV antibodies within 10 min without cross-reactions with antibodies against other canine viruses. The test strips could detect anti-JEV in serum with dilution up to 640 times, showing high sensitivity. The coincidence rate with the NT test was higher than 96.6%. Among 586 serum samples from dogs in Shanghai examined using the ICS test, 179 (29.98%) were found to be positive for JEV antibodies, and the high seropositivity of JEV in dogs in China was significantly correlated with the season and living environment. In summary, we developed an accurate and economical ICS for the rapid detection of anti-JEV in dog serum samples with great potential for the surveillance of JEV in dogs.