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Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release.IMPORTANCEResearch on the biology and pathogenicity of certain caliciviruses, such as Norovirus and Sapovirus, is hindered by the lack of easy-to-use cell culture system. Feline calicivirus (FCV), which grows effectively in cell lines, is used as a substitute. At present, there is limited understanding of the genome release mechanism in caliciviruses. Our findings suggest that FCV uses VP2 to pierce the endosome membrane for genome release and provide new insights into the calicivirus gRNA release mechanism.
Assuntos
Calicivirus Felino , Proteínas do Capsídeo , Endossomos , RNA Viral , Animais , Gatos , Infecções por Caliciviridae/virologia , Infecções por Caliciviridae/metabolismo , Calicivirus Felino/genética , Calicivirus Felino/metabolismo , Calicivirus Felino/fisiologia , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Endossomos/virologia , Endossomos/metabolismo , Genoma Viral , Lipossomos/metabolismo , RNA Viral/metabolismo , RNA Viral/genética , Liberação de VírusRESUMO
Mammalian reovirus (reovirus) is a multilayered, turreted member of Reoviridae characterized by transcription of dsRNA genome within the innermost capsid shell. Here, we present high-resolution in situ structures of reovirus transcriptase complex in an intact double-layered virion, and in the uncoated single-layered core particles in the unloaded, reloaded, pre-elongation, and elongation states, respectively, obtained by cryo-electron microscopy and sub-particle reconstructions. At the template entry of RNA-dependent RNA polymerase (RdRp), the RNA-loading region gets flexible after uncoating resulting in the unloading of terminal genomic RNA and inactivity of transcription. However, upon adding transcriptional substrates, the RNA-loading region is recovered leading the RNAs loaded again. The priming loop in RdRp was found to play a critical role in regulating transcription, which hinders the elongation of transcript in virion and triggers the rearrangement of RdRp C-terminal domain (CTD) during elongation, resulting in splitting of template-transcript hybrid and opening of transcript exit. With the integration of these structures, a transcriptional model of reovirus with five states is proposed. Our structures illuminate the RdRp activation and regulation of the multilayered turreted reovirus.
Assuntos
RNA Viral , Reoviridae , Animais , Microscopia Crioeletrônica , RNA Viral/genética , Reoviridae/genética , RNA Polimerase Dependente de RNA/genética , Capsídeo , Mamíferos/genéticaRESUMO
Activation of small molecules is considered to be a central concern in the theoretical investigation of environment- and energy-related catalytic conversions. Sub-nanostructured frustrated Lewis pairs (FLPs) have been an emerging research hotspot in recent years due to their advantages in small molecule activation. Although the progress of catalytic applications of FLPs is increasingly reported, the fundamental theories related to the structural formation, site regulation, and catalytic mechanism of FLPs have not yet been fully developed. Given this, it is attempted to demonstrate the underlying theory of FLPs formation, corresponding regulation methods, and its activation mechanism on small molecules using CeO2 as the representative metal oxide. Specifically, this paper presents three fundamental principles for constructing FLPs on CeO2 surfaces, and feasible engineering methods for the regulation of FLPs sites are presented. Furthermore, cases where typical small molecules (e.g., hydrogen, carbon dioxide, methane oxygen, etc.) are activated over FLPs are analyzed. Meanwhile, corresponding future challenges for the development of FLPs-centered theory are presented. The insights presented in this paper may contribute to the theories of FLPs, which can potentially provide inspiration for the development of broader environment- and energy-related catalysis involving small molecule activation.
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Pregnancy heightens susceptibility to influenza A virus (IAV) infection, thereby increasing the risk of severe pneumonia and maternal mortality. It also raises the chances of adverse outcomes in offspring, such as fetal growth restriction, preterm birth, miscarriage, and stillbirth in offsprings. However, the underlying mechanisms behind these effects remain largely unknown. Syncytiotrophoblast cells, crucial in forming the placental barrier, nutrient exchange and hormone secretion, have not been extensively studied for their responses to IAV. In our experiment, we used Forskolin-treated BeWo cells to mimic syncytiotrophoblast cells in vitro, and infected them with H1N1, H5N1 and H7N9 virus stains. Our results showed that syncytiotrophoblast cells, with their higher intensity of sialic acid receptors, strongly support IAV infection and replication. Notably, high-dose viral infection and prolonged exposure resulted in a significant decrease in fusion index, as well as gene and protein expression levels associated with trophoblast differentiation, ß-human chorionic gonadotropin secretion, estrogen and progesterone biosynthesis, and nutrient transport. In pregnant BALB/c mice infected with the H1N1 virus, we observed significant decreases in trophoblast differentiation and hormone secretion gene expression levels. IAV infection also resulted in preterm labor, fetal growth restriction, and increased maternal and fetal morbidity and mortality. Our findings indicate that IAV infection in syncytiotrophoblastic cells can result in adverse pregnancy outcomes by altering trophoblast differentiation, suppressing of ß-hCG secretion, and disrupting placental barrier function.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae , Resultado da Gravidez , Trofoblastos , Feminino , Trofoblastos/virologia , Gravidez , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Camundongos , Infecções por Orthomyxoviridae/virologia , Influenza Humana/virologia , Linhagem Celular , Virus da Influenza A Subtipo H5N1/fisiologia , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Complicações Infecciosas na Gravidez/virologia , Placenta/virologia , Replicação ViralRESUMO
The continuous antigenic variation of influenza A viruses remains a major hurdle for vaccine selection; however, the molecular determinants and mechanisms of antigenic change remain largely unknown. In this study, two escape mutants were generated by serial passages of the Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV) A/swine/Henan/11/2005 (HeN11) in the presence of two neutralizing monoclonal antibodies (mAbs) against the hemagglutinin (HA) protein, which were designated HeN11-2B6-P5 and HeN11-4C7-P8, respectively. The HeN11-2B6-P5 mutant simultaneously harbored the N190D and I230M substitutions in HA, whereas HeN11-4C7-P8 harbored the M269R substitution in HA (H3 numbering). The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the representative viruses. The results indicate that residues 190 and 269 are key determinants of viral antigenic variation. In particular, the N190D mutation had the greatest antigenic impact, as determined by the HI assay. Further studies showed that both HeN11-2B6-P5 and HeN11-4C7-P8 maintained the receptor-binding specificity of the parent virus, although the single mutation N190D decreased the binding affinity for the human-type receptor. The replicative ability in vitro of HeN11-2B6-P5 was increased, whereas that of HeN11-4C7-P8 was decreased. These findings extend our understanding of the antigenic evolution of influenza viruses under immune pressure and provide insights into the functional effects of amino acid substitutions near the receptor-binding site and the interplay among receptor binding, viral replication, and antigenic drift. IMPORTANCE The antigenic changes that occur continually in the evolution of influenza A viruses remain a great challenge for the effective control of disease outbreaks. Here, we identified three amino acid substitutions (at positions 190, 230, and 269) in the HA of EA H1N1 SIVs that determine viral antigenicity and result in escape from neutralizing monoclonal antibodies. All three of these substitutions have emerged in nature. Of note, residues 190 and 230 have synergistic effects on receptor binding and antigenicity. Our findings provide a better understanding of the functional effects of amino acid substitutions in HA and their consequences for the antigenic drift of influenza viruses.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Evasão da Resposta Imune , Vírus da Influenza A Subtipo H1N1 , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais , Deriva e Deslocamento Antigênicos , Antígenos Virais/genética , Hemaglutininas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Mutação , SuínosRESUMO
African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Vírus da Febre Suína Africana/genética , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Epitopos/genética , Camundongos , SuínosRESUMO
Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively.
Assuntos
Proteínas Munc18/fisiologia , Organelas/metabolismo , Proteínas de Protozoários/fisiologia , Toxoplasma/metabolismo , Fibroblastos , Células HEK293 , HumanosRESUMO
Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.
Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Circoviridae/virologia , Circovirus/metabolismo , Circovirus/ultraestrutura , Microscopia Crioeletrônica , Epitopos , Genótipo , Conformação Proteica , Suínos , Doenças dos Suínos/virologiaRESUMO
Efficient human-to-human transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission are still not fully understood. In this study, we compared the respiratory droplet transmissibilities of four H7N9 viruses that are genetic closely related and found that these viruses have dissimilar transmissibilities in guinea pigs: A/Anhui/1/2013 (AH/1) transmitted efficiently, whereas the other three viruses did not transmit. The three nontransmissible viruses have one to eight amino acid differences compared with the AH/1 virus. To investigate which of these amino acids is important for transmission, we used reverse genetics to generate a series of reassortants and mutants in the AH/1 background and tested their transmissibility in guinea pigs. We found that the neuraminidase (NA) of the nontransmissible virus A/chicken/Shanghai/S1053/2013 had low enzymatic activity that impaired the transmission of AH/1 virus, and three amino acid mutations-V292I and K627E in PB2 and D156E in M1-independently abolished the transmission of the AH/1 virus. We further found that an NA reassortant and three single-amino-acid mutants replicated less efficiently than the AH/1 virus in A549 cells and that the amino acid at position 156 of M1 affected the morphology of H7N9 viruses. Our study identifies key amino acids in PB2 and M1 that play important roles in H7N9 inï¬uenza virus transmission and provides new insights into the transmissibility of inï¬uenza virus.IMPORTANCE Efficient transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission remain poorly understood. H7N9 influenza viruses, which emerged in 2013 in China, have caused over 1,560 human infection cases, showing clear pandemic potential. Previous studies have shown that the H7N9 viruses differ in their transmissibility in animal models. In this study, we found two amino acids in PB2 (292V and 627K) and one in M1 (156D) that are extremely important for H7N9 virus transmission. Of note, PB2 292V and M1 156D appear in most H7N9 viruses, and the PB2 627K mutation could easily occur when the H7N9 virus replicates in humans. Our study thus identifies new amino acids that are important for influenza virus transmission and suggests that just a few key amino acid changes can render the H7N9 virus transmissible in mammals.
Assuntos
Subtipo H7N9 do Vírus da Influenza A/genética , Neuraminidase/genética , Infecções por Orthomyxoviridae/transmissão , RNA Polimerase Dependente de RNA/genética , Vírus Reordenados/genética , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Células A549 , Substituição de Aminoácidos , Animais , Expressão Gênica , Cobaias , Humanos , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Mutação , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , RNA Polimerase Dependente de RNA/metabolismo , Vírus Reordenados/metabolismo , Vírus Reordenados/patogenicidade , Genética Reversa , Relação Estrutura-Atividade , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Equine influenza virus (EIV) causes severe acute respiratory disease in horses. Currently, the strains belonging to the H3N8 subtype are divided into two clades, Florida clade 1 (FC1) and Florida clade 2 (FC2), which emerged in 2002. Both FC1 and FC2 clades were reported in Asian and Middle East countries in the last decade. In this study, we described the evolution, epidemiology, and molecular characteristic of the EIV lineages, with focus on those detected in Asia from 2007 to 2017. The full genome phylogeny showed that FC1 and FC2 constituted separate and divergent lineages, without evidence of reassortment between the clades. While FC1 evolved as a single lineage, FC2 showed a divergent event around 2004 giving rise to two well-supported and coexisting sublineages, European and Asian. Furthermore, two different spread patterns of EIV in Asian countries were identified. The FC1 outbreaks were caused by independent introductions of EIV from the Americas, with the Asian isolates genetically similar to the contemporary American lineages. On the other hand, the FC2 strains detected in Asian mainland countries conformed to an autochthonous monophyletic group with a common ancestor dated in 2006 and showed evidence of an endemic circulation in a local host. Characteristic aminoacidic signature patterns were detected in all viral proteins in both Asian-FC1 and FC2 populations. Several changes were located at the top of the HA1 protein, inside or near antigenic sites. Further studies are needed to assess the potential impact of these antigenic changes in vaccination programs.IMPORTANCE The complex and continuous antigenic evolution of equine influenza viruses (EIVs) remains a major hurdle for vaccine development and the design of effective immunization programs. The present study provides a comprehensive analysis showing the EIV evolutionary dynamics, including the spread and circulation within the Asian continent and its relationship to global EIV populations over a 10-year period. Moreover, we provide a better understanding of EIV molecular evolution in Asian countries and its consequences on the antigenicity. The study underscores the association between the global horse movement and the circulation of EIV in this region. Understanding EIV evolution is imperative in order to mitigate the risk of outbreaks affecting the horse industry and to help with the selection of the viral strains to be included in the formulation of future vaccines.
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Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Vírus da Influenza A Subtipo H3N8/classificação , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Filogenia , Animais , Ásia , Surtos de Doenças , Evolução Molecular , Cavalos , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H7N7/classificação , Filogeografia , Proteínas Virais/genéticaRESUMO
Autophagy functions as an intrinsic antiviral defense. However, some viruses can subvert or even enhance host autophagic machinery to increase viral replication and pathogenesis. The role of autophagy during avibirnavirus infection, especially late stage infection, remains unclear. In this study, infectious bursal disease virus (IBDV) was used to investigate the role of autophagy in avibirnavirus replication. We demonstrated IBDV induction of autophagy as a significant increase in puncta of LC3+ autophagosomes, endogenous levels of LC3-II, and ultrastructural characteristics typical of autophagosomes during the late stage of infection. Induction of autophagy enhances IBDV replication, whereas inhibition of autophagy impairs viral replication. We also demonstrated that IBDV infection induced autophagosome-lysosome fusion, but without active degradation of their contents. Moreover, inhibition of fusion or of lysosomal hydrolysis activity significantly reduced viral replication, indicating that virions utilized the low-pH environment of acidic organelles to facilitate viral maturation. Using immuno-transmission electron microscopy (TEM), we observed that a large number of intact IBDV virions were arranged in a lattice surrounded by p62 proteins, some of which lay between virions. Additionally, many virions were encapsulated within the vesicular membranes, with an obvious release stage observed by TEM. The autophagic endosomal pathway facilitates low-pH-mediated maturation of viral proteins and membrane-mediated release of progeny virions.IMPORTANCE IBDV is the most extensively studied virus in terms of molecular characteristics and pathogenesis; however, mechanisms underlying the IBDV life cycle require further exploration. The present study demonstrated that autophagy enhances viral replication at the late stage of infection, and the autophagy pathway facilitates IBDV replication complex function and virus assembly, which is critical to completion of the virus life cycle. Moreover, the virus hijacks the autophagic vacuoles to mature in an acidic environment and release progeny virions in a membrane-mediated cell-to-cell manner. This autophagic endosomal pathway is proposed as a new mechanism that facilitates IBDV maturation, release, and reinternalization. This report presents a concordance in exit strategies among some RNA and DNA viruses, which exploit autophagy pathway for their release from cells.
Assuntos
Autofagia , Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/fisiologia , Doenças das Aves Domésticas/virologia , Vacúolos/virologia , Animais , Infecções por Birnaviridae/virologia , Linhagem Celular , Embrião de Galinha , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Inata , Transdução de Sinais , Vacúolos/fisiologia , Internalização do Vírus , Liberação de Vírus , Replicação ViralRESUMO
During March 25-May 5, 2014, we investigated 11 outbreaks of peste des petits ruminants in Heilongjiang Province, China. We found that the most likely source of the outbreaks was animals from livestock markets in Shandong. Peste des petits ruminants viruses belonging to lineages II and IV were detected in sick animals.
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Peste dos Pequenos Ruminantes/epidemiologia , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/classificação , Vírus da Peste dos Pequenos Ruminantes/genética , Animais , China/epidemiologia , Surtos de Doenças , História do Século XXI , Tipagem de Sequências Multilocus , Peste dos Pequenos Ruminantes/história , Filogenia , RNA ViralRESUMO
Reticuloendotheliosis virus (REV), classified as a gammaretrovirus, has a variety of hosts, including chickens, ducks, geese, turkeys, and wild birds. REV causes a series of pathological syndromes, especially the immunosuppression of the host, which may lead to an increased susceptibility to other pathogens, thus greatly damaging the poultry industry. Mixed infections of REV and Marek's disease virus (MDV) have been reported in many countries, including China. Previous reports revealed that MDV vaccines were not efficacious, and even less-virulent MDV strains would cause some losses due to mixed infections with REV. Additionally, contaminants in the MDV vaccine might be the main source of REV. In this study, two clinical samples were collected from two flocks of chickens that were diagnosed with MDV. Subsequently, two REV isolates were obtained from the clinical samples. The isolates, named CY1111 and SY1209, were further confirmed through an indirect immunofluorescence assay and electron microscopy. Complete genome sequences of the two REV strains were determined to test the relationship between them and other REV strains. Phylogenetic trees showed that the two REV strains were closely related to most REV strains that were isolated from a variety of hosts. Therefore, REVs might spread freely among these hosts under natural conditions. Additionally, most REV strains in China were in the same clade. The present work offers some information regarding REV in China.
Assuntos
Coinfecção/veterinária , Coinfecção/virologia , Genoma Viral , Doenças das Aves Domésticas/virologia , Vírus da Reticuloendoteliose/genética , Vírus da Reticuloendoteliose/isolamento & purificação , Infecções por Retroviridae/veterinária , Animais , Galinhas , China , Análise por Conglomerados , Herpesvirus Galináceo 2/isolamento & purificação , Doença de Marek/complicações , Microscopia , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Infecções por Retroviridae/complicações , Infecções por Retroviridae/virologia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
BACKGROUND: Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine. RESULTS: We generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15-20 µg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4-6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. CONCLUSION: These results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.
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Vírus da Encefalite Japonesa (Espécie)/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Linhagem Celular , Cricetinae , Feminino , Vacinas contra Encefalite Japonesa/biossíntese , Vacinas contra Encefalite Japonesa/genética , Vacinas contra Encefalite Japonesa/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
A novel influenza A/H1N1 virus, emerging from Mexico and the United States in the spring of 2009, caused the pandemic human infection of 2009-2010. The haemagglutinin (HA) glycoprotein is the major surface antigen of influenza A virus and plays an important role in viral infection. In this study, three hybridoma cell lines secreting specific monoclonal antibodies (Mabs) against the HA protein of pandemic influenza A/H1N1 2009 virus were generated with the recombinant plasmid pCAGGS-HA as an immunogen. Using Pepscan analysis, the binding sites of these Mabs were identified in a linear region of the HA protein. Further, refined mapping was conducted using truncated peptides expressed as GST-fusion proteins in E. coli. We found that the (250)VPRYA(254) motif was the minimal determinant of the linear epitope that could be recognized by the Mabs. Alignment with sequences from the databases showed that the amino acid residues of this epitope were highly conserved among all pandemic A/H1N1 2009 viruses as well as the classical swine H1N1 viruses isolated to date. These results provide additional insights into the antigenic structure of the HA protein and virus-antibody interactions at the amino acid level, which may assist in the development of specific diagnostic methods for influenza viruses.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Mapeamento de Epitopos , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Foot-and-mouth disease virus (FMDV) loses infectivity and immunogenicity due to its disassembly in culture environments below pH 6.8. To study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of inactivated FMD vaccines during the manufacturing process, type O FMDV mutants with increased resistance to acid inactivation were selected, and the genes encoding their capsid proteins were sequenced. Three amino acid substitutions (VP1 N17D, VP2 D86A, and VP4 S73N) were found in all of the mutants. When these substitutions were introduced into seven infectious FMDV clones alone or combined, a single amino acid substitution in the VP1 protein, N17D, which also appears in type C FMDV acid-resistant mutants, was found to be responsible for the increased resistance to acid inactivation for type O FMDV. In addition, although viral fitness was reduced under standard culture conditions, viral growth kinetics and virulence were not significantly altered in the rescued mutant virus rN17D with the VP1 N17D substitution. Importantly, the N17D substitution could confer improved immunogenicity to the mutant virus rN17D under acidic conditions compared with its parental virus O/YS/CHA/05. These results demonstrate that the N17D substitution in VP1 is the molecular determinant of the acid-resistant phenotype in type O FMDV, indicating the potential for use of this substitution to improve the acid stability of inactivated FMD vaccines during the vaccine production process.
Assuntos
Ácidos/toxicidade , Farmacorresistência Viral , Vírus da Febre Aftosa/efeitos dos fármacos , Vírus da Febre Aftosa/genética , Viabilidade Microbiana/efeitos dos fármacos , Seleção Genética , Substituição de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Cricetinae , Análise Mutacional de DNA , Vírus da Febre Aftosa/isolamento & purificação , Vírus da Febre Aftosa/fisiologia , Concentração de Íons de Hidrogênio , Análise de Sequência de DNA , Virulência , Cultura de Vírus , Replicação ViralRESUMO
This research investigates the recognition of basketball techniques actions through the implementation of three-dimensional (3D) Convolutional Neural Networks (CNNs), aiming to enhance the accurate and automated identification of various actions in basketball games. Initially, basketball action sequences are extracted from publicly available basketball action datasets, followed by data preprocessing, including image sampling, data augmentation, and label processing. Subsequently, a novel action recognition model is proposed, combining 3D convolutions and Long Short-Term Memory (LSTM) networks to model temporal features and capture the spatiotemporal relationships and temporal information of actions. This facilitates the facilitating automatic learning of the spatiotemporal features associated with basketball actions. The model's performance and robustness are further improved through the adoption of optimization algorithms, such as adaptive learning rate adjustment and regularization. The efficacy of the proposed method is verified through experiments conducted on three publicly available basketball action datasets: NTURGB + D, Basketball-Action-Dataset, and B3D Dataset. The results indicate that this approach achieves outstanding performance in basketball technique action recognition tasks across different datasets compared to two common traditional methods. Specifically, when compared to the frame difference-based method, this model exhibits a significant accuracy improvement of 15.1%. When compared to the optical flow-based method, this model demonstrates a substantial accuracy improvement of 12.4%. Moreover, this method showcases strong robustness, accurately recognizing actions under diverse lighting conditions and scenes, achieving an average accuracy of 93.1%. The research demonstrates that the method reported here effectively captures the spatiotemporal relationships of basketball actions, thereby providing reliable technical assessment tools for basketball coaches and players.
Assuntos
Basquetebol , Redes Neurais de Computação , Reconhecimento Automatizado de Padrão , Gravação em Vídeo , Humanos , Basquetebol/classificação , Basquetebol/fisiologia , Conjuntos de Dados como Assunto , Movimento , Reprodutibilidade dos Testes , Reconhecimento Automatizado de Padrão/métodosRESUMO
Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.
Assuntos
Alphavirus , Animais , Humanos , Alphavirus/genética , Alphavirus/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , RNA Viral/genética , RNA Guia de Sistemas CRISPR-Cas , Genômica , Vírion/genéticaRESUMO
Developing inexpensive and efficient catalysts for biomass hydrogenation or hydrodeoxygenation (HDO) is essential for efficient energy conversion. Transition metal phosphides (TMPs), with the merits of abundant active sites, unique physicochemical properties, tunable component structures, and excellent catalytic activities, are recognized as promising biomass hydrogenation or HDO catalytic materials. Nevertheless, the biomass hydrogenation or HDO catalytic applications of TMPs are still limited by various complexities and inherent performance bottlenecks, and thus their future development and utilization remain to be systematically sorted out and further explored. This review summarizes the current popular strategies for the preparation of TMPs. Subsequently, based on the structural and electronic properties of TMPs, the catalytic activity origins of TMPs in biomass hydrogenation or HDO is elucidated. Additionally, the application of TMPs in efficient biomass hydrogenation or HDO catalysis, as well as highly targeted multiscale strategies to enhance the catalytic performance of TMPs, are comprehensively described. Finally, large-scale amplification synthesis, rational construction of TMP-based catalysts and in-depth study of the catalytic mechanism are also mentioned as challenges and future directions in this research field. Expectedly, this review can provide professional and targeted guidance for the rational design and practical application of TMPs biomass hydrogenation or HDO catalysts.
RESUMO
Skin could protect our body and regenerate itself to against dysfunctional and disfiguring scars when faced with external injury. As wound dressings, hydrogels are biocompatible, hydrophilic and have a 3D structure similar to the extracellular matrix (ECM). In particular, hydrogels with drug-releasing capabilities are in acute wound healing. In this paper, photocrosslinked hydrogels served as wound dressing based on sodium carboxymethylcellulose (CMC) were prepared to promote wound healing. Photocrosslinked hydrogels were prepared by grafting lysine and allyl glycidyl ether (AGE) onto CMC and encapsulating curcumin (Cur). The synthesized hydrogels had the unique 3D porous structure with a swelling ratio up to 1300 % in aqueous solution. The drug release ratios of the hydrogels were 20.8 % in acid environment, and 14.4 % in alkaline environment. Notably, the hydrogels showed good biocompatibility and antibacterial properties and also exhibited the ability to accelerate the process of skin wound healing while prevent inflammation and scar formation when applied to a mouse skin wound model. As a result, the prepared hydrogels Gel-CLA@Cur showed great potential in wound healing.