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1.
Arch Virol ; 166(10): 2937-2942, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34347169

RESUMO

The genus Gyrovirus was assigned to the family Anelloviridae in 2017 with only one recognized species, Chicken anemia virus. Over the last decade, many diverse viruses related to chicken anemia virus have been identified but not classified. Here, we provide a framework for the classification of new species in the genus Gyrovirus and communicate the establishment of nine new species. We adopted the 'Genus + freeform epithet' binomial system for the naming of these species.


Assuntos
Gyrovirus/classificação , Terminologia como Assunto , Anelloviridae/classificação , Anelloviridae/genética , Animais , Proteínas do Capsídeo/genética , Vírus da Anemia da Galinha/classificação , Vírus da Anemia da Galinha/genética , DNA Viral/genética , Bases de Dados Genéticas , Genoma Viral/genética , Gyrovirus/genética , Humanos , Filogenia , Análise de Sequência de DNA
2.
Arch Virol ; 166(10): 2927-2935, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34350513

RESUMO

The highly diverse virus family Picornaviridae presently comprises 68 approved genera with 158 species plus many unassigned viruses. In order to better match picornavirus taxonomy to the functional and genomic groupings between genera, the establishment of five subfamilies (Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae and Heptrevirinae) is proposed. The subfamilies are defined by phylogenetic analyses of 3CD (precursor of virus-encoded proteinase and polymerase) and P1 (capsid protein precursor) coding sequences and comprise between 7 and 22 currently approved virus genera. Due to the high within-subfamily and between-subfamily divergences of the picornavirus genera, p-distance estimates are unsuited for the demarcation of subfamilies. Members of the proposed subfamilies typically show some commonalities in their genome organisations, including VP1/2A cleavage mechanisms and possession of leader proteins. Other features, such as internal ribosomal entry site types, are more variable within and between members of genera. Some subfamilies are characterised by homology of proteins 1A, 2A, 2B and 3A encoded by members, which do not belong to the canon of orthologous picornavirus proteins. The proposed addition of a subfamily layer to the taxonomy of picornaviruses provides a valuable additional organisational level to the family that acknowledges the existence of higher-level evolutionary groupings of its component genera.


Assuntos
Genoma Viral/genética , Filogenia , Picornaviridae/classificação , Proteases Virais 3C/genética , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Evolução Molecular , Genômica , Sítios Internos de Entrada Ribossomal/genética , Picornaviridae/genética , Análise de Sequência de DNA
3.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445463

RESUMO

Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.


Assuntos
Antivirais/química , Proteínas do Capsídeo/química , Capsídeo/química , Enterovirus Humano A/química , Simulação de Acoplamento Molecular , Silimarina/química , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Farmacorresistência Viral Múltipla/genética , Enterovirus Humano A/genética , Humanos , Mutação , Estrutura Secundária de Proteína
4.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445426

RESUMO

The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cisteína Proteases/metabolismo , Nodaviridae/metabolismo , Palaemonidae/virologia , Animais , Sítios de Ligação , Proteínas do Capsídeo/química , Simulação por Computador , Desenvolvimento de Medicamentos , Regulação Viral da Expressão Gênica , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Análise de Sequência de Proteína
5.
Viruses ; 13(7)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34372536

RESUMO

Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina-based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry.


Assuntos
Doenças dos Cavalos/virologia , Cavalos/virologia , Rotavirus/patogenicidade , Animais , Proteínas do Capsídeo/genética , Diarreia/etiologia , Diarreia/virologia , Surtos de Doenças/veterinária , Fezes/virologia , Kentucky , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rotavirus/classificação , Infecções por Rotavirus/veterinária
6.
Viruses ; 13(7)2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34372555

RESUMO

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein-RNA condensates that may be formed via liquid-liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.


Assuntos
Rotavirus/genética , Rotavirus/metabolismo , Compartimentos de Replicação Viral/metabolismo , Animais , Proteínas do Capsídeo/genética , Citoplasma/virologia , Citosol/metabolismo , Humanos , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Infecções por Rotavirus/virologia , Proteínas não Estruturais Virais/metabolismo , Compartimentos de Replicação Viral/fisiologia , Montagem de Vírus , Replicação Viral/genética
7.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361120

RESUMO

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Dependovirus/genética , Endopeptidases/imunologia , Epitopos/imunologia , Vetores Genéticos/administração & dosagem , Proteínas de Membrana/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/metabolismo , Transdução Genética
8.
Nat Commun ; 12(1): 4320, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262046

RESUMO

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas , Norovirus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Variação Antigênica , Sítios de Ligação , Antígenos de Grupos Sanguíneos/metabolismo , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/metabolismo , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Mapeamento de Epitopos , Genótipo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Norovirus/genética , Ligação Proteica , Domínios Proteicos
9.
Vaccine ; 39(35): 5015-5024, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34303562

RESUMO

Foot-and-mouth disease (FMD) is a global burden on the livestock industry. The causative agent, FMD virus (FMDV), is highly infectious and exists in seven distinct serotypes. Vaccination remains the most effective control strategy in endemic regions and current FMD vaccines are made from inactivated preparations of whole virus. The inherent instability of FMDV and the emergence of new strains presents challenges to efficacious vaccine development. Currently, vaccines available in East Africa are comprised of relatively historic strains with unreported stabilities. As an initial step to produce an improved multivalent FMD vaccine we have identified naturally stable East African FMDV strains for each of the A, O, SAT1 and SAT2 serotypes and investigated their potential for protecting ruminants against strains that have recently circulated in East Africa. Interestingly, high diversity in stability between and within serotypes was observed, and in comparison to non-African A serotype viruses reported to date, the East African strains tested in this study are less stable. Candidate vaccine strains were adapted to propagation in BHK-21 cells with minimal capsid changes and used to generate vaccinate sera that effectively neutralised a panel of FMDV strains selected to improve FMD vaccines used in East Africa. This work highlights the importance of combining tools to predict and assess FMDV vaccine stability, with cell culture adaptation and serological tests in the development of FMD vaccines.


Assuntos
Vírus da Febre Aftosa , Febre Aftosa , Vacinas Virais , Animais , Proteínas do Capsídeo/genética , Febre Aftosa/prevenção & controle , Sorogrupo
10.
Anal Chem ; 93(30): 10403-10410, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34291903

RESUMO

Adeno-associated viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, post-translational modifications (PTMs), and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized peptide mapping-based workflow that provides thorough characterization within 1 day. This workflow is also highly reproducible due to its simplicity having very few steps and is easy to perform proteolytic digestion utilizing thermally stable pepsin, which is active at 70 °C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low-abundance VP1 capsid protein with a digestion process taking only 10 min to prepare and 45 min to complete the digestion.


Assuntos
Capsídeo , Dependovirus , Proteínas do Capsídeo/genética , Dependovirus/genética , Digestão , Humanos , Mapeamento de Peptídeos
11.
Antimicrob Agents Chemother ; 65(10): e0103921, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34228546

RESUMO

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection and an attractive therapeutic target for antivirals. Here, we report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is 1 of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between reverse transcription and proviral DNA integration, without any effect on RT activity in vitro, suggesting that this compound may affect HIV-1 core disassembly (uncoating) as well as a CA inhibitor, PF74. Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and noncovalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues. These results indicate that ACAi-028 is a new CA inhibitor by binding to the novel hydrophobic pocket in CA-NTD. This study demonstrates that a compound, ACAi-028, targeting the hydrophobic pocket should be a promising anti-HIV-1 inhibitor.


Assuntos
Fármacos Anti-HIV , HIV-1 , Fármacos Anti-HIV/farmacologia , Capsídeo , Proteínas do Capsídeo/genética , Humanos , Fenilalanina/farmacologia , Replicação Viral
12.
Sheng Wu Gong Cheng Xue Bao ; 37(7): 2435-2442, 2021 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-34327908

RESUMO

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.


Assuntos
Vírus da Febre Aftosa , Febre Aftosa , Vacinas de Partículas Semelhantes a Vírus , Vacinas Virais , Aminoácidos , Animais , Proteínas do Capsídeo/genética , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/genética , Cinética , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas Virais/genética
13.
BMC Infect Dis ; 21(1): 682, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34261441

RESUMO

BACKGROUND: Norovirus (NoV) is a major cause of viral acute gastroenteritis (AGE) in children worldwide. Epidemiological analysis with respect to the virus strains is limited in China. This study aimed to investigate the prevalence, patterns, and molecular characteristics of NoV infection among children with AGE in China. METHODS: A total 4848 stool samples were collected from children who were admitted with AGE in Tianjin Children's Hospital from August 2018 to July 2020. NoV was preliminarily detected using real-time reverse transcription polymerase chain reaction (RT-PCR). Partial sequences of the RNA-dependent RNA polymerase (RdRp) and capsid genes of positive samples were amplified by conventional RT-PCR and then sequenced. The NoV genotype was determined by online Norovirus Typing Tool Version 2.0, and phylogenetic analysis was conducted using MEGA 6.0. RESULTS: The prevalence of NoV was 26.4% (1280/4848). NoV was detected in all age groups, with the 7-12 months group having the highest detection rate (655/2014, 32.5%). NoV was detected during most part of the year with higher frequency in winter than other seasons. Based on the genetic analysis of RdRp, GII. Pe was the most predominant genotype detected at 70.7% (381/539) followed by GII.P12 at 25.4% (137/539). GII.4 was the most predominant capsid genotype detected at 65.3% (338/518) followed by GII.3 at 26.8% (139/518). Based on the genetic analysis of RdRp and capsid sequences, the strains were clustered into 10 RdRp-capsid genotypes: GII.Pe-GII.4 Sydney 2012 (65.5%), GII.P12-GII.3 (27.2%), GII.P16-GII.2 (1.8%), GII.P12-GII.2 (0.2%), GII.P17-GII.17 (1.1%), GII.Pe-GII.3 (1.8%), GII.Pe-GII.2 (1.1%), GII.Pe-GII.1 (0.4%), GII.16-GII.4 Sydney 2012 (0.7%), and GII.P7-GII.6 (0.2%). The predominant NoV genotypes changed from GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 between August 2018 and July 2019 to GII.Pe-GII.4 Sydney 2012 and GII.P16-GII.2 between August 2019 and July 2020. The patients with GII.Pe-GII.4 Sydney 2012 genotype were more likely to suffer from vomiting symptom than those with GII.P12-GII.3. CONCLUSIONS: NoV is an important pathogen responsible for viral AGE among children in China. GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 were major recombinant genotypes. Knowledge of circulating genotypes and seasonal trends is of great importance for disease prevention and surveillance.


Assuntos
Infecções por Caliciviridae , Gastroenterite , Norovirus , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/fisiopatologia , Proteínas do Capsídeo/genética , Criança , China/epidemiologia , Feminino , Gastroenterite/diagnóstico , Gastroenterite/epidemiologia , Gastroenterite/virologia , Hospitais Pediátricos/estatística & dados numéricos , Humanos , Lactente , Masculino , Norovirus/classificação , Norovirus/genética , Norovirus/isolamento & purificação , Norovirus/fisiologia , Prevalência , RNA Viral/isolamento & purificação
14.
Viruses ; 13(7)2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202573

RESUMO

Adenovirus vector-based genetic vaccines have emerged as a powerful strategy against the SARS-CoV-2 health crisis. This success is not unexpected because adenoviruses combine many desirable features of a genetic vaccine. They are highly immunogenic and have a low and well characterized pathogenic profile paired with technological approachability. Ongoing efforts to improve adenovirus-vaccine vectors include the use of rare serotypes and non-human adenoviruses. In this review, we focus on the viral capsid and how the choice of genotypes influences the uptake and subsequent subcellular sorting. We describe how understanding capsid properties, such as stability during the entry process, can change the fate of the entering particles and how this translates into differences in immunity outcomes. We discuss in detail how mutating the membrane lytic capsid protein VI affects species C viruses' post-entry sorting and briefly discuss if such approaches could have a wider implication in vaccine and/or vector development.


Assuntos
Adenovírus Humanos/imunologia , Adenovírus Humanos/fisiologia , Capsídeo/metabolismo , Vetores Genéticos , Vacinas Virais/imunologia , Internalização do Vírus , Imunidade Adaptativa , Adenovírus Humanos/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Ensaios Clínicos como Assunto , Humanos , Imunidade Inata , Camundongos , SARS-CoV-2/imunologia
15.
Acta Virol ; 65(2): 192-199, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34130470

RESUMO

We have previously reported that bovine papillomavirus type 1 (BPV1) can replicate its genome and produces infectious virus-like particles in short-term BPV1 virion-infected Sacharomyces cerevisiae (Zhao and Frazer, 2002). Here, we report viral RNA transcription and L1 capsid protein expression in long-term BPV1 virion-infected S. cerevisiae culture. Northern blot hybridization showed that viral RNA was detected in long-term BPV1-infected S. cerevisiae cultures (82-108 days). The levels of the viral RNA transcription varied significantly over the long time period, which showed active transcription at an early stage (Day 3 to Day 16), weak transcription at a middle stage (Day 23 to Day 45) and stable transcription at the late stage of culture (Day 55 to Day 82/85/95). Three major BPV1 transcripts of 4.3, 2.6 and 1.8 Kb were identified, with 4.3 Kb a minor transcript and the 1.8 Kb the most prominent transcript compared with the 2.6 Kb species. Immunoblotting showed that L1 capsid protein was expressed, with its variable amounts corresponding to the levels of RNA transcription over the time period. 35S-methionine/cysteine labeling and immunoprecipitation proved that the detected L1 protein was newly synthesized in BPV1-infected S. cerevisiae cultures. 33.3-54.2% of the cell colonies expressed L1 protein. Thus, the S. cerevisiae system, as a promising model, may be used not only for the study of virus like particle formation of BPV1 in vitro, but also for further functional analysis of individual viral genes in BPV1 life cycle. Keywords: BPV1; viral RNA transcription; expression of L1 capsid protein; virion-infected Saccharomyces cerevisiae.


Assuntos
Papillomavirus Bovino 1 , Papillomavirus Bovino 1/genética , Capsídeo , Proteínas do Capsídeo/genética , Saccharomyces cerevisiae/genética , Vírion
16.
Genes (Basel) ; 12(6)2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071769

RESUMO

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.


Assuntos
Resistência à Doença , Inativação Gênica , Vírus Eruptivo da Ameixa/genética , Prunus/genética , Biotecnologia/métodos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Engenharia Genética/métodos , Vírus Eruptivo da Ameixa/patogenicidade , Prunus/virologia , Transgenes
17.
Methods Mol Biol ; 2323: 171-197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086281

RESUMO

The method development to analyze direct RNA-protein interaction is of high importance as not many homogeneous assay formats are available.The discovery of fluorescent light-up aptamers (FLAPs), short RNA aptamers that switch the fluorescence of small, cell-permeable, and nontoxic organic chromophores on, paved the road for their utilization in direct RNA -protein interactions. The combination with fluorescent proteins as biological fluorophores enabled the development of homogeneous assays that are in principle even encodable on genomic level.Here the rules and methods to design a homogeneous in vitro assay for the detection and quantification of a direct RNA -protein interaction will be described. The design and application of a homogeneous assay to observe and quantify the interaction of the Pseudomonas aeruginosa bacteriophage coat protein 7 (PP7) with its binding RNA sequence (pp7-RNA) will be shown. For this, the Spinach-DFHBI aptamer as RNA fusion and the red fluorescent mCherry as protein fusion was used.The methods presented here do not require any chemical modification of proteins or RNAs which make them relatively easy to use and to adopt on other systems. As all fluorophores are fusion tags to the according biomolecules, standard cloning strategies and molecular biology technologies are sufficient and make this method available for a broad community of researchers.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Bacteriófagos/química , Sequência de Bases , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cromatografia em Gel/métodos , Clonagem Molecular/métodos , Dimerização , Proteínas Luminescentes/genética , Conformação de Ácido Nucleico , Ligação Proteica , RNA/isolamento & purificação , RNA/metabolismo
18.
Arch Virol ; 166(8): 2273-2278, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34059971

RESUMO

Feline panleukopenia virus (FPV) is a highly contagious infectious pathogen of cats globally. However, there is no information on the molecular identification and characterization of FPV in Bangladesh. Here, 8.16% (8/98) and 18.37% (18/98) of diarrheic cats tested positive for FPV by an immunochromatography (IC) test and PCR, respectively. The IC test showed 44.44% sensitivity and 100% specificity in comparison with PCR. Our newly sequenced Bangladeshi FPV strain (MN826076) showed the highest (99.71%) sequence identity to strains from the United Arab Emirates (UAE). Strain MN826076 contained two characteristic amino acid variations in VP2 identifying it as an FPV strain: valine at position 103 and aspartic acid at position 323. Phylogenetically, the VP2 of strain MN826076 was found to be closely related to 19 FPV strains, sharing the same clade.


Assuntos
Diarreia/veterinária , Diarreia/virologia , Vírus da Panleucopenia Felina/classificação , Panleucopenia Felina/diagnóstico , Substituição de Aminoácidos , Animais , Bangladesh , Proteínas do Capsídeo/genética , Gatos , China , Cromatografia de Afinidade , Vírus da Panleucopenia Felina/genética , Vírus da Panleucopenia Felina/isolamento & purificação , Filogenia , Filogeografia , Portugal , Sensibilidade e Especificidade , Tailândia , Emirados Árabes Unidos
19.
Arch Virol ; 166(8): 2225-2234, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091782

RESUMO

In order to discover the causes of a coxsackievirus B4 (CV-B4)-associated hand, foot, and mouth disease (HFMD) outbreak and to study the evolutionary characteristics of the virus, we sequenced isolates obtained during an outbreak for comparative analysis with previously sequenced strains. Phylogenetic and evolutionary dynamics analysis was performed to examine the genetic characteristics of CV-B4 in China and worldwide. Phylogenetic analysis showed that CV-B4 originated from a common ancestor in Shandong. CV-B4 strains isolated worldwide could be classified into genotypes A-E based on the sequence of the VP1 region. All CV-B4 strains in China belonged to genotype E. The global population diversity of CV-B4 fluctuated substantially over time, and CV-B4 isolated in China accounted for a significant increase in the diversity of CV-B4. The average nucleotide substitution rate in VP1 of Chinese CV-B4 (5.20 × 10-3 substitutions/site/year) was slightly higher than that of global CV-B4 (4.82 × 10-3 substitutions/site/year). This study is the first to investigate the evolutionary dynamics of CV-B4 and its association with an HFMD outbreak. These findings explain both the 2011 outbreak and the global increase in CV-B4 diversity. In addition to improving our understanding of a major outbreak, these findings provide a basis for the development of surveillance strategies.


Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano B/classificação , Doença de Mão, Pé e Boca/virologia , Polimorfismo de Nucleotídeo Único , China , Enterovirus Humano B/genética , Enterovirus Humano B/isolamento & purificação , Evolução Molecular , Humanos , Tipagem Molecular , Taxa de Mutação , Filogenia , Análise de Sequência de RNA
20.
BMC Infect Dis ; 21(1): 446, 2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001016

RESUMO

BACKGROUND: Coxsackievirus A21 (CVA21), a member of Enterovirus C from the Picornaviridae family, has been associated with respiratory illnesses in humans. METHODS: A molecular epidemiological investigation of CVA21 was conducted among patients presenting with acute upper respiratory illnesses in the ambulatory settings between 2012 and 2014 in Kuala Lumpur, Malaysia. RESULTS: Epidemiological surveillance of acute respiratory infections (n = 3935) showed low-level detection of CVA21 (0.08%, 1.4 cases/year) in Kuala Lumpur, with no clear seasonal distribution. Phylogenetic analysis of the new complete genomes showed close relationship with CVA21 strains from China and the United States. Spatio-temporal mapping of the VP1 gene determined 2 major clusters circulating worldwide, with inter-country lineage migration and strain replacement occurring over time. CONCLUSIONS: The study highlights the emerging role of CVA21 in causing sporadic acute respiratory outbreaks.


Assuntos
Infecções por Coxsackievirus/diagnóstico , Enterovirus/genética , Variação Genética , Infecções Respiratórias/diagnóstico , Adolescente , Adulto , Proteínas do Capsídeo/classificação , Proteínas do Capsídeo/genética , Infecções por Coxsackievirus/epidemiologia , Infecções por Coxsackievirus/virologia , Surtos de Doenças , Enterovirus/classificação , Enterovirus/isolamento & purificação , Feminino , Humanos , Malásia/epidemiologia , Masculino , Pessoa de Meia-Idade , Filogenia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia
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