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1.
PLoS Pathog ; 16(9): e1008803, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956403

RESUMO

The Dearing isolate of Mammalian orthoreovirus (T3D) is a prominent model of virus-host relationships and a candidate oncolytic virotherapy. Closely related laboratory strains of T3D, originating from the same ancestral T3D isolate, were recently found to exhibit significantly different oncolytic properties. Specifically, the T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we discover that T3DPL and T3DTD also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3DTD induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3DPL. Using mono-reassortants with intermediate replication kinetics and pharmacological inhibitors of reovirus replication, IFN responses were found to inversely correlate with kinetics of virus replication. In other words, slow-replicating T3D strains induce more IFN signalling than fast-replicating T3D strains. Paradoxically, during co-infections by T3DPL and T3DTD, there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3DTD. Using silencing and knock-out of RIG-I to impede IFN, we found that IFN induction does not affect the first round of reovirus replication but does prevent cell-cell spread in a paracrine fashion. Accordingly, during co-infections, T3DPL continues to replicate robustly despite activation of IFN by T3DTD. Using gene expression analysis, we discovered that reovirus can also induce a subset of genes in a RIG-I and IFN-independent manner; these genes were induced more by T3DPL than T3DTD. Polymorphisms in reovirus σ3 viral protein were found to control activation of RIG-I/ IFN-independent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing replication kinetics and by modifying viral protein activity, such that two closely related T3D strains can induce opposite cytokine landscapes.


Assuntos
Proteínas do Capsídeo/metabolismo , Interferons/metabolismo , Polimorfismo Genético , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores do Ácido Retinoico/metabolismo , Infecções por Reoviridae/virologia , Replicação Viral , Proteínas do Capsídeo/genética , Citocinas , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Orthoreovirus de Mamíferos/fisiologia , RNA de Cadeia Dupla/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Receptores do Ácido Retinoico/genética , Infecções por Reoviridae/genética , Infecções por Reoviridae/metabolismo , Transdução de Sinais
2.
Nat Commun ; 11(1): 4693, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943634

RESUMO

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.


Assuntos
Alphavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Genômica , RNA Viral/genética , Alphavirus/genética , Alphavirus/ultraestrutura , Animais , Sítios de Ligação , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Vírus Chikungunya/genética , Chlorocebus aethiops , Modelos Moleculares , Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/química , Vírus da Floresta de Semliki/metabolismo , Células Vero , Montagem de Vírus , Replicação Viral
3.
Nat Commun ; 11(1): 3849, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737300

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr Virus (EBV) establish life-long infections and are associated with malignancies. Striking geographic variation in incidence and the fact that virus alone is insufficient to cause disease, suggests other co-factors are involved. Here we present epidemiological analysis and genome-wide association study (GWAS) in 4365 individuals from an African population cohort, to assess the influence of host genetic and non-genetic factors on virus antibody responses. EBV/KSHV co-infection (OR = 5.71(1.58-7.12)), HIV positivity (OR = 2.22(1.32-3.73)) and living in a more rural area (OR = 1.38(1.01-1.89)) are strongly associated with immunogenicity. GWAS reveals associations with KSHV antibody response in the HLA-B/C region (p = 6.64 × 10-09). For EBV, associations are identified for VCA (rs71542439, p = 1.15 × 10-12). Human leucocyte antigen (HLA) and trans-ancestry fine-mapping substantiate that distinct variants in HLA-DQA1 (p = 5.24 × 10-44) are driving associations for EBNA-1 in Africa. This study highlights complex interactions between KSHV and EBV, in addition to distinct genetic architectures resulting in important differences in pathogenesis and transmission.


Assuntos
Anticorpos Antivirais/biossíntese , Resistência à Doença/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Henipavirus/genética , Interações Hospedeiro-Patógeno/genética , Sarcoma de Kaposi/genética , Adolescente , Adulto , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Coinfecção , Infecções por Vírus Epstein-Barr/epidemiologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , HIV/genética , HIV/imunologia , HIV/patogenicidade , Cadeias alfa de HLA-DQ/genética , Cadeias alfa de HLA-DQ/imunologia , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , População Rural , Sarcoma de Kaposi/epidemiologia , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/virologia , Uganda/epidemiologia , População Urbana
4.
Proc Natl Acad Sci U S A ; 117(33): 20211-20222, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747554

RESUMO

The giant tiger prawn (Penaeus monodon) is a decapod crustacean widely reared for human consumption. Currently, viruses of two distinct lineages of parvoviruses (PVs, family Parvoviridae; subfamily Hamaparvovirinae) infect penaeid shrimp. Here, a PV was isolated and cloned from Vietnamese P. monodon specimens, designated Penaeus monodon metallodensovirus (PmMDV). This is the first member of a third divergent lineage shown to infect penaeid decapods. PmMDV has a transcription strategy unique among invertebrate PVs, using extensive alternative splicing and incorporating transcription elements characteristic of vertebrate-infecting PVs. The PmMDV proteins have no significant sequence similarity with other PVs, except for an SF3 helicase domain in its nonstructural protein. Its capsid structure, determined by cryoelectron microscopy to 3-Å resolution, has a similar surface morphology to Penaeus stylirostris densovirus, despite the lack of significant capsid viral protein (VP) sequence similarity. Unlike other PVs, PmMDV folds its VP without incorporating a ßA strand and displayed unique multimer interactions, including the incorporation of a Ca2+ cation, attaching the N termini under the icosahedral fivefold symmetry axis, and forming a basket-like pentamer helix bundle. While the PmMDV VP sequence lacks a canonical phospholipase A2 domain, the structure of an EDTA-treated capsid, determined to 2.8-Å resolution, suggests an alternative membrane-penetrating cation-dependent mechanism in its N-terminal region. PmMDV is an observed example of convergent evolution among invertebrate PVs with respect to host-driven capsid structure and unique as a PV showing a cation-sensitive/dependent basket structure for an alternative endosomal egress.


Assuntos
Evolução Biológica , Proteínas do Capsídeo/genética , Densovirus/genética , Penaeidae/virologia , Animais , Regulação Viral da Expressão Gênica , Genoma Viral
5.
PLoS Pathog ; 16(8): e1008732, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750093

RESUMO

Rotavirus is a major cause of gastroenteritis in children, with infection typically inducing high levels of protective antibodies. Antibodies targeting the middle capsid protein VP6 are particularly abundant, and as VP6 is only exposed inside cells, neutralisation must be post-entry. However, while a system of poly immune globulin receptor (pIgR) transcytosis has been proposed for anti-VP6 IgAs, the mechanism by which VP6-specific IgG mediates protection remains less clear. We have developed an intracellular neutralisation assay to examine how antibodies neutralise rotavirus inside cells, enabling comparison between IgG and IgA isotypes. Unexpectedly we found that neutralisation by VP6-specific IgG was much more efficient than by VP6-specific IgA. This observation was highly dependent on the activity of the cytosolic antibody receptor TRIM21 and was confirmed using an in vivo model of murine rotavirus infection. Furthermore, mice deficient in only IgG and not other antibody isotypes had a serious deficit in intracellular antibody-mediated protection. The finding that VP6-specific IgG protect mice against rotavirus infection has important implications for rotavirus vaccination. Current assays determine protection in humans predominantly by measuring rotavirus-specific IgA titres. Measurements of VP6-specific IgG may add to existing mechanistic correlates of protection.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Imunoglobulina G/imunologia , Infecções por Rotavirus/imunologia , Rotavirus/fisiologia , Animais , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Rotavirus/genética , Infecções por Rotavirus/virologia , Especificidade da Espécie
6.
Arch Virol ; 165(10): 2301-2309, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757056

RESUMO

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.


Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Bombyx/virologia , Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Baculoviridae/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/imunologia , Soros Imunes/química , Imunogenicidade da Vacina , Larva/virologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
7.
Arch Virol ; 165(10): 2367-2372, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757058

RESUMO

Mammalian orthoreoviruses (MRVs) infect almost all mammals, and there are some reports on MRVs in China. In this study, a novel strain was identified, which was designated as HLJYC2017. The results of genetic analysis showed that MRV HLJYC2017 is a reassortant strain. According to biological information analysis, different serotypes of MRV contain specific amino acid insertions and deletions in the σ1 protein. Neutralizing antibody epitope analysis revealed partial cross-protection among MRV1, MRV2, and MRV3 isolates from China. L3 gene recombination in MRV was identified for the first time in this study. The results of this study provide valuable information on MRV reassortment and evolution.


Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Orthoreovirus de Mamíferos/genética , Vírus Reordenados/genética , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/veterinária , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , China/epidemiologia , Quirópteros , Cervos , Fezes/virologia , Expressão Gênica , Mutação INDEL , Camundongos , Epidemiologia Molecular , Orthoreovirus de Mamíferos/classificação , Orthoreovirus de Mamíferos/imunologia , Orthoreovirus de Mamíferos/isolamento & purificação , Filogenia , RNA Viral/genética , Vírus Reordenados/classificação , Vírus Reordenados/imunologia , Vírus Reordenados/isolamento & purificação , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/virologia , Sorogrupo , Suínos
8.
Arch Virol ; 165(10): 2291-2299, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32754877

RESUMO

The multimammate mouse (Mastomys natalensis; M. natalensis) serves as the main reservoir for the zoonotic arenavirus Lassa virus (LASV), and this has led to considerable investigation into the distribution of LASV and other related arenaviruses in this host species. In contrast to the situation with arenaviruses, the presence of other viruses in M. natalensis remains largely unexplored. In this study, herpesviruses and polyomaviruses were identified and partially characterized by PCR methods, sequencing, and phylogenetic analysis. In tissues sampled from M. natalensis populations in Côte d'Ivoire and Mali, six new DNA viruses (four betaherpesviruses, one gammaherpesvirus and one polyomavirus) were identified. Phylogenetic analysis based on glycoprotein B amino acid sequences showed that the herpesviruses clustered with cytomegaloviruses and rhadinoviruses of multiple rodent species. The complete circular genome of the newly identified polyomavirus was amplified by PCR. Amino acid sequence analysis of the large T antigen or VP1 showed that this virus clustered with a known polyomavirus from a house mouse (species Mus musculus polyomavirus 1). These two polyomaviruses form a clade with other rodent polyomaviruses, and the newly identified virus represents the third known polyomavirus of M. natalensis. This study represents the first identification of herpesviruses and the discovery of a novel polyomavirus in M. natalensis. In contrast to arenaviruses, we anticipate that these newly identified viruses represent a low zoonotic risk due to the normally highly restricted specificity of members of these two DNA virus families to their individual mammalian host species.


Assuntos
Genoma Viral , Infecções por Herpesviridae/epidemiologia , Herpesviridae/genética , Filogenia , Infecções por Polyomavirus/epidemiologia , Polyomavirus/genética , Doenças dos Roedores/epidemiologia , África ao Sul do Saara/epidemiologia , Animais , Antígenos Virais de Tumores/genética , Proteínas do Capsídeo/genética , Reservatórios de Doenças/virologia , Herpesviridae/classificação , Herpesviridae/isolamento & purificação , Infecções por Herpesviridae/virologia , Especificidade de Hospedeiro , Tipagem Molecular , Murinae/virologia , Polyomavirus/classificação , Polyomavirus/isolamento & purificação , Infecções por Polyomavirus/virologia , Doenças dos Roedores/virologia , Proteínas do Envelope Viral/genética
9.
Arch Virol ; 165(9): 2021-2028, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32601957

RESUMO

Since 2006, Egypt has been affected by eleven various foot-and-mouth disease virus (FMDV) lineages. Accordingly, the nucleotide sequences of the 1D gene and the genes encoding the external capsid protein of some isolates of serotype O (the most predominant epidemic serotype in the country) collected from 2004 to 2017 were determined. All of these viruses (including the vaccine strain) belonged to serotype O, topotype ME-SA, and lineage Sharquia-72, and their sequences were of 98.6-98.9% identical to that of strain O1/Sharquia/EGY/72 (DQ164871), and differed from cultured and clinical (D197E) virus strains. The characteristic sites on the surface of the structural proteins of the Egyptian serotype O, topotype ME-SA viruses were located at residues 138 and 198 of VP1, residue 132 of VP2, and residues 56 and 104 of VP3. Furthermore, a phylogenetic tree revealed that Sharquia-72 was the only lineage present in Egypt for many decades prior to 2007. Unfortunately, however, during the last decade, five lineages of two separate topotypes of FMDV serotype O were detected in Egypt. Lineages Sharquia-72 and PanAsia-2 belong to topotype ME-SA and show ~ 14.5 to 17.5% intra-lineage divergence. In addition, lineages Qal-13, Ism-16, and Alx-17 cluster within topotype EA-3 and show ~ 4.5 to 15% intra-lineage diversity. The predecessors of the Egyptian EA-3 viruses are likely to have been from Sudan. Finally, at least a penta- or hexavalent vaccine comprising strains representing the endemic FMDV topotypes should be implemented on a wide scale in Egypt, which could combat the incursion of new lineages.


Assuntos
Proteínas do Capsídeo/genética , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/virologia , Animais , Búfalos , Bovinos , Doenças dos Bovinos/virologia , Egito , Vírus da Febre Aftosa/classificação , Vírus da Febre Aftosa/genética , Filogenia , Análise de Sequência de DNA
10.
Arch Virol ; 165(9): 2065-2071, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32613291

RESUMO

Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:216). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterite Viral do Vison/prevenção & controle , Vírus da Enterite do Vison/imunologia , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas do Capsídeo/administração & dosagem , Expressão Gênica , Vison/imunologia , Vison/virologia , Enterite Viral do Vison/imunologia , Enterite Viral do Vison/virologia , Vírus da Enterite do Vison/genética , Vírus da Enterite do Vison/patogenicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência
11.
Nat Commun ; 11(1): 3279, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32606306

RESUMO

Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the three-fold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/classificação , Sistema Nervoso Central/virologia , Microscopia Crioeletrônica , DNA Viral/análise , DNA Viral/genética , Dependovirus/classificação , Dependovirus/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Filogenia , Sorogrupo , Transdução Genética , Montagem de Vírus/genética
12.
Nat Commun ; 11(1): 3505, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665593

RESUMO

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.


Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Linfócitos T CD4-Positivos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Viral/genética , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Microscopia de Fluorescência , Virologia , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
13.
BMC Infect Dis ; 20(1): 488, 2020 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-32646445

RESUMO

BACKGROUND: Washington University polyomavirus (WUPyV) is a novel human polyomavirus detected in childwith acute respiratory infection in 2007. However, the relationship between WUPyV and respiratory diseases has yet to be established for lacking of a suitable in vitro culture system. METHODS: To isolate WUPyV with human airway epithelial (HAE) cells, the positive samples were incubated in HAE, and then the nucleic acid, VP1 protein and virions were detected using real-time PCR, immunofluorescence and electron microscopy respectively. RESULTS: The result showed that WUPyV could replicate effectively in HAE cells and virions with typical polyomavirus characteristics could be observed. Additionally, the entire genome sequence of the isolated strain (BJ0771) was obtained and phylogenetic analysis indicated that BJ0771 belongs to gene cluster I. CONCLUSIONS: Our findings demonstrated clinical WUPyV strain was successfully isolated for the first time in the world and this will help unravel the etiology and pathogenic mechanisms of WUPyV in respiratory infection diseases.


Assuntos
Células Epiteliais/virologia , Infecções por Polyomavirus/diagnóstico , Infecções por Polyomavirus/virologia , Polyomavirus/genética , Polyomavirus/isolamento & purificação , Mucosa Respiratória/patologia , Infecções Respiratórias/diagnóstico , Adolescente , Proteínas do Capsídeo/genética , Polaridade Celular , Células Cultivadas , Criança , Pré-Escolar , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Família Multigênica , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/virologia , Vírion/genética , Replicação Viral , Sequenciamento Completo do Genoma
14.
Proc Natl Acad Sci U S A ; 117(30): 17992-18001, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669438

RESUMO

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a "kissing" interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor's inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.


Assuntos
Antivirais/química , Proteínas do Capsídeo/química , Vírus da Dengue , Modelos Moleculares , Conformação Proteica , Sequência de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação , Proteínas do Capsídeo/genética , Vírus da Dengue/efeitos dos fármacos , Mutação , Nucleocapsídeo/química , Nucleocapsídeo/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
15.
Arch Virol ; 165(10): 2389-2392, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32699979

RESUMO

A novel tobamovirus, brugmansia latent virus (BrLV), was discovered during a study of brugmansia (Brugmansia spp.) in the living collections held at the Royal Botanic Gardens, Kew. Here, we report the complete genome sequence of BrLV, which is 6,397 nucleotides long and contains the four open reading frames (RNA-dependent RNA polymerase, methyltransferase/helicase, movement, and coat proteins) typical of tobamoviruses. The complete genome sequence of BrLV shares 69.7% nucleotide sequence identity with brugmansia mild mottle virus (BrMMV) and 66.7 to 68.7% identity with other tobamoviruses naturally infecting members of the Solanaceae plant family. Phylogenetic analysis of the complete genome nucleotide sequence and the deduced amino acid sequences of the four tobamovirus proteins place BrLV in a subcluster with BrMMV within the Solanaceae-infecting tobamovirus subgroup as a new species.


Assuntos
Brugmansia/virologia , Proteínas do Capsídeo/genética , Genoma Viral , RNA Viral/genética , Tobamovirus/genética , Sequência de Bases , Sequência Conservada , Metiltransferases/genética , Fases de Leitura Aberta , Filogenia , Doenças das Plantas/virologia , RNA Replicase/genética , Alinhamento de Sequência , Tobamovirus/classificação , Tobamovirus/isolamento & purificação , Reino Unido , Sequenciamento Completo do Genoma
16.
Arch Virol ; 165(10): 2213-2227, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32666145

RESUMO

In this study, we investigated the epidemiology and molecular characteristics of enteroviruses associated with severe hand, foot and mouth disease (HFMD) in Shenzhen, China, during 2014-2018. A total of 137 fecal specimens from patients with severe HFMD were collected. Enterovirus (EV) types were determined using real-time reverse transcription polymerase chain reaction (RT-PCR), RT nested PCR, and sequencing. Sequences were analyzed using bioinformatics programs. Of 137 specimens tested, 97 (70.8%), 12 (8.8%), and 10 (7.3%) were positive for EV-A71, coxsackievirus A6 (CVA6), and CVA16, respectively. Other pathogens detected included CVA2 (2.9%, 4/137), CVA10 (2.9%, 4/137), CVA5 (0.7%, 1/137), echovirus 6 (E6) (0.7%, 1/137) and E18 (0.7%, 1/137). The most frequent complication in patients with proven EV infections was myoclonic jerk, followed by aseptic encephalitis, tachypnea, and vomiting. The frequencies of vomiting and abnormal eye movements were higher in EV-A71-infected patients than that in CVA6-infected or CVA16-infected patients. Molecular phylogeny based on the complete VP1 gene revealed no association between the subgenotype of the virus and disease severity. Nevertheless, 12 significant mutations that were likely to be associated with virulence or the clinical phenotype were observed in the 5'UTR, 2Apro, 2C, 3A, 3Dpol and 3'UTR of CVA6. Eight significant mutations were observed in the 5'UTR, 2B, 3A, 3Dpol and 3'UTR of CVA16, and 10 significant mutations were observed in the 5'UTR, VP1, 3A and 3Cpro of CVA10. In conclusion, EV-A71 is still the main pathogen causing severe HFMD, although other EV types can also cause severe complications. Potential virulence or phenotype-associated sites were identified in the genomes of CVA6, CVA16, and CVA10.


Assuntos
Proteínas do Capsídeo/genética , Encefalite/epidemiologia , Enterovirus Humano C/genética , Doença de Mão, Pé e Boca/epidemiologia , Mioclonia/epidemiologia , Taquipneia/epidemiologia , Vômito/epidemiologia , Criança , Pré-Escolar , China/epidemiologia , Encefalite/diagnóstico , Encefalite/fisiopatologia , Encefalite/virologia , Enterovirus Humano C/classificação , Enterovirus Humano C/isolamento & purificação , Fezes/virologia , Feminino , Expressão Gênica , Genótipo , Doença de Mão, Pé e Boca/diagnóstico , Doença de Mão, Pé e Boca/fisiopatologia , Doença de Mão, Pé e Boca/virologia , Humanos , Lactente , Recém-Nascido , Masculino , Epidemiologia Molecular , Mutação , Mioclonia/diagnóstico , Mioclonia/fisiopatologia , Mioclonia/virologia , Fenótipo , Filogenia , Índice de Gravidade de Doença , Taquipneia/diagnóstico , Taquipneia/fisiopatologia , Taquipneia/virologia , Virulência , Vômito/diagnóstico , Vômito/fisiopatologia , Vômito/virologia
17.
PLoS One ; 15(6): e0235280, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584905

RESUMO

Rotavirus infection is the most common cause of viral diarrhea in infants and young children but uncommon and usually asymptomatic in adults. In the winter of 2017-2018, a large-scale outbreak of rotavirus in both children and adults was reported in Thailand. The current study focused on the prevalence, genotyping, and molecular characterization of rotavirus infections in Thai adults from July 2016 to December 2019. In 2,598 stool samples collected from adult residents of Bangkok (aged #x2265; 15 years) with acute gastroenteritis, rotavirus was detected via real-time RT-PCR analysis of the VP6 gene. G, P and I genotypes were determined by direct sequencing of VP7, VP4, and VP6 genes, respectively. Our results showed 8.7% (226/2,598) of stool samples were positive for rotavirus. The incidence of rotavirus was high during the winter season of 2017-2018 (17.7%) compared to another studied periods (4.5% between July 2016- October 2017 and 2.8% between March 2018- December 2019). Nucleotide sequencing of VP7 and VP4 revealed G3P[8] as the predominant strain (33.2%,75/226), followed by G9P[8] (17.3%,39/226), and G2P[4] (15.0%,34/226). Uncommon G and P combinations were additionally detected at low frequencies. VP6 sequencing was conducted to discriminate I genotype between the Wa and DS-1 genogroup. The unusual DS-1-like G3P[8] strain was most prevalent amomg rotavirus strains detected in this study (29.6%, 67/226), and the corresponding VP7 sequences showed high nucleotide identity with unusual DS-1-like globally circulating strains. Our study demonstrates that rotavirus outbreaks in adults are attributable not only to high prevalence of RV infection but also the unusual DS-like genogroup. The collective findings reinforce the importance of investigating rotavirus diagnosis in adults suffering from acute gastroenteritis and taking appropriate preventive measures.


Assuntos
Infecções por Rotavirus/diagnóstico , Rotavirus/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais/classificação , Antígenos Virais/genética , Proteínas do Capsídeo/classificação , Proteínas do Capsídeo/genética , Fezes/virologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Prevalência , RNA Viral/química , RNA Viral/metabolismo , Rotavirus/classificação , Rotavirus/isolamento & purificação , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/virologia , Tailândia/epidemiologia , Adulto Jovem
18.
PLoS Pathog ; 16(6): e1008588, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584886

RESUMO

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , DNA Viral/metabolismo , Vírion/metabolismo , Replicação Viral , Células A549 , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/patologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , DNA Viral/genética , Humanos , Vírion/genética
19.
Arch Virol ; 165(9): 2003-2011, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32594321

RESUMO

Porcine circovirus 3 (PCV3) is a recently identified virus that is associated with reproductive failure, porcine dermatitis and nephropathy syndrome, and multi-systemic inflammation. To investigate the molecular epidemic characteristics and genetic evolution of PCV3 in northern China, a commercial TaqMan-based real-time quantitative PCR kit was used to detect PCV3 in 435 tissue specimens collected from pigs with various clinical signs from 105 different swine farms in northern China. The results showed that 48 out of 105 (45.7%) farms and 97 out of 435 (22.3%) samples tested positive for PCV3. Of the 97 PCV3-positive samples, 80 (82.5%) tested positive for other pathogens. PCV3 was found more frequently in pigs with reproductive failure than in those with other clinical signs. This study is the first to detect PCV3 in Tianjin. The complete genome sequences of six PCV3 isolates and the capsid (Cap) protein gene sequences of 11 isolates were determined. Based on the predicted amino acids at positions 24 and 27 of the Cap protein and their evolutionary relationships, the 17 PCV3 strains obtained from northern China and 49 reference strains downloaded from the GenBank database were divided into four major groups (3a-3d). An analysis of selection pressure and polymorphism indicated that the PCV3 Cap protein seems to be evolving under balancing selection, that the population is in dynamic equilibrium, and that no population expansion occurred during the study period. Our results provide new information about the molecular epidemiology and evolution of PCV3.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/classificação , Circovirus/isolamento & purificação , Filogenia , Doenças dos Suínos/virologia , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , China/epidemiologia , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Circovirus/genética , Epidemiologia Molecular , Suínos , Doenças dos Suínos/epidemiologia
20.
J Med Microbiol ; 69(7): 960-970, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32510304

RESUMO

Introduction. Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1-2 years. Although not all infected women develop detectable HPV antibodies, about 60-70 % seroconvert and retain their antibodies at low levels.Aim. We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology. We used baseline data for women participating in the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson's r was used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results. Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65-0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion. HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.


Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/diagnóstico , Adulto , Anticorpos Antivirais/sangue , Brasil , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Colo do Útero/virologia , Estudos de Coortes , Feminino , Papillomavirus Humano 16/patogenicidade , Humanos , Complexo Antígeno L1 Leucocitário/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Carga Viral/métodos , Vírion/imunologia
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