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1.
PLoS Pathog ; 15(10): e1008057, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31671153

RESUMO

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.


Assuntos
Infecções por Astroviridae/imunologia , Infecções por Astroviridae/veterinária , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Mamastrovirus/fisiologia , Tropismo Viral/genética , Animais , Células CACO-2 , Linhagem Celular , Enterócitos/virologia , Gastroenterite/virologia , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/virologia , Intestino Delgado/citologia , Intestino Delgado/virologia , Mamastrovirus/genética , Mamastrovirus/imunologia , Células Vero , Tropismo Viral/imunologia
2.
PLoS Pathog ; 15(6): e1007790, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194854

RESUMO

Bourbon virus (BRBV) is an emerging tick-borne RNA virus in the orthomyxoviridae family that was discovered in 2014. Although fatal human cases of BRBV have been described, little is known about its pathogenesis, and no antiviral therapies or vaccines exist. We obtained serum from a fatal case in 2017 and successfully recovered the second human infectious isolate of BRBV. Next-generation sequencing of the St. Louis isolate of BRBV (BRBV-STL) showed >99% nucleotide identity to the original reference isolate. Using BRBV-STL, we developed a small animal model to study BRBV-STL tropism in vivo and evaluated the prophylactic and therapeutic efficacy of the experimental antiviral drug favipiravir against BRBV-induced disease. Infection of Ifnar1-/- mice lacking the type I interferon receptor, but not congenic wild-type animals, resulted in uniformly fatal disease 6 to 10 days after infection. RNA in situ hybridization and viral yield assays demonstrated a broad tropism of BRBV-STL with highest levels detected in liver and spleen. In vitro replication and polymerase activity of BRBV-STL were inhibited by favipiravir. Moreover, administration of favipiravir as a prophylaxis or as post-exposure therapy three days after infection prevented BRBV-STL-induced mortality in immunocompromised Ifnar1-/- mice. These results suggest that favipiravir may be a candidate treatment for humans who become infected with BRBV.


Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Infecções por Orthomyxoviridae/prevenção & controle , Pirazinas/farmacologia , Thogotovirus/imunologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/imunologia , Thogotovirus/patogenicidade , Células Vero , Tropismo Viral/efeitos dos fármacos , Tropismo Viral/genética , Tropismo Viral/imunologia
3.
PLoS One ; 14(2): e0212882, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30818365

RESUMO

BACKGROUND: The association between X4 virus and an increased risk of non-AIDS-events has been reported. Morbidity/mortality due to non-AIDS events, which are properly predicted by the CD4/CD8 ratio and VACS index, have become particularly remarkable in HIV-infected patients receiving effective combined antiretroviral therapy (cART). METHODS: We verified the validity of the syllogism: as HIV-tropism (CRT) contributes to the onset of non-AIDS events which are successfully predicted by the CD4/CD8 ratio and VACS index, then CRT correlates with these two variables. The CD4/CD8 ratio and VACS index at baseline and overtime were analyzed according to CRT tested before the first successful cART regimen in newly-diagnosed patients. RESULTS: Patients with R5 variants had a significantly lower baseline VACS percentage risk [mean (95%CI):18.2%(16.1-20.3) vs 24.3%(18.2-22.5), p = 0.002] and higher baseline CD4/CD8 ratio [mean (95%CI):0.43 (0.38-0.47) vs 0.28 (0.19-0.36), p = 0.002] than non-R5 patients. After an initial drop, VACS increased again in R5 and non-R5 patients and the two trend curves almost overlapped. The CD4/CD8 ratio had an increasing trend in both R5 and non-R5 patients; however, even though non-R5 patients had a greater gain of CD4+, they maintained a lower CD4/CD8 ratio at any time point. CONCLUSION: Our study confirms an association between pre-therapy CRT, CD4/CD8 ratio and VACS. A successful cART regimen positively affects the CD4/CD8 ratio; however, the disadvantage conferred by a non-R5 CRT is maintained overtime. The restoration of VACS in all patients could be directly due to variables included in the VACS calculation and to factors that adversely influence these variables.


Assuntos
Infecções por HIV/imunologia , HIV-1 , Adulto , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Relação CD4-CD8 , Estudos de Coortes , Feminino , Variação Genética , Infecções por HIV/complicações , Infecções por HIV/mortalidade , HIV-1/genética , HIV-1/fisiologia , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Receptores CCR5/genética , Receptores CXCR4/genética , Fatores de Risco , Veteranos , Tropismo Viral/genética , Tropismo Viral/imunologia
4.
PLoS One ; 14(3): e0213160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30856186

RESUMO

BACKGROUND AND AIMS: We investigated the conditioning roles of viral tropism and other variables on plasma HIV RNA levels after 6 months of combination antiretroviral therapy (cART) in an HIV-infected Italian naïve population using regression tree, random forest regression, and path analysis (PA). Patients in this multicenter observational study were treated with all antiviral drugs that are currently recommended as first-line therapies. METHODS: Adult patients with chronic HIV infection were enrolled at the beginning of first-line cART (T0). The main variables were age, gender, tropism, "lcd4_0" and "lcd4_6" (log10 CD4+counts at T0 and after 6 months of cART, respectively), and "lrna0" (log10 HIV RNA at T0). Regression tree and random forest analyses were applied. The predictive effect on lrna6 (log10-transformed plasma HIV RNA after 6 months of cART) was also investigated via PA (x4->lcd4_0->lrna0->lrna6) with a treatment selection step included as a dependent (mediator) variable for each third drug and, as predictive covariates, age, female, x4_10, x4_5, lcd4_0, and lrna0. Tropism was assessed in plasma using the Geno2pheno algorithm with 2 false positive rate (FPR) cut-offs: 5% (x4_5) and 10% (x4_10). RESULTS: The study included 571 subjects (21% x4_10 and 10.7% x4_5). The only important predictor of lrna6 was lrna0, and a positive indirect effect of bearing X4 virus in plasma was suggested. A significant direct positive effect of protease inhibitors on lrna6 was found (p = 0.022), and a significant negative effect of integrase strand transfer inhibitor (INSTI) was also detected (p = 0.003 for FPR ≤ 5% and p = 0.01 for FPR < 10%). PA predicted mean residual viremias of 40 copies/mL without INSTI and 3 copies/mL with INSTI. CONCLUSIONS: PA indicated a possible indirect role of HIV tropism on lrna6 with both FPR < 10% and ≤ 5%. Patients treated with INSTI had a predicted residual viremia of 3 copies/mL.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/genética , RNA Viral/sangue , Adulto , Algoritmos , Contagem de Linfócito CD4 , Doença Crônica , Feminino , Genótipo , HIV-1/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Resultado do Tratamento , Tropismo Viral/genética
5.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728251

RESUMO

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is characterized by genetic instability and different antigenic properties. The nonstructural protein 3A is a primary determinant of the tropism and virulence of Cathay topotype FMDVs. However, several other determinants are also speculated to be involved in viral tropism and virulence. Deletion of 43 nucleotides (nt) in the pseudoknot (PK) region of the 5' untranslated region (UTR) has been found to coexist with the identified 3A deletion in Cathay topotype FMDV genomes. In this study, we isolated an O/ME-SA/PanAsia lineage FMDV strain, O/GD/CHA/2015, that includes an 86-nt deletion in the PK region and shows a porcinophilic phenotype. To investigate the potential role of the PK region in viral pathogenicity, we generated a recombinant FMDV strain with an incomplete PK region and compared its virulence and pathogenesis to the intact FMDV strain in swine and bovines. Deletion of the 86 nt in the PKs had no major effects on the pathogenicity of the virus in swine but significantly attenuated its ability to infect bovine cells and cattle, indicating that the PK region is a newly discovered determinant of viral tropism and virulence. The role of the 43-nt deletion existing in the Cathay topotype FMDV was also investigated by evaluating the infection properties of genetically engineered viruses. Consistently, the 43-nt deletion in the PK region significantly decreased the pathogenicity of the virus in bovines. Overall, our findings suggest that the PK region deletion occurred naturally in the FMDV genome and that the PK region is highly associated with viral host range and functions as a novel determinant for FMDV pathogenesis.IMPORTANCE This study demonstrates that the deletion in the PK region occurred naturally in the FMDV genome. The isolated O/ME-SA/PanAsia lineage FMDV with an 86-nt deletion in the PK region showed a pig-adapted characteristic that could cause clinical signs in swine but not bovines. Compared to the wild-type FMDV strain, which possesses full infection capacity in both swine and bovines, the recombinant virus with the 86-nt deletion in the PK region is deficient in causing disease in bovines. Deletion of the previously reported 43 nt in the PK region also led to significantly decreased pathogenicity of FMDV in bovines. This study indicates that the PK region is a novel determinant of the tropism and virulence of FMDV.


Assuntos
Regiões 5' não Traduzidas , Sequência de Bases , Vírus da Febre Aftosa/genética , Genoma Viral , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Tropismo Viral/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Febre Aftosa/genética , Febre Aftosa/metabolismo , Vírus da Febre Aftosa/patogenicidade , Suínos , Proteínas não Estruturais Virais/metabolismo
6.
Nature ; 567(7746): 109-112, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787439

RESUMO

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.


Assuntos
Quirópteros/virologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Especificidade de Hospedeiro , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Zoonoses/imunologia , Zoonoses/virologia , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Galinhas/genética , Galinhas/imunologia , Quirópteros/genética , Quirópteros/imunologia , Quirópteros/metabolismo , Feminino , Perfilação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Especificidade de Hospedeiro/genética , Especificidade de Hospedeiro/imunologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Sistema Respiratório/virologia , Suínos/genética , Suínos/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Replicação Viral , Zoonoses/genética , Zoonoses/metabolismo
7.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30487276

RESUMO

Elite and viremic HIV controllers are able to control their HIV infection and maintain undetectable or low-level viremia in the absence of antiretroviral treatment. Despite extensive studies, the immune factors responsible for such exclusive control remain poorly defined. We identified a cohort of 14 HIV controllers that suffered an abrupt loss of HIV control (LoC) to investigate possible mechanisms and virological and immunological events related to the sudden loss of control. The in-depth analysis of these subjects involved the study of cell tropism of circulating virus, evidence for HIV superinfection, cellular immune responses to HIV, as well as an examination of viral adaptation to host immunity by Gag sequencing. Our data demonstrate that a poor capacity of T cells to mediate in vitro viral suppression, even in the context of protective HLA alleles, predicts a loss of viral control. In addition, the data suggest that inefficient viral control may be explained by an increase of CD8 T-cell activation and exhaustion before LoC. Furthermore, we detected a switch from C5- to X4-tropic viruses in 4 individuals after loss of control, suggesting that tropism shift might also contribute to disease progression in HIV controllers. The significantly reduced inhibition of in vitro viral replication and increased expression of activation and exhaustion markers preceding the abrupt loss of viral control may help identify untreated HIV controllers that are at risk of losing control and may offer a useful tool for monitoring individuals during treatment interruption phases in therapeutic vaccine trials.IMPORTANCE A few individuals can control HIV infection without the need for antiretroviral treatment and are referred to as HIV controllers. We have studied HIV controllers who suddenly lose this ability and present with high in vivo viral replication and decays in their CD4+ T-cell counts to identify potential immune and virological factors that were responsible for initial virus control. We identify in vitro-determined reductions in the ability of CD8 T cells to suppress viral control and the presence of PD-1-expressing CD8+ T cells with a naive immune phenotype as potential predictors of in vivo loss of virus control. The findings could be important for the clinical management of HIV controller individuals, and it may offer an important tool to anticipate viral rebound in individuals in clinical studies that include combination antiretroviral therapy (cART) treatment interruptions and which, if not treated quickly, could pose a significant risk to the trial participants.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Tropismo Viral/fisiologia , Adulto , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Coortes , Feminino , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Carga Viral/fisiologia , Tropismo Viral/genética , Viremia/imunologia , Replicação Viral/efeitos dos fármacos
8.
Virology ; 526: 138-145, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30388629

RESUMO

Subgroup C Avian Metapneumoviruses (AMPV-C) has two lineages, one mostly in turkeys and one mostly in ducks. To investigate the molecular basis of AMPV-C host tropism, a reverse genetics system for a duck AMPV-C virus was developed. A recombinant copy and a recombinant virus in which the SH protein had been exchanged for that of a turkey AMPV-C were rescued. No change in cytopathogenic effect or replication profile in vitro were observed for either virus compared to the wild type. In SPF Muscovy ducks the wild type and its recombinant copy were equally pathogenic. Exchanging the SH in the recombinant copy produced the same results. In SPF turkeys, neither recombinant virus was pathogenic, although both showed a low level of replication. Thus, from the current model, it appears that AMPV-C SH proteins derived from the different species are compatible and that turkey SH does not affect duck AMPV-C pathogenicity.


Assuntos
Metapneumovirus/fisiologia , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/virologia , Vírus Reordenados/fisiologia , Proteínas Virais/metabolismo , Tropismo Viral/genética , Animais , Efeito Citopatogênico Viral , Patos , Metapneumovirus/genética , Metapneumovirus/patogenicidade , Infecções por Paramyxoviridae/virologia , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Genética Reversa , Perus , Proteínas Virais/genética , Replicação Viral
9.
Proc Natl Acad Sci U S A ; 116(1): 239-244, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30559208

RESUMO

HIV-1 evolved into various genetic subtypes and circulating recombinant forms (CRFs) in the global epidemic. The same subtype or CRF is usually considered to have similar phenotype. Being one of the world's major CRFs, CRF01_AE infection was reported to associate with higher prevalence of CXCR4 (X4) viruses and faster CD4 decline. However, the underlying mechanisms remain unclear. We identified eight phylogenetic clusters of CRF01_AE in China and hypothesized that they may have different phenotypes. In the National HIV Molecular Epidemiology Survey, we discovered that people infected by CRF01_AE cluster 4 had significantly lower CD4 counts (391 vs. 470, P < 0.0001) and higher prevalence of X4-using viruses (17.1% vs. 4.4%, P < 0.0001) compared with those infected by cluster 5. In an MSM cohort, X4-using viruses were only isolated from seroconvertors in cluster 4, which was associated with low a CD4 count within the first year of infection (141 vs. 440, P = 0.003). Using a coreceptor binding model, we identified unique V3 signatures in cluster 4 that favor CXCR4 use. We demonstrate that the HIV-1 phenotype and pathogenicity can be determined at the phylogenetic cluster level in the same subtype. Since its initial spread to humans from chimpanzees, estimated to be the first half of the 20th century, HIV-1 continues to undergo rapid evolution in larger and more diverse populations. The divergent phenotype evolution of two major CRF01_AE clusters highlights the importance of monitoring the genetic evolution and phenotypic shift of HIV-1 to provide early warning of the appearance of more pathogenic strains.


Assuntos
Contagem de Linfócito CD4 , HIV-1/genética , China/epidemiologia , Progressão da Doença , Estudos de Associação Genética , Infecções por HIV/epidemiologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Epidemiologia Molecular , Filogenia , Receptores de HIV/genética , Relação Estrutura-Atividade , Tropismo Viral/genética
10.
PLoS One ; 13(12): e0204099, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30586365

RESUMO

Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile® assay detected CXCR4-using virus pre-treatment in 16/20 participants failing with CXCR4-using virus. Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1-5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key 'signature' mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain-specific mutational patterns.


Assuntos
Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , HIV-1/genética , Maraviroc/administração & dosagem , Filogenia , Tropismo Viral/genética , Adulto , Feminino , Infecções por HIV/patologia , Humanos , Masculino , Maraviroc/efeitos adversos , Pessoa de Meia-Idade , Falha de Tratamento , Tropismo Viral/efeitos dos fármacos
11.
Elife ; 72018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30520725

RESUMO

Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).


Assuntos
Células Epiteliais/imunologia , Edição de Genes/métodos , HIV-1/genética , Interações Hospedeiro-Patógeno , Proteínas Nucleares/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/imunologia , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Humanos , Interferon-alfa/farmacologia , Lentivirus/genética , Lentivirus/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/imunologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Transdução de Sinais , Células THP-1 , Tropismo Viral/genética , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
12.
Sci Rep ; 8(1): 13408, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194325

RESUMO

Deep sequencing of live-attenuated viral vaccines has focused on vaccines in current use. Here we report characterization of a discontinued live yellow fever (YF) vaccine associated with severe adverse events. The French neurotropic vaccine (FNV) strain of YF virus was derived empirically in 1930 by 260 passages of wild-type French viscerotropic virus (FVV) in mouse brain. The vaccine was administered extensively in French-speaking Africa until discontinuation in 1982, due to high rates of post-vaccination encephalitis in children. Using rare archive strains of FNV, viral RNAs were sequenced and analyzed by massively parallel, in silico methods. Diversity and specific population structures were compared in reference to the wild-type parental strain FVV, and between the vaccine strains themselves. Lower abundance of polymorphism content was observed for FNV strains relative to FVV. Although the vaccines were of lower diversity than FVV, heterogeneity between the vaccines was observed. Reversion to wild-type identity was variably observed in the FNV strains. Specific population structures were recovered from vaccines with neurotropic properties; loss of neurotropism in mice was associated with abundance of wild-type RNA populations. The analysis provides novel sequence evidence that FNV is genetically unstable, and that adaptation of FNV contributed to the neurotropic adverse phenotype.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo Genético , Vacina contra Febre Amarela/genética , Febre Amarela , Vírus da Febre Amarela/genética , África/epidemiologia , Animais , Criança , Pré-Escolar , Encefalomielite Aguda Disseminada/epidemiologia , Encefalomielite Aguda Disseminada/genética , Humanos , Camundongos , Análise de Sequência de DNA , Tropismo Viral/genética , Febre Amarela/epidemiologia , Febre Amarela/genética , Febre Amarela/prevenção & controle , Vacina contra Febre Amarela/administração & dosagem , Vacina contra Febre Amarela/efeitos adversos , Vírus da Febre Amarela/patogenicidade
13.
PLoS Pathog ; 14(8): e1007190, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30075025

RESUMO

Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient's respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus's dissemination.


Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano A/fisiologia , Doença de Mão, Pé e Boca/virologia , Heparitina Sulfato/metabolismo , Tropismo Viral/genética , Animais , Enterovirus Humano A/patogenicidade , Doença de Mão, Pé e Boca/genética , Doença de Mão, Pé e Boca/metabolismo , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Mutação , Receptores Depuradores/metabolismo , Replicação Viral/genética
14.
Gene Ther ; 25(5): 331-344, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30013187

RESUMO

While the mammalian orthoreovirus type 3 dearing (reovirus T3D) infects many different tumour cells, various cell lines resist the induction of reovirus-mediated cell death. In an effort to increase the oncolytic potency, we introduced transgenes into the S1 segment of reovirus T3D. The adenovirus E4orf4 gene was selected as transgene since the encoded E4orf4 protein induces cell death in transformed cells. The induction of cell death by E4orf4 depends in part on its binding to phosphatase 2A (PP2A). In addition to the S1-E4orf4 reovirus, two other reoviruses were employed in our studies. The reovirus rS1-RFA encodes an E4orf4 double-mutant protein that cannot interact with PP2A and the rS1-iLOV virus encoding the fluorescent marker iLOV as a reporter. The replacement of the codons for the junction adhesion molecule-A (JAM-A) binding head domain of the truncated spike protein blocks the entry of these recombinant viruses via the reovirus receptor JAM-A. Instead these viruses rely on internalization via binding to sialic acids on the cell surface. This expands their tropism and allows infection of JAM-A-deficient tumour cells. Here we not only demonstrate the feasibility of this approach but also established that the cytolytic activity of these recombinant viruses is largely transgene independent.


Assuntos
Orthoreovirus Mamífero 3/fisiologia , Proteínas Virais/fisiologia , Tropismo Viral/genética , Linhagem Celular , Humanos , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/metabolismo , Infecções por Reoviridae/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29997207

RESUMO

UL148 is a viral endoplasmic reticulum (ER)-resident glycoprotein that contributes to human cytomegalovirus (HCMV) cell tropism. The influence of UL148 on tropism correlates with its potential to promote the expression of glycoprotein O (gO), a viral envelope glycoprotein that participates in a heterotrimeric complex with glycoproteins H and L that is required for infectivity. In an effort to gain insight into the mechanism, we used mass spectrometry to identify proteins that coimmunoprecipitate from infected cells with UL148. This approach led us to identify an interaction between UL148 and SEL1L, a factor that plays key roles in ER-associated degradation (ERAD). In pulse-chase experiments, gO was less stable in cells infected with UL148-null mutant HCMV than during wild-type infection, suggesting a potential functional relevance for the interaction with SEL1L. To investigate whether UL148 regulates gO abundance by influencing ERAD, small interfering RNA (siRNA) silencing of either SEL1L or its partner, Hrd1, was carried out in the context of infection. Knockdown of these ERAD factors strongly enhanced levels of gO but not other viral glycoproteins, and the effect was amplified in the presence of UL148. Furthermore, pharmacological inhibition of ERAD showed similar results. Silencing of SEL1L during infection also stabilized an interaction of gO with the ER lectin OS-9, which likewise suggests that gO is an ERAD substrate. Taken together, our results identify an intriguing interaction of UL148 with the ERAD machinery and demonstrate that gO behaves as a constitutive ERAD substrate during infection. These findings have implications for understanding the regulation of HCMV cell tropism.IMPORTANCE Viral glycoproteins in large part determine the cell types that an enveloped virus can infect and hence play crucial roles in transmission and pathogenesis. The glycoprotein H/L heterodimer (gH/gL) is part of the conserved membrane fusion machinery that all herpesviruses use to enter cells. In human cytomegalovirus (HCMV), gH/gL participates in alternative complexes in virions, one of which is a trimer of gH/gL with glycoprotein O (gO). Here, we show that gO is constitutively degraded during infection by the endoplasmic reticulum-associated degradation (ERAD) pathway and that UL148, a viral factor that regulates HCMV cell tropism, interacts with the ERAD machinery and slows gO decay. Since gO is required for cell-free virus to enter new host cells but dispensable for cell-associated spread that resists antibody neutralization, our findings imply that the posttranslational instability of a viral glycoprotein provides a basis for viral mechanisms to modulate tropism and spread.


Assuntos
Citomegalovirus/genética , Retículo Endoplasmático/virologia , Glicoproteínas de Membrana/genética , Proteínas/genética , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Tropismo Viral/genética , Células Cultivadas , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Retículo Endoplasmático/fisiologia , Células Epiteliais/virologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Humanos , Mutação com Perda de Função , Espectrometria de Massas , Glicoproteínas de Membrana/metabolismo , RNA Interferente Pequeno , Ubiquitina-Proteína Ligases/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Tropismo Viral/fisiologia , Internalização do Vírus
17.
Papillomavirus Res ; 5: 180-191, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29723682

RESUMO

Human papillomaviruses (HPVs) co-evolve slowly with the human host and each HPV genotype displays epithelial tropisms. We assessed the evolution of intra HPV genotype variants within samples, and their association to anogenital site, cervical cytology and HIV status. Variability in the L1 gene of 35 HPV genotypes was characterized phylogenetically using maximum likelihood, and portrayed by phenotype. Up to a thousand unique variants were identified within individual samples. In-depth analyses of the most prevalent genotypes, HPV16, HPV18 and HPV52, revealed that the high diversity was dominated by a few abundant variants. This suggests high intra-host mutation rates. Clades of HPV16, HPV18 and HPV52 were associated to anatomical site and HIV co-infection. Particularly, we observed that one HPV16 clade was specific to vaginal cells and one HPV52 clade was specific to anal cells. One major HPV52 clade, present in several samples, was strongly associated with cervical neoplasia. Overall, our data suggest that tissue tropism and HIV immunosuppression are strong shapers of HPV evolution.


Assuntos
Alphapapillomavirus/genética , Variação Genética , Tropismo Viral/genética , Adulto , Alphapapillomavirus/classificação , Canal Anal/citologia , Canal Anal/virologia , Proteínas do Capsídeo/genética , Colo do Útero/citologia , Colo do Útero/virologia , Coinfecção/virologia , Evolução Molecular , Feminino , Genótipo , Infecções por HIV/complicações , Humanos , Mutação , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Filogenia , Vagina/citologia , Vagina/virologia , Adulto Jovem
18.
J Neurovirol ; 24(4): 439-453, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29687407

RESUMO

Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~ 50,000 high-quality full-length HIV envelope sequences (> 2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.


Assuntos
Encéfalo/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Tropismo Viral/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Adulto , Infecções por HIV/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Macrófagos/virologia , Masculino , Filogenia , Provírus/genética , Receptores CXCR4
19.
Medicine (Baltimore) ; 97(13): e0176, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29595649

RESUMO

With limited and low-genetic barrier drugs used for the prevention of mother-to-child transmission (PMTCT) of HIV in sub-Saharan Africa, vertically transmitted HIV-1 drug-resistance (HIVDR) is concerning and might prompt optimal pediatric strategies.The aim of this study was to ascertain HIVDR and viral-tropism in majority and minority populations among Cameroonian vertically infected children.A comparative analysis among 18 HIV-infected children (7 from PMTCT-exposed mothers and 11 from mothers without PMTCT-exposure) was performed. HIVDR and HIV-1 co-receptor usage was evaluated by analyzing sequences obtained by both Sanger sequencing and ultra-deep 454-pyrosequencing (UDPS), set at 1% threshold.Overall, median (interquartile range) age, viremia, and CD4 count were 6 (4-10) years, 5.5 (4.9-6.0) log10 copies/mL, and 526 (282-645) cells/mm, respectively. All children had wild-type viruses through both Sanger sequencing and UDPS, except for 1 PMTCT-exposed infant harboring minority K103N (8.31%), born to a mother exposed to AZT+3TC+NVP. X4-tropic viruses were found in 5 of 15 (33.3%) children (including 2 cases detected only by UDPS). Rate of X4-tropic viruses was 0% (0/6) below 5 years (also as minority species), and became relatively high above 5 years (55.6% [5/9], P = .040. X4-tropic viruses were higher with CD4 ≤15% (4/9 [44.4%]) versus CD4 >15% (1/6 [16.7%], P = .580); similarly for CD4 ≤200 (3/4 [75%]) versus CD4 >200 (2/11 [18.2%] cells/mm, P = .077.NGS has the ability of excluding NRTI- and NNRTI-mutations as minority species in all but 1 children, thus supporting the safe use of these drug-classes in those without such mutations, henceforth sparing ritonavir-boosted protease inhibitors or integrase inhibitors for the few remaining cases. In children under five years, X4-tropic variants would be rare, suggesting vertical-transmission with CCR5-tropic viruses and possible maraviroc usage at younger ages.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Infecções por HIV/transmissão , HIV-1/genética , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Fármacos Anti-HIV/administração & dosagem , Contagem de Linfócito CD4 , Camarões , Criança , Pré-Escolar , Feminino , Infecções por HIV/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , RNA Viral , Tropismo Viral/genética
20.
J Virol ; 92(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29563289

RESUMO

In this study, we have characterized quasispecies dynamics and the evolution of viral tropism in naive HIV-1-infected patients treated with a short course of maraviroc monotherapy (ClinicalTrials.gov registration no. NCT01060618) independently of the tropism of the infecting virus. We randomly selected 20 patients infected with viruses displaying different basal tropisms-10 carrying R5 and 10 carrying dual/mixed X4 (DM/X4) viruses-at recruitment as determined by phenotypic assay (Trofile). Evolution of viral quasiespecies at the end of treatment was determined by ultradeep sequencing of the V3 region using a 454 Life Sciences Platform and geno2pheno (g2p) algorithm for viral tropism prediction. The false-positive rate (FPR) that defines the probability of classifying an R5 virus falsely as X4 was set at 10%. X4-specific HIV-1 viral load (VL) was calculated from sequences with an FPR of <3.75%. Virological response as defined as >1-log10 copies/ml reduction in VL was detected in 70% of patients independently of the basal tropism of the infecting virus. Viral tropism remained stable, and nonsignificant differences in FPR values before and after treatment were found for the majority of patients in both tropism groups. Only three patients (one with R5 and two with DM/X4 viruses) showed an increased (>1 log) X4 VL, and one patient harboring a DM/X4-tropic virus displayed a significant reduction in FPR values at the end of treatment. Fast changes in the composition of viral populations were observed in all patients after 10 days of maraviroc (MVC) monotherapy treatment, and a complete replacement of viral quasiespecies was found in 3/10 patients carrying R5-using viruses and 4/10 patients carrying DM/X4-using viruses.IMPORTANCE Initiation of treatment with maraviroc requires previous determination of viral tropism by genotypic or phenotypic methods because of the risk of treatment failure and selection of DM/X4-tropic variants. In this study, we confirm previous work showing that the virologic response to maraviroc is independent of basal tropism. By deep-sequencing analysis, we determined that fast changes in viral populations were due to the emergence of minority variants in some patients whereas in others generation of new strains was detected. The risk of DM/X4 selection was very low as FPR values remained stable, and only one patient showed a detrimental switch to DM/X4 variants. Our data show that some DM/X4 viruses are sensitive to maraviroc treatment probably because only a low proportion of DM/X4 viruses use preferentially the X4 receptor and contain authentically maraviroc-resistant viruses that are not accurately detected by current assays.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Cicloexanos/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , Triazóis/uso terapêutico , Tropismo Viral/genética , Adulto , Antagonistas dos Receptores CCR5/farmacologia , Feminino , Infecções por HIV/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Maraviroc , Pessoa de Meia-Idade , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Adulto Jovem
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