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2.
Nat Commun ; 12(1): 1383, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654063

RESUMO

In this study, we investigate the seroprevalence of SARS-CoV-2 antibodies among blood donors in the cities of Wuhan, Shenzhen, and Shijiazhuang in China. From January to April 2020, 38,144 healthy blood donors in the three cities were tested for total antibody against SARS-CoV-2 followed by pseudotype SARS-CoV-2 neutralization tests, IgG, and IgM antibody testing. Finally, a total of 398 donors were confirmed positive. The age- and sex-standardized SARS-CoV-2 seroprevalence among 18-60 year-old adults (18-65 year-old in Shenzhen) was 2.66% (95% CI: 2.24%-3.07%) in Wuhan, 0.033% (95% CI: 0.0029%-0.267%) in Shenzhen, and 0.0028% (95% CI: 0.0001%-0.158%) in Shijiazhuang, respectively. Female sex and older-age were identified to be independent risk factors for SARS-CoV-2 seropositivity among blood donors in Wuhan. As most of the population of China remained uninfected during the early wave of the COVID-19 pandemic, effective public health measures are still certainly required to block viral spread before a vaccine is widely available.


Assuntos
SARS-CoV-2/patogenicidade , Anticorpos Antivirais/sangue , Doadores de Sangue/estatística & dados numéricos , COVID-19/sangue , COVID-19/epidemiologia , COVID-19/imunologia , China/epidemiologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Testes de Neutralização , Prevalência , Fatores de Risco , SARS-CoV-2/imunologia
3.
Small Methods ; 5(2): 2001031, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33614907

RESUMO

The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed. In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

4.
Emerg Microbes Infect ; 9(1): 2105-2113, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32893735

RESUMO

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
5.
Eur Respir J ; 56(2)2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32430429

RESUMO

BACKGROUND: Timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a prerequisite for treatment and prevention. The serology characteristics and complement diagnosis value of the antibody test to RNA test need to be demonstrated. METHOD: Serial sera of 80 patients with PCR-confirmed coronavirus disease 2019 (COVID-19) were collected at the First Affiliated Hospital of Zhejiang University, Hangzhou, China. Total antibody (Ab), IgM and IgG antibodies against SARS-CoV-2 were detected, and the antibody dynamics during the infection were described. RESULTS: The seroconversion rates for Ab, IgM and IgG were 98.8%, 93.8% and 93.8%, respectively. The first detectible serology marker was Ab, followed by IgM and IgG, with a median seroconversion time of 15, 18 and 20 days post exposure (d.p.e.) or 9, 10 and 12 days post onset (d.p.o.), respectively. The antibody levels increased rapidly beginning at 6 d.p.o. and were accompanied by a decline in viral load. For patients in the early stage of illness (0-7 d.p.o), Ab showed the highest sensitivity (64.1%) compared with IgM and IgG (33.3% for both; p<0.001). The sensitivities of Ab, IgM and IgG increased to 100%, 96.7% and 93.3%, respectively, 2 weeks later. When the same antibody type was detected, no significant difference was observed between enzyme-linked immunosorbent assays and other forms of immunoassays. CONCLUSIONS: A typical acute antibody response is induced during SARS-CoV-2 infection. Serology testing provides an important complement to RNA testing in the later stages of illness for pathogenic-specific diagnosis and helpful information to evaluate the adapted immunity status of patients.


Assuntos
Betacoronavirus , Técnicas de Laboratório Clínico , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Adulto , Idoso , COVID-19 , Teste para COVID-19 , China , Infecções por Coronavirus/complicações , Feminino , Hospitalização , Humanos , Período de Incubação de Doenças Infecciosas , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/complicações , SARS-CoV-2 , Sensibilidade e Especificidade , Soroconversão , Avaliação de Sintomas , Fatores de Tempo , Carga Viral
6.
Cell Host Microbe ; 27(2): 249-261.e5, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32027857

RESUMO

Hand, foot, and mouth disease is a common childhood illness primarily caused by coxsackievirus A16 (CVA16), for which there are no current vaccines or treatments. We identify three CVA16-specific neutralizing monoclonal antibodies (nAbs) with therapeutic potential: 18A7, 14B10, and NA9D7. We present atomic structures of these nAbs bound to all three viral particle forms-the mature virion, A-particle, and empty particle-and show that each Fab can simultaneously occupy the mature virion. Additionally, 14B10 or NA9D7 provide 100% protection against lethal CVA16 infection in a neonatal mouse model. 18A7 binds to a non-conserved epitope present in all three particles, whereas 14B10 and NA9D7 recognize broad protective epitopes but only bind the mature virion. NA9D7 targets an immunodominant site, which may overlap the receptor-binding site. These findings indicate that CVA16 vaccines should be based on mature virions and that these antibodies could be used to discriminate optimal virion-based immunogens.


Assuntos
Anticorpos Neutralizantes , Enterovirus Humano A/imunologia , Doença de Mão, Pé e Boca/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/ultraestrutura , Proteínas do Capsídeo/imunologia , Linhagem Celular , Microscopia Crioeletrônica , Enterovirus/imunologia , Enterovirus/ultraestrutura , Enterovirus Humano A/ultraestrutura , Doença de Mão, Pé e Boca/imunologia , Doença de Mão, Pé e Boca/prevenção & controle , Humanos , Camundongos , Vacinas Virais/imunologia , Vírion/imunologia
7.
ACS Infect Dis ; 6(5): 811-819, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31840495

RESUMO

Zika virus (ZIKV) is a mosquito-borne flavivirus that has been associated with neuropathology in fetuses and adults, imposing a serious health concern. Therefore, the development of a vaccine is a global health priority. Notably, neutralization tests have a significant value for vaccine development and virus diagnosis. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) is a common neutralization method for ZIKV. However, this method has some drawbacks, such as being time-consuming and labor-intensive and having low-throughput, which precludes its application in the detection of large numbers of specimens. To improve this problem, we developed a neutralization test based on an enzyme-linked immunospot assay (Nt-ELISPOT) for ZIKV and performed the assay in a 96-well format. A monoclonal antibody (mAb), 11C11, with high affinity and reactivity to ZIKV was used to detect ZIKV-infected cells. To optimize this method, the infectious dose of ZIKV was set at a multiplicity of infection (MOI) of 0.0625, and a detection experiment was performed after incubating for 24 h. As a result, under these conditions, the Nt-ELISPOT had good consistency with the traditional Nt-CPE to measure neutralizing titers of sera and neutralizing antibodies. Additionally, three neutralizing antibodies against ZIKV were screened by this method. Overall, we successfully developed an efficient neutralization test for ZIKV that is high-throughput and rapid. This Nt-ELISPOT can potentially be applied to detecting neutralizing titers of large numbers of specimens in vaccine evaluation and neutralizing antibody screening for ZIKV.


Assuntos
Anticorpos Antivirais , ELISPOT , Testes de Neutralização , Infecção por Zika virus , Zika virus , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Zika virus/imunologia , Infecção por Zika virus/diagnóstico
8.
J Biol Chem ; 294(49): 18742-18755, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666336

RESUMO

The centrosome is a cytoplasmic nonenveloped organelle functioning as one of the microtubule-organizing centers and composing a centriole center surrounded by pericentriolar material (PCM) granules. PCM consists of many centrosomal proteins, including PCM1 and centrosomal protein 131 (CEP131), and helps maintain centrosome stability. Zika virus (ZIKV) is a flavivirus of the family Flaviviridae whose RNA and viral particles are replicated in the cytoplasm. However, how ZIKV interacts with host cell components during its productive infection stage is incompletely understood. Here, using several primate cell lines, we report that ZIKV infection disrupts and disperses the PCM granules. We demonstrate that PCM1- and CEP131-containing granules are dispersed in ZIKV-infected cells, whereas the centrioles remain intact. We found that ZIKV does not significantly alter cellular skeletal proteins, and, hence, these proteins may not be involved in the interaction between ZIKV and centrosomal proteins. Moreover, ZIKV infection decreased PCM1 and CEP131 protein, but not mRNA, levels. We further found that the protease inhibitor MG132 prevents the decrease in PCM1 and CEP131 levels and centriolar satellite dispersion. Therefore, we hypothesized that ZIKV infection induces proteasomal PCM1 and CEP131 degradation and thereby disrupts the PCM granules. Supporting this hypothesis, we show that ZIKV infection increases levels of mind bomb 1 (MIB1), previously demonstrated to be an E3 ubiquitin ligase for PCM1 and CEP131 and that ZIKV fails to degrade or disperse PCM in MIB1-ko cells. Our results imply that ZIKV infection activates MIB1-mediated ubiquitination that degrades PCM1 and CEP131, leading to PCM granule dispersion.


Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Infecção por Zika virus/metabolismo , Animais , Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células HEK293 , Humanos , Immunoblotting , Ubiquitina-Proteína Ligases/genética , Células Vero , Zika virus , Infecção por Zika virus/genética
9.
Oncotarget ; 10(35): 3209-3226, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31143369

RESUMO

Due to the small number of molecular targets in viruses and the rapid evolution of viral genes, it is very challenging to develop specific antiviral drugs. Viruses require host factors to translate their transcripts, and targeting the host factor(s) offers a unique opportunity to develop broad antiviral drugs. It is well documented that some viruses utilize a host protein, Receptor for Activated C Kinase 1 (RACK1), to translate their mRNAs using a viral mRNA secondary structure known as the Internal Ribosomal Entry Site (IRES). RACK1 is essential for the translation of many viruses including hepatitis C (HCV), polio, Drosophila C (DCV), Dengue, Cricket Paralysis (CrpV), and vaccinia viruses. In addition, HIV-1 and Herpes Simplex virus (HSV-1) are known to use IRES as well. Therefore, host RACK1 protein is an attractive target for developing broad antiviral drugs. Depletion of the host's RACK1 will potentially inhibit virus replication. This background study has led us to the development of novel antiviral therapeutics, such as RACK1 inhibitors. By utilizing the crystal structure of the RACK1A protein from the model plant Arabidopsis and using a structure based drug design method, dozens of small compounds were identified that could potentially bind to the experimentally determined functional site of the RACK1A protein. The SPR assays showed that the small compounds bound strongly to recombinant RACK1A protein. Here we provide evidence that the drugs show high efficacy in inhibition of HSV-1 proliferation in a HEp-2 cell line. The drug showed similar efficacy as the available anti-herpes drug acyclovir and showed supralinear effect when applied in a combinatorial manner. As an increasing number of viruses are reported to use host RACK1 proteins, and more than 100 diverse animals and plant disease-causing viruses are known to use IRES-based translation, these drugs can be established as host-targeted broad antiviral drugs.

10.
Gut ; 68(11): 2044-2056, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30700543

RESUMO

OBJECTIVE: Developing a small animal model that accurately delineates the natural history of hepatitis B virus (HBV) infection and immunopathophysiology is necessary to clarify the mechanisms of host-virus interactions and to identify intervention strategies for HBV-related liver diseases. This study aimed to develop an HBV-induced chronic hepatitis and cirrhosis mouse model through transplantation of human bone marrow mesenchymal stem cells (hBMSCs). DESIGN: Transplantation of hBMSCs into Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice with fulminant hepatic failure (FHF) induced by hamster-anti-mouse CD95 antibody JO2 generated a liver and immune cell dual-humanised (hBMSC-FRGS) mouse. The generated hBMSC-FRGS mice were subjected to assessments of sustained viremia, specific immune and inflammatory responses and liver pathophysiological injury to characterise the progression of chronic hepatitis and cirrhosis after HBV infection. RESULTS: The implantation of hBMSCs rescued FHF mice, as demonstrated by robust proliferation and transdifferentiation of functional human hepatocytes and multiple immune cell lineages, including B cells, T cells, natural killer cells, dendritic cells and macrophages. After HBV infection, the hBMSC-FRGS mice developed sustained viremia and specific immune and inflammatory responses and showed progression to chronic hepatitis and liver cirrhosis at a frequency of 55% after 54 weeks. CONCLUSION: This new humanised mouse model recapitulates the liver cirrhosis induced by human HBV infection, thus providing research opportunities for understanding viral immune pathophysiology and testing antiviral therapies in vivo.


Assuntos
Modelos Animais de Doenças , Hepatite B Crônica/etiologia , Cirrose Hepática/etiologia , Transplante de Células-Tronco Mesenquimais , Animais , Hepatite B Crônica/patologia , Humanos , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID
11.
Appl Microbiol Biotechnol ; 103(4): 1931-1938, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30617817

RESUMO

Coxsackievirus A10 (CVA10) recently has become one of the major pathogens of hand, foot, and mouth disease (HFMD) in children worldwide, but no cure or vaccine against CVA10 is available yet. Serological evaluation of herd immunity to CVA10 will promote the development of vaccine. The traditional neutralization assay based on inhibition of cytopathic effect (Nt-CPE) is a common method for measuring neutralizing antibody titer against CVA10, which is time-consuming and labor-intensive. In this study, an efficient neutralization test based on a monoclonal antibody (mAb) 3D1 against CVA10, called Elispot-based neutralization test (Nt-Elispot), was developed. In the Nt-Elispot, the mAb 3D1 labeled with horseradish peroxidase (HRP) was used to detect the CVA10-infected RD cells at a 1:4000 dilution and the optimal infectious dose of CVA10 was set at 105 TCID50/well when combined with a fixed incubation time of 14 h. Compared with the Nt-CPE, the Nt-Elispot method effectively shortened the detection period and presented a good correlativity with it. Using the Nt-Elispot, a total of 123 sera from healthy children were tested for neutralizing antibody against CVA10, demonstrating that the overall seroprevalence was 49.3% (54/123) and the geometric mean titer (GMT) had been calculated as 574.2. Furthermore, 2 anti-CVA10 neutralizing mAbs were obtained by screening via the Nt-Elispot. Overall, the established Nt-Elispot could be used as an efficient and high-throughput method for evaluating immunity to CVA10 and screening the neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Enterovirus/imunologia , Doença de Mão, Pé e Boca/imunologia , Testes de Neutralização/métodos , Pré-Escolar , Ensaios de Triagem em Larga Escala/métodos , Humanos , Lactente , Estudos Soroepidemiológicos
12.
Nat Microbiol ; 4(1): 124-133, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30397341

RESUMO

Enterovirus D68 (EV-D68) undergoes structural transformation between mature, cell-entry intermediate (A-particle) and empty forms throughout its life cycle. Structural information for the various forms and antibody-bound capsids will facilitate the development of effective vaccines and therapeutics against EV-D68 infection, which causes childhood respiratory and paralytic diseases worldwide. Here, we report the structures of three EV-D68 capsid states representing the virus at major phases. We further describe two original monoclonal antibodies (15C5 and 11G1) with distinct structurally defined mechanisms for virus neutralization. 15C5 and 11G1 engage the capsid loci at icosahedral three-fold and five-fold axes, respectively. To block viral attachment, 15C5 binds three forms of capsids, and triggers mature virions to transform into A-particles, mimicking engagement by the functional receptor ICAM-5, whereas 11G1 exclusively recognizes the A-particle. Our data provide a structural and molecular explanation for the transition of picornavirus capsid conformations and demonstrate distinct mechanisms for antibody-mediated neutralization.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Complexo Antígeno-Anticorpo/ultraestrutura , Capsídeo/imunologia , Enterovirus/imunologia , Animais , Anticorpos Monoclonais/ultraestrutura , Complexo Antígeno-Anticorpo/imunologia , Capsídeo/ultraestrutura , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/metabolismo
13.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30258013

RESUMO

To countermeasure the host cellular intrinsic defense, cytomegalovirus (CMV) and herpes simplex viruses (HSV) have evolved the ability to disperse nuclear domain 10 (ND10, aka PML body). However, mechanisms underlying their action on ND10 differ. HSV infection produces ICP0, which degrades the ND10-forming protein PML. Human CMV (HCMV) infection expresses IE1 that deSUMOylates PML to result in dispersion of ND10. It has been demonstrated that HSV ICP0 degraded only the SUMOylated PML, so we hypothesized that HCMV IE1 can protect PML from degradation by ICP0. HCMV IE1-expressing cell lines (U-251 MG-IE1 and HELF-IE1) were used for infection of HSV-1 or transfection of ICP0-expressing plasmid. Multilabeling by immunocytochemistry assay and protein examination by Western blot assay were performed to determine the resultant fate of PML caused by ICP0 in the presence or absence of HCMV IE1. Here, we report that deSUMOylation of human PML (hPML) by HCMV IE1 was incomplete, as mono-SUMOylated PML remained in the IE1-expressing cells, which is consistent with the report by E. M. Schilling, M. Scherer, N. Reuter, J. Schweininger, et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). As expected, we found that IE1 protected PML from degradation by ICP0 or HSV-1 infection. An in vitro study found that IE1 with mutation of L174P failed to deSUMOylate PML and did not protect PML from degradation by ICP0; hence, we conclude that the deSUMOylation of PML is important for IE1 to protect PML from degradation by ICP0. In addition, we revealed that murine CMV failed to deSUMOylate and to protect the HSV-mediated degradation of hPML, and that HCMV failed to deSUMOylate and protect the HSV-mediated degradation of mouse PML. However, IE1-expressing cells did not enhance wild-type HSV-1 replication but significantly increased ICP0-defective HSV-1 replication at a low multiplicity of infection. Therefore, our results uncovered a host-virus functional interaction at the posttranslational level.IMPORTANCE Our finding that HCMV IE1 protected hPML from degradation by HSV ICP0 is important, because the PML body (aka ND10) is believed to be the first line of host intrinsic defense against herpesviral infection. How the infected viruses overcome the nuclear defensive structure (PML body) has not been fully understood. Herpesviral proteins, ICP0 of HSV and IE1 of CMV, have been identified to interact with PML. Here, we report that HCMV IE1 incompletely deSUMOylated PML, resulting in the mono-SUMOylated PML, which is consistent with the report of Schilling et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). The mono-SUMOylated PML was subjected to degradation by HSV ICP0. However, it was protected by IE1 from degradation by ICP0 or HSV-1 infection. In contrast, IE1 with L174P mutation lost the function of deSUMOylating PML and failed to protect the degradation of the mono-SUMOylated PML. Whether the mono-SUMOylated PML has any defensive function against viral infection will be further investigated.


Assuntos
Infecções por Citomegalovirus/metabolismo , Herpes Simples/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Proteólise , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos , Proteína da Leucemia Promielocítica/química , Proteína da Leucemia Promielocítica/genética , Ubiquitina-Proteína Ligases/genética , Replicação Viral
14.
Emerg Microbes Infect ; 7(1): 144, 2018 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-30097574

RESUMO

The human liver chimeric mouse with primary human hepatocytes (PHHs) engraftment has been demonstrated to be a useful animal model to study hepatitis B virus (HBV) pathogenesis and evaluate anti-HBV drugs. However, the disadvantages of using PHHs include the inability for cellular expansion in vitro, limited donor availability, individual differences, and ethical issues, necessitating the development of alternatives. To obtain in vitro expandable hepatocytes, we optimized the hepatic differentiation procedure of the human liver progenitor cell line, HepaRG, using four functional small molecules (4SM) and enriched the precursor hepatocyte-like cells (HLCs). HepaRG cells of different hepatic differentiation states were engrafted to immunodeficient mice (FRGS) with weekly 4SM treatment. The HepaRG-engrafted mice were challenged with HBV and/or treated with several antivirals to evaluate their effects. We demonstrated that the 4SM treatment enhanced hepatic differentiation and promoted cell proliferation capacity both in vitro and in vivo. Mice engrafted with enriched HepaRG of prehepatic differentiation and treated with 4SM displayed approximately 10% liver chimerism at week 8 after engraftment and were maintained at this level for another 16 weeks. Therefore, we developed a HepaRG-based human liver chimeric mouse model: HepaRG-FRGS. Our experimental results showed that the liver chimerism of the mice was adequate to support chronic HBV infection for 24 weeks and to evaluate antivirals. We also demonstrated that HBV infection in HepaRG cells was dependent on their hepatic differentiation state and liver chimerism in vivo. Overall, HepaRG-FRGS mice provide a novel human liver chimeric mouse model to study chronic HBV infection and evaluate anti-HBV drugs.


Assuntos
Quimera/virologia , Modelos Animais de Doenças , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Hepatócitos/virologia , Fígado/virologia , Animais , Diferenciação Celular , Linhagem Celular , Quimera/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/fisiopatologia , Hepatócitos/citologia , Humanos , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Replicação Viral
15.
Front Microbiol ; 9: 908, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867819

RESUMO

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/-SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs. Highlights:    (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver.    (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle.    (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo. Research in Context  To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses.

16.
Viruses ; 10(1)2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29361773

RESUMO

Zika virus (ZIKV) has been defined as a teratogenic pathogen behind the increased number of cases of microcephaly in French Polynesia, Brazil, Puerto Rico, and other South American countries. Experimental studies using animal models have achieved tremendous insight into understanding the viral pathogenesis, transmission, teratogenic mechanisms, and virus-host interactions. However, the animals used in published investigations are mostly interferon (IFN)-compromised, either genetically or via antibody treatment. Herein, we studied ZIKV infection in IFN-competent mice using African (MR766) and Asian strains (PRVABC59 and SZ-WIV01). After testing four different species of mice, we found that BALB/c neonatal mice were resistant to ZIKV infection, that Kunming, ICR and C57BL/6 neonatal mice were fatally susceptible to ZIKV infection, and that the fatality of C57BL/6 neonates from 1 to 3 days old were in a viral dose-dependent manner. The size and weight of the brain were significantly reduced, and the ZIKV-infected mice showed neuronal symptoms such as hind-limb paralysis, tremor, and poor balance during walking. Pathologic and immunofluorescent experiments revealed that ZIKV infected different areas of the central nervous system (CNS) including gray matter, hippocampus, cerebral cortex, and spinal cord, but not olfactory bulb. Interestingly, ZIKV replicated in multiple organs and resulted in pathogenesis in liver and testis, implying that ZIKV infection may engender a high health risk in neonates by postnatal infection. In summary, we investigated ZIKV pathogenesis using an animal model that is not IFN-compromised.


Assuntos
Sistema Nervoso Central/virologia , Imunocompetência , Replicação Viral , Infecção por Zika virus/fisiopatologia , Zika virus/patogenicidade , Animais , Animais Recém-Nascidos/virologia , Encéfalo/patologia , Encéfalo/virologia , Sistema Nervoso Central/patologia , Córtex Cerebral/patologia , Córtex Cerebral/virologia , Modelos Animais de Doenças , Hipocampo/patologia , Hipocampo/virologia , Fígado/patologia , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Bulbo Olfatório , Paralisia , Testículo/patologia , Testículo/virologia , Zika virus/fisiologia
17.
Nat Commun ; 8(1): 505, 2017 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-28894095

RESUMO

Coxsackievirus A6 (CVA6) has recently emerged as a major cause of hand, foot and mouth disease in children worldwide but no vaccine is available against CVA6 infections. Here, we demonstrate the isolation of two forms of stable CVA6 particles-procapsid and A-particle-with excellent biochemical stability and natural antigenicity to serve as vaccine candidates. Despite the presence (in A-particle) or absence (in procapsid) of capsid-RNA interactions, the two CVA6 particles have essentially identical atomic capsid structures resembling the uncoating intermediates of other enteroviruses. Our near-atomic resolution structure of CVA6 A-particle complexed with a neutralizing antibody maps an immune-dominant neutralizing epitope to the surface loops of VP1. The structure-guided cell-based inhibition studies further demonstrate that these loops could serve as excellent targets for designing anti-CVA6 vaccines.Coxsackievirus A6 (CVA6) causes hand, foot and mouth disease in children. Here the authors present the CVA6 procapsid and A-particle cryo-EM structures and identify an immune-dominant neutralizing epitope, which can be exploited for vaccine development.


Assuntos
Anticorpos Neutralizantes/química , Microscopia Crioeletrônica/métodos , Enterovirus Humano A/química , Animais , Capsídeo/química , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Epitopos , Humanos , Imageamento Tridimensional , Camundongos , Proteínas Virais/química , Proteínas Virais/metabolismo , Vacinas Virais/química , Vacinas Virais/imunologia
18.
Gene ; 628: 117-128, 2017 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-28720531

RESUMO

Zika virus (ZIKV) encodes a precursor protein (also called polyprotein) of about 3424 amino acids that is processed by proteases to generate 10 mature proteins and a small peptide. In the present study, we characterized the chemical features, suborganelle distribution and potential function of each protein using Flag-tagged protein expression system. Western blot analysis revealed the molecular weight of the proteins and the polymerization of E, NS1, and NS3 proteins. In addition, we performed multi-labeled fluorescent immunocytochemistry and subcellular fractionation to determine the subcellular localization of these proteins in host cells. We found that 1) the capsid protein colocalizes with 3 different cellular organelles: nucleoli, Golgi apparatus, and lipid droplet; NS2b and NS4a are associated with the Golgi apparatus; 2) the capsid and NS1proteins distribute in both cytoplasm and nucleus, NS5 is a nuclear protein; 3) NS3 protein colocalizes with tubulin and affects Lamin A; 4) Envelope, PrM, and NS2a proteins co-localize with the endoplasmic reticulum; 5) NS1 is associated with autophagosomes and NS4b is related to early endosome; 6) NS5 forms punctate structures in the nucleus that associate with splicing compartments shown by SC35, leading to reduction of SC35 protein level and trafficking of SC35 from the nucleus to the cytoplasm. These data suggest that ZIKV generates 10 functional viral proteins that exhibit distinctive subcellular distribution in host cells.


Assuntos
Proteínas Virais/genética , Zika virus/genética , Animais , Autofagia , Nucléolo Celular/virologia , Núcleo Celular/virologia , Chlorocebus aethiops , Clonagem Molecular , Citoplasma/virologia , Retículo Endoplasmático/virologia , Endossomos/virologia , Genes Virais , Vetores Genéticos , Complexo de Golgi/virologia , Células HEK293 , Humanos , Lamina Tipo A/metabolismo , Transfecção , Tubulina (Proteína)/metabolismo , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/genética
19.
Antiviral Res ; 144: 247-255, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28625478

RESUMO

Epidemiological data indicate that coxsackievirus A10 (CVA10) has become one of the main causative agents of hand, foot and mouth disease (HFMD) and in recent years has often been found to co-circulate with other enteroviruses, which poses a challenge for the prevention and control of HFMD. Although most CVA10-associated HFMD cases present mild symptoms, severe manifestations and even death can also occur. However, the study of the pathogenesis and the development of drugs and vaccines for CVA10 infection are still far from complete. In this study, we established a neonatal mouse model for anti-viral evaluation and characterized the pathology of CVA10 infection. To develop the mouse model, both inbred and outbred mouse strains were used to compare their sensitivity to CVA10 infection; then, one-day-old BALB/c mice were selected and inoculated intraperitoneally with a CVA10 clinical strain, CVA10-FJ-01. Clinical symptoms, such as wasting, hind-limb paralysis and even death were observed in the CVA10-infected mice. Moreover, pathological examination and immunohistochemistry staining showed that severe myonecrosis with inflammatory infiltration was observed in CVA10-infected mice, indicating that CVA10 exhibited strong tropism to muscle tissue. Using real-time PCR, we also found that the viral load in the blood and muscle was higher than that in other organs/tissues at different time points post-infection, suggesting that CVA10 had a strong tropism to mice muscle and that viremic spread may also contribute to the death of the CVA10-infected mice. Additionally, to evaluate the neonatal mouse model of CVA10 infection, female mice were immunized with formalin-inactivated CVA10 and then allowed to mate after the third immunization. The results showed that maternal antibodies could protect mice against CVA10 infection. In summary, the results demonstrated that the neonatal mice model was a useful tool for evaluating the protective effects of CVA10 vaccines and anti-viral reagents.


Assuntos
Antivirais/administração & dosagem , Infecções por Coxsackievirus/tratamento farmacológico , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Enterovirus/patogenicidade , Animais , Animais Recém-Nascidos , Sangue/virologia , Infecções por Coxsackievirus/virologia , Camundongos Endogâmicos BALB C , Miosite/patologia , Miosite/virologia , Necrose/patologia , Carga Viral , Tropismo Viral
20.
BMC Infect Dis ; 17(1): 239, 2017 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-28359304

RESUMO

BACKGROUND: Two lineages of Zika virus (ZIKV) have been classified according to the phylogenetic analysis: African and Asian lineages. It is unclear whether differences exist between the two strains in host cell permissiveness, this information is important for understanding viral pathogenesis and designing anti-viral strategies. METHODS: In the present study, we comparatively studied the permissive spectrum of human cells for both the African (MR766) and Asian strains (PRVABC59) using an RNA in situ hybridization (RISH) to visualize RNA replication, an immunofluorescence technology, and a western blot assay to determine viral protein production, and a real-time RT-PCR to examine viral RNA multiplication level. The experiments were undertaken in the condition of cell culture. RESULTS: We identified several human cell lines, including fibroblast, epithelial cells, brain cells, stem cells, and blood cells that are susceptible for the infection of both Asian and African strains. We did not find any differences between the MR766 and the PRVABC59 in the permissiveness, infection rate, and replication modes. Inconsistent to a previous report (Hamel et al. JVI 89:8880-8896, 2015), using RISH or real-time RT-PCR, we found that human foreskin fibroblast cells were not permissive for ZIKV infection. Instead, human lung fibroblast cells (MRC-5) were fully permissive for ZIKV infection. Surprisingly, a direct interaction of ZIKV RNA with envelop (E) protein (a structure protein) was demonstrated by an RNA chromatin immunoprecipitation (ChIP) assay. Three binding sites were identified in the ZIKV RNA genome for the interaction with the E protein. CONCLUSION: Our results imply that the E protein may be important for viral RNA replication, and provide not only the information of ZIKV permissiveness that guides the usage of human cells for the ZIKV studies, but also the insight into the viral RNA-E protein interaction that may be targeted for intervention by designing small molecule drugs.


Assuntos
RNA Viral , Proteínas do Envelope Viral/genética , Zika virus/genética , Zika virus/patogenicidade , África , Ásia , Western Blotting , Linhagem Celular , Marcadores Genéticos , Humanos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/genética
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