RESUMO
Hepatitis E virus (HEV) causes self-limited acute hepatitis in immunocompetent individuals and can establish chronic infection in solid organ transplant recipients taking immunosuppressive drugs. A well characterized small animal model is needed to understand HEV pathogenesis. In this study, we established a robust model to study acute and persistent HEV infection using Mongolian gerbils (Meriones unguiculatus) with or without immunosuppression. Gerbils were implanted subcutaneously with continuous release tacrolimus pellet to induce immunosuppression. Gerbils with or without tacrolimus treatment were inoculated with HEV intraperitoneally. Viremia, fecal virus shedding, serum antibody and ALT levels, liver histopathological lesions, hepatocyte apoptosis, and liver macrophage distribution were assessed. Mild to moderate self-limited hepatitis and IgM and IgG antibody responses against HEV ORF2 were observed in immunocompetent gerbils. Levels of HEV-specific IgM responses were higher and lasted longer in immunocompetent gerbils with higher peak viremia. Persistent viremia and fecal virus shedding with either weak, or absent HEV antibody levels were seen in immunosuppressed gerbils. Following HEV infection, serum ALT levels were increased, with lower and delayed peaks observed in immunosuppressed compared to immunocompetent gerbils. In immunocompetent gerbils, foci of apoptotic hepatocytes were detected that were distributed with inflammatory infiltrates containing CD68+ macrophages. However, these foci were absent in immunosuppressed gerbils. The immunosuppressed gerbils showed no inflammation with no increase in CD68+ macrophages despite high virus replication in liver. Our findings suggest adaptive immune responses are necessary for inducing hepatocyte apoptosis, CD68+ macrophage recruitment, and inflammatory cell infiltration in response to HEV infection. Our studies show that Mongolian gerbils provide a promising model to study pathogenesis during acute and persistent HEV infection.
Assuntos
Vírus da Hepatite E , Hepatite E , Animais , Humanos , Vírus da Hepatite E/genética , Gerbillinae , Tacrolimo , Viremia , GenótipoRESUMO
Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 (CHI3L1), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest age-dependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression.
Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Adolescente , Adulto , Envelhecimento/fisiologia , Biomarcadores/metabolismo , Proteína 1 Semelhante à Quitinase-3/fisiologia , Quitinases/metabolismo , Feminino , Expressão Gênica/genética , Hepacivirus/patogenicidade , Células Estreladas do Fígado/patologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fígado/citologia , MasculinoRESUMO
BACKGROUND: The emergence of Zika virus (ZIKV) in 2015 to 2016 created a global public health crisis and an urgent need for accurate detection assays. Nucleic acid testing (NAT) is the most specific and sensitive technology for early detection of ZIKV. Various NAT protocols have been created, but until recently, assessment of assay performance and comparative studies were hampered by the lack of available standards and reference reagents. STUDY DESIGN AND METHODS: The Center for Biologics Evaluation and Research/Food and Drug Administration responded to this crisis with the generation of two ZIKV-RNA reference reagents (ZIKV-RRs) for use in the development, validation, and assessment of performance of ZIKV-NAT assays. These reagents were produced from heat-inactivated (HI) ZIKV culture supernatant stock from two strains (PRVABC59 and FSS13025) diluted in dialyzed, defibrinated human plasma and lyophilized for evaluation in collaborative studies. The liquid, HI stock had been shared with the Paul-Ehrlich-Institute (Germany) and were included in the collaborative validation studies for the World Health Organization International Standard for ZIKV (WHO ZIKV IS). RESULTS: NAT-detectable units (NDUs)/mL were determined in a collaborative study that led to the assignment of 5.77 log NDUs/mL for PRVABC59 and 5.54 log NDUs/mL for FSS13025 as the final concentrations of the FDA ZIKV-RRs. CONCLUSION: We have established well-characterized reference reagents for ZIKV to facilitate evaluation of existing NAT assays and development of novel ZIKV assays which are correlated to that of the First WHO ZIKV IS. Vials of the ZIKV-RRs are available to qualified organizations upon request.
Assuntos
Surtos de Doenças , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/normas , Infecção por Zika virus/epidemiologia , Zika virus/genética , Animais , Chlorocebus aethiops , Criopreservação , Liofilização , Humanos , Indicadores e Reagentes , Técnicas de Amplificação de Ácido Nucleico/métodos , Saúde Pública , Estabilidade de RNA , RNA Viral/sangue , RNA Viral/isolamento & purificação , Padrões de Referência , Sensibilidade e Especificidade , Células Vero , Viremia/sangue , Viremia/diagnóstico , Inativação de Vírus , Organização Mundial da Saúde , Zika virus/isolamento & purificação , Infecção por Zika virus/sangue , Infecção por Zika virus/diagnósticoRESUMO
Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1-4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin-streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2-3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.
Assuntos
Vírus da Hepatite E , Hepatite E , Animais , Humanos , Vírus da Hepatite E/genética , Gerbillinae , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Anti-Hepatite , RNA/metabolismoRESUMO
BACKGROUND: To harmonize assays for detection of HEV RNA, a World Health Organization International Standard (WHO IS) was established. The WHO IS represents the highest order standard for HEV RNA but is limited in quantity. Secondary standards are needed to limit the use of WHO IS and minimize the need to replace it. OBJECTIVE: Establish secondary standards for HEV NAAT assays and to calibrate these against the WHO IS. METHODS: Stocks of genotype 3 HEV were prepared using both cell lysates and cell culture supernatants to produce non-enveloped and quasi-enveloped virus stocks, respectively. Both stocks were heat-inactivated, diluted in negative human plasma, and lyophilized to produce two candidate secondary standards: HEV-RR (non-enveloped virus) and HEV-RR.1 (quasi-enveloped virus). Both candidate standards were characterized and calibrated against the WHO IS for HEV RNA in an international collaborative study. RESULTS: The collaborative study returned a total of 15 data sets, with different RNA extraction and amplification methods. The estimated mean values relative to the WHO IS (250,000 IU/ml) are 229,000 IU/ml and 355,000 IU/ml for HEV-RR and HEV-RR.1, respectively. CONCLUSION: We have established two secondary standards for HEV RNA calibrated against the WHO IS. These standards are non-infectious and stable under different storage temperatures.
Assuntos
Vírus da Hepatite E , Humanos , Vírus da Hepatite E/genética , RNA Viral/genética , Cooperação Internacional , Padrões de Referência , TecnologiaRESUMO
Zika virus (ZIKV) caused a public health threat in the United States in 2016, leading to rapid development and implementation of blood screening assays for ZIKV RNA. Several ZIKV sequences from clinical cases have been reported, but none from asymptomatic/pre-symptomatic infections. We isolated and sequenced ZIKV from asymptomatic/pre-symptomatic blood donor (ABD-ZIKV) samples and compared with reported clinical sequences. Twelve ABD-ZIKV isolates were produced from 67 cultivated samples, and isolates were genetically similar among themselves. Most isolates shared mutations with the clinical isolate PRVABC59 2015, whereas two ABD-ZIKV isolates shared specific mutations with U.S. clinical isolates from 2016. The ABD-ZIKV strains clustered into two distinct subclades: one comprised mostly ABD-ZIKV from Puerto Rico, and another one comprised ABD-ZIKV from Florida and QTX-02 isolate (Puerto Rico). In this study, we showed the circulation of two slightly distinct virus strains among Puerto Rico blood donors, one of which was also reported in Florida.
Assuntos
Doadores de Sangue , Filogenia , Infecção por Zika virus/sangue , Zika virus/genética , Zika virus/isolamento & purificação , Adulto , Doenças Transmissíveis Emergentes/sangue , Doenças Transmissíveis Emergentes/epidemiologia , Florida/epidemiologia , Genômica , Humanos , Porto Rico/epidemiologia , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/epidemiologiaRESUMO
Infections with dengue virus (DENV), West Nile virus (WNV) and Zika virus (ZIKV) usually present similar mild symptoms at early stages, and most infections (~80%) are asymptomatic. However, these infections may progress to severe disease with different clinical manifestations. In this study we attempted to identify unique characteristics for each infection at the presymptomatic/asymptomatic stage of infection and compared levels of soluble immune markers that have been shown to be altered during clinical course of these viral infections. Levels of soluble markers were determined by Luminex-based assays or by ELISA in plasma samples from asymptomatic blood donors who were reactive for RNA from DENV (n = 71), WNV (n = 52) or ZIKV (n = 44), and a control or non-infected (NI) group (n = 22). Results showed that even in the absence of symptoms, increased interleukin (IL) levels of IL-12, IL-17, IL-10, IL-5, CXCL9, E-Selectin and ST2/IL-1R4; and decreased levels of IL-13 and CD40 were found in all flavivirus group samples, compared to those from NI donors. DENV-infected donors demonstrated variation in expression of IL-1ra and IL-2; WNV-infected donors demonstrated variation in expression of IL-1ra, P-Selectin, IL-4 and IL-5; ZIKV-infected donors demonstrated variation in expression of IL-1ra, P-Selectin, IL-4, RANK-L, CD40L and C3a. The findings suggest that, even in the presymptomatic/asymptomatic phase of the infection, different immunomodulation profiles were associated with DENV, WNV and ZIKV infections.
Assuntos
Biomarcadores/sangue , Vírus da Dengue/imunologia , Dengue/sangue , Febre do Nilo Ocidental/sangue , Vírus do Nilo Ocidental/imunologia , Infecção por Zika virus/sangue , Zika virus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções Assintomáticas , Reações Cruzadas/imunologia , Dengue/imunologia , Humanos , Febre do Nilo Ocidental/imunologia , Infecção por Zika virus/imunologiaRESUMO
In 2015, Zika virus (ZIKV) appeared as an emerging pathogen, generating a global and urgent need for accurate diagnostic devices. During this public health crisis, several nucleic acid testing (NAT)-based Zika assays were submitted to the US Food and Drug Administration (FDA) for Emergency Use Authorization. The FDA's Center for Devices and Radiological Health, in collaboration with the FDA's Center for Biologics Evaluation and Research, responded to this Zika emergency by developing and producing a reference panel (RP) for Zika RNA (Zika FDA-RP) suitable for performance assessment of ZIKV NAT-based in vitro diagnostic devices. Reference panels are a fundamental tool for performance assessment of molecular tests. The panel is composed of five vials: two different heat-inactivated ZIKV strains (PRVABC59 and FSS13025) in concentrated stocks and three blinded concentrations prepared from those strains. The Zika FDA-RP was shared with developers who had devices in the final stages of validation. In vitro diagnostic developers tested the Zika FDA-RP using the FDA-provided protocol. Depending on sample type, 85% (12/14) of the NAT assays had analytical sensitivities between 500 and 5000 RNA NAT-detectable units/mL (NDUs/mL). One device showed better performance (100 NDUs/mL), and another one showed lower performance (10,000 to 30,000 NDUs/mL). Vials of the Zika FDA-RP are available on request to developers who have interacted with the FDA through the review process.
Assuntos
RNA Viral/genética , Infecção por Zika virus/diagnóstico , Zika virus/genética , Humanos , Técnicas de Diagnóstico Molecular/normas , Saúde Pública , Kit de Reagentes para Diagnóstico , Padrões de Referência , Estados Unidos , United States Food and Drug Administration , Infecção por Zika virus/virologiaRESUMO
INTRODUCTION: Chagas disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), is the fourth most important tropical disease, which affects approximately 7 million people worldwide. The mechanisms involved in the development of this disease are not completely well understood. An important protective role of regulatory T cells (Treg) in Chagas disease has been observed; however, the specific mechanisms remain unclear. We evaluated apoptosis as a possible mechanism mediated by Treg cells (CD4+CD25HighFOXP3+) to orchestrate the immune response in chronic Chagas disease. METHODS AND RESULTS: Patients with Chagas disease were grouped as the indeterminate (IND; asymptomatic patients with Chagas disease; nâ¯=â¯10) and dilated cardiomyopathy (CARD; nâ¯=â¯10). Healthy T. cruzi-negative individuals (NI; nâ¯=â¯10) were included as a control group. In order to evaluate the apoptotic cell profile, the expression of PD1, PD1L, CD39, CD95, CD95L molecules were investigated. We also evaluated the proportion of CD14+ cells expressing caspase 3. The IND group presented a substantially higher expression of CD39 by Treg cells as compared to the CARD group. On the other hand, the CARD group showed higher expression of PD-1 by Treg cells than both NI and IND groups. Significant positive correlations were observed between Treg CD95L+ cells and CD14 cells expressing caspase 3 as well as between Treg CD39 cells and CD14+ Caspase3+ cells in the IND group. CONCLUSION: Our data indicate that the expressions of different molecules that induce apoptosis are associated with suppressive mechanisms mediated by Treg cells and suggest a possible role for PD1 and PDL1 molecules in the morbidity of chronic Chagas disease.
Assuntos
Antígeno B7-H1/metabolismo , Cardiomiopatia Dilatada/sangue , Cardiomiopatia Chagásica/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/imunologia , Trypanosoma cruzi/imunologia , Adulto , Idoso , Antígenos de Protozoários/imunologia , Apoptose/imunologia , Apirase/metabolismo , Antígenos CD4/metabolismo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Testes SorológicosRESUMO
We report here the complete genome sequences of two Zika virus strains (FSS13025 and PRVABC59) used for formulation of CBER/FDA RNA reference reagents and lot release panels for use with nucleic acid technology (NAT) testing.