During infection with the hepatitis A virus (HAV), most patients develop mild or asymptomatic disease. However, a small number of patients develop serious, life-threatening hepatitis. We investigated this variability in disease severity by examining 30 Argentinean patients with HAV-induced acute liver failure in a case-control, cross-sectional, observational study. We found that HAV-induced severe liver disease was associated with a 6-amino-acid insertion in TIM1/HAVCR1 (157insMTTTVP), the gene encoding the HAV receptor. This polymorphism was previously shown to be associated with protection against asthma and allergic diseases and with HIV progression. In binding assays, the TIM-1 protein containing the 157insMTTTVP insertion polymorphism bound HAV more efficiently. When expressed by human natural killer T (NKT) cells, this long form resulted in greater NKT cell cytolytic activity against HAV-infected liver cells, compared with the shorter TIM-1 protein without the polymorphism. To our knowledge, the 157insMTTTVP polymorphism in TIM1 is the first genetic susceptibility factor shown to predispose to HAV-induced acute liver failure. Furthermore, these results suggest that HAV infection has driven the natural selection of shorter forms of the TIM-1 protein, which binds HAV less efficiently, thereby protecting against severe HAV-induced disease, but which may predispose toward inflammation associated with asthma and allergy.
Sensitive detection of pathogens is crucial for early disease diagnosis and quarantine, which is of tremendous need in controlling severe and fatal illness epidemics such as of Ebola virus (EBOV) disease. Serology assays can detect EBOV‐specific antigens and antibodies cost‐effectively without sophisticated equipment; however, they are less sensitive than reverse transcriptase polymerase chain reaction (RT‐PCR) tests. Herein, a 3D plasmonic nanoantenna assay sensor is developed as an on‐chip immunoassay platform for ultrasensitive detection of Ebola virus (EBOV) antigens. The EBOV sensor exhibits substantial fluorescence intensity enhancement in immunoassays compared to flat gold substrate. The nanoantenna‐based biosensor successfully detects EBOV soluble glycoprotein (sGP) in human plasma down to 220 fg mL−1, a significant 240 000‐fold sensitivity improvement compared to the 53 ng mL−1 EBOV antigen detection limit of the existing rapid EBOV immunoassay. In a mock clinical trial, the sensor detects sGP‐spiked human plasma samples at two times the limit of detection with 95.8% sensitivity. The results combined highlight the nanosensor's extraordinary capability of detecting EBOV antigen at ultralow concentration compared to existing immunoassay methods. It is a promising next‐generation bioassay platform for early‐stage disease diagnosis and pathogen detection for both public health and national security applications.
Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGPFc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use.
Human wild-type (wt) hepatitis A virus (HAV), the causative agent of acute hepatitis, barely grows in cell culture and in the process accumulates attenuating and cell culture-adapting mutations. This genetic instability of wt HAV in cell culture is a major roadblock to studying HAV pathogenesis and producing live vaccines that are not overly attenuated for humans. To develop a robust cell culture system capable of supporting the efficient growth of wt HAV, we transfected different cell lines with in vitro RNA transcripts of wt HAV containing the blasticidin resistance gene. Blasticidin-resistant colonies grew only in transfected Huh7 cells and produced infectious virus. HAV was genetically stable in Huh7 cells for at least nine serial passages and did not accumulate attenuating or cell culture-adapting mutations. Treatment with alpha interferon A/D cured the blasticidin-resistant Huh7 cells of the HAV infection. The cured cells, termed Huh7-A-I cells, did not contain virus or HAV antigens and were sensitive to blasticidin. Huh7-A-I cells were more permissive than parental cells for wt HAV infection, including a natural isolate from a human stool sample, and produced 10-fold-more infectious particles. This is the first report of a cell line that allows the genetically stable growth of human wt HAV. The viral vectors and cells described here should allow better insight into the pathogenesis of HAV and the development of attenuated vaccines. The cell lines susceptible to wt HAV growth may also be used to detect and isolate infectious virus from patient and environmental samples.Hepatitis A virus (HAV) is a member of the Picornaviridae that causes acute hepatitis in humans, a worldwide preventable infectious disease. In the United States, approximately 25,000 cases of HAV are reported each year, and an estimated average of 263,000 HAV cases occur annually when corrected for underreporting and asymptomatic infections (17). HAV is a nonenveloped virus that contains a 7.5-kb single-stranded positive-sense genomic RNA encapsidated in an icosahedral 27-to 32-nm particle. The viral RNA has a 5Ј-end nontranslated region of approximately 750 bases containing an internal ribosome entry site (IRES) (39), and a 3Ј-end short nontranslated region followed by a poly(A) tail. The viral protease 3C pro cleaves the HAV polyprotein into smaller structural (VP0, VP3, and VP1-2A) and nonstructural (2B, 2C, 3A, 3B, 3C, and 3D) proteins (references 25, 27, and 31 and references therein), and a cellular protease cleaves the VP1-2A precursor (24).Human wild-type (wt) HAV rarely grows in cell culture, is genetically unstable, and requires several weeks to months in culture before it can be detected (4,7,14,19). HAV has been adapted to a variety of primate (3, 9, 18, 34, 38) and nonprimate (10, 16) cell lines, but this process results in the accumulation of cell culture-adapting and attenuating mutations (4,5,9,11,20,34). During adaptation to cell culture, two main hot-spot mutation areas in the HAV genome were identified, one located in the...
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