Highly pathogenic human coronaviruses associated with a severe respiratory syndrome, including Middle East respiratory syndrome coronavirus (MERS-CoV), have recently emerged. The MERS-CoV epidemic ...started in 2012 and is still ongoing, with a mortality rate of approximately 35%. No vaccine is available against MERS-CoV and therapeutic options for MERS-CoV infections are limited to palliative and supportive care. A search for specific antiviral treatments is urgently needed. Coronaviruses are enveloped viruses, with the spike proteins present on their surface responsible for virus entry into the target cell. Lectins are attractive anti-coronavirus candidates because of the highly glycosylated nature of the spike protein. We tested the antiviral effect of griffithsin (GRFT), a lectin isolated from the red marine alga Griffithsia sp. against MERS-CoV infection. Our results demonstrate that while displaying no significant cytotoxicity, griffithsin is a potent inhibitor of MERS-CoV infection. Griffithsin also inhibits entry into host cells of particles pseudotyped with the MERS-CoV spike protein, suggesting that griffithsin inhibits spike protein function during entry. Spike proteins have a dual function during entry, they mediate binding to the host cell surface and also the fusion of the viral envelope with host cell membrane. Time course experiments show that griffithsin inhibits MERS-CoV infection at the binding step. In conclusion, we identify griffithsin as a potent inhibitor of MERS-CoV infection at the entry step.
•We analyze the anti-MERS-CoV potential of the lectin griffithsin.•Griffithsin inhibits MERS-CoV infection at the entry step.•Griffithsin inhibits binding of MERS-CoV to the cell surface potentially by interacting with spike protein glycans.
Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that ...antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy, the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals.
More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV ...infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry, we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice. Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo. Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.
In most exposed individuals, hepatitis C virus (HCV) establishes a chronic infection; this long-term infection in turn contributes to the development of liver diseases such as cirrhosis and ...hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. We demonstrate that three broadly nAbs-AR3A, AR3B, and AR4A-delivered with adeno-associated viral vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a therapeutic approach to interfere with HCV infection by exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes to sustain a chronic infection.
Natural waters are contaminated globally with pharmaceuticals including many antibiotics. In this study, we assessed the acquisition of antimicrobial resistance in the culturable intestinal ...microbiota of rainbow trout (
Oncorhynchus mykiss
) exposed for 6 months to sub-inhibitory concentrations of sulfamethoxazole (SMX), one of the most prevalent antibiotics in natural waters. SMX was tested at three concentrations: 3000 µg/L, a concentration that had no observed effect (NOEC) on the in vitro growth of fish intestinal microbiota; 3 µg/L, a theoretical predicted no effect concentration (PNEC) for long-term studies in natural environments; and 0.3 µg/L, a concentration detected in many surveys of surface waters from various countries including the USA. In two independent experiments, the emergence of phenotypic resistance and an increased prevalence of bacteria carrying a sulfonamide-resistance gene (
sul
1) were observed in SMX-exposed fish. The emergence of phenotypic resistance to1000 mg/L SMX was significant in fish exposed to 3 µg/L SMX and was in large part independent of
sul
resistance genes. The prevalence of bacteria carrying the
sul
1 resistance gene increased significantly in the culturable intestinal microbiota of SMX-exposed fish, but the
sul
1-positive population was in large part susceptible to 1000 mg/L SMX, suggesting that the gene confers a lower resistance level or a growth advantage. The increased prevalence of
sul
1 bacteria was observed in all groups of SMX-exposed fish. Overall, this study suggests that fish exposed long-term to waters contaminated with low levels of antibiotics serve as reservoir of antimicrobial resistant genes and of resistant bacteria, a potential threat to public health.
Humanized mice have emerged as a promising model to study human immunity in vivo. Although they are susceptible to many pathogens exhibiting an almost exclusive human tropism, human immune responses ...to infection remain functionally impaired. It has recently been demonstrated that the expression of HLA molecules improves human immunity to lymphotropic virus infections in humanized mice. However, little is known about the extent of functional human immune responses in nonlymphoid tissues, such as in the liver, and the role of HLA expression in this context. Therefore, we analyzed human antiviral immunity in humanized mice during a hepatotropic adenovirus infection. We compared immune responses of conventional humanized NOD SCID IL-2Rγ-deficient (NSG) mice to those of a novel NOD SCID IL-2Rγ-deficient strain transgenic for both HLA-A*0201 and a chimeric HLA-DR*0101 molecule. Using a firefly luciferase-expressing adenovirus and in vivo bioluminescence imaging, we demonstrate a human T cell-dependent partial clearance of adenovirus-infected cells from the liver of HLA-transgenic humanized mice. This correlated with liver infiltration and activation of T cells, as well as the detection of Ag-specific humoral and cellular immune responses. When infected with a hepatitis C virus NS3-expressing adenovirus, HLA-transgenic humanized mice mounted an HLA-A*0201-restricted hepatitis C virus NS3-specific CD8(+) T cell response. In conclusion, our study provides evidence for the generation of partial functional antiviral immune responses against a hepatotropic pathogen in humanized HLA-transgenic mice. The adenovirus reporter system used in our study may serve as simple in vivo method to evaluate future strategies for improving human intrahepatic immune responses in humanized mice.
Abstract Hepatitis C virus (HCV) remains a major medical problem. In-depth study of HCV pathogenesis and immune responses is hampered by the lack of suitable small animal models. The narrow host ...range of HCV remains incompletely understood. We demonstrate that the entire HCV life-cycle can be recapitulated in mouse cells. We show that antiviral signaling interferes with HCV RNA replication in mouse cells. We were able to infect mouse cells expressing human CD81 and occludin (OCLN)—the minimal set of entry factor factors required for HCV uptake into mouse cells. Infected mouse cells sustain HCV RNA replication in the presence of miR122 and release infectious particles when mouse apoE is supplied. Our data demonstrate that the barriers of HCV interspecies transmission can be overcome by engineering a suitable cellular environment and provide a blue-print towards constructing a small animal model for HCV infection.
Mouse handling and restraint affect behavior, physiology, and animal welfare, yet little information is available on how various mouse restraint methods affect cardiovascular parameters. We validated ...the use of a smartphone-based ECG sys- tem in mice by performing simultaneous smartphone
and telemetry ECG recordings in conscious, restrained mice and in anesthetized mice. We observed that mice held in standard immobilizing restraint ("scruffing") experienced severe bradycardia. Mice of both sexes and 4 different strains (BALB/cJ, C57BL/6J, DBA/2J, and FVB/nJ) were restrained
by 3 handlers using 3 different restraint methods: light restraint; 3-finger restraint, which creates a dorsal transverse fold of skin; and the standard immobilizing restraint, which creates a dorsal longitudinal fold of skin that results in a crease on the ventral neck. Regardless of the
handler, immobilizing restraint, but not 3-finger restraint, produced severe bradycardia with irregular rhythm in all 4 strains and both sexes, with an average decrease in heart rate of 31%, or 211 bpm, and a maximal decrease of 79%, or 542 bpm. When evaluated using telemetry, immobilizing
restraint produced severe arrhythmias such as junctional and ventricular escape rhythms, and second- and third-degree atrioventricular block. Sinus pauses were observed for an average of 4 min, but up to 6.8 min after release from immobilizing restraint. Atropine administration to C57BL/6J
mice attenuated immobilizing restraint-induced bradycardia, supporting the hypothesis that pressure on cervical baroreceptors during stretching of the neck skin results in a vagally-mediated reflex bradycardia. Because of these profound cardiovascular effects, we recommend using the light
or 3-finger restraint and avoiding or minimizing the use of immobilization restraint while handling mice.
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