ABSTRACT
Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.
Subject(s)
Disease Models, Animal , Encephalitis Viruses, Tick-Borne/pathogenicity , Encephalitis, Tick-Borne/virology , Hemorrhagic Fevers, Viral/virology , Macaca nemestrina , Animals , Chlorocebus aethiops , Cytokines/blood , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/immunology , Encephalitis, Tick-Borne/immunology , Encephalitis, Tick-Borne/pathology , Female , HEK293 Cells , Hemorrhagic Fevers, Viral/immunology , Hemorrhagic Fevers, Viral/pathology , Humans , Lymph Nodes/virology , Vero Cells , ViremiaABSTRACT
[This corrects the article DOI: 10.1371/journal.ppat.1006219.].
ABSTRACT
Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.
Subject(s)
Zika Virus Infection/pathology , Zika Virus Infection/virology , Animals , Cell Separation , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , In Situ Hybridization , Macaca mulatta , Male , Neutralization Tests , Polymerase Chain Reaction , Viremia/virology , Zika VirusABSTRACT
Chikungunya virus (CHIKV) is a reemerging alphavirus that causes acute febrile illness and severe joint pain in humans. Although acute symptoms often resolve within a few days, chronic joint and muscle pain can be long lasting. In the last decade, CHIKV has caused widespread outbreaks of unprecedented scale in the Americas, Asia, and the Indian Ocean island regions. Despite these outbreaks and the continued expansion of CHIKV into new areas, mechanisms of chikungunya pathogenesis and disease are not well understood. Experimental animal models are indispensable to the field of CHIKV research. The most commonly used experimental animal models of CHIKV infection are mice and nonhuman primates; each model has its advantages for studying different aspects of CHIKV disease. This review will provide an overview of animal models used to study CHIKV infection and disease and major advances in our understanding of chikungunya obtained from studies performed in these models.
Subject(s)
Chikungunya Fever , Chikungunya virus/pathogenicity , Disease Models, Animal , Animals , Chikungunya Fever/virology , Chikungunya virus/immunology , Chikungunya virus/physiology , Mice , PrimatesABSTRACT
Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.
Subject(s)
COVID-19 , Interferon Type I , Humans , Mice , Animals , Cytokines , SARS-CoV-2 , Mice, Transgenic , Inflammation/genetics , Disease Models, Animal , LungABSTRACT
Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type-specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Encephalitis Viruses, Tick-Borne/immunology , Encephalitis, Tick-Borne/immunology , Viral Vaccines/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , Cell Line , Chlorocebus aethiops , Cross Reactions/immunology , Encephalitis Viruses, Tick-Borne/drug effects , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/physiology , Encephalitis, Tick-Borne/prevention & control , Encephalitis, Tick-Borne/virology , Epitopes/immunology , HEK293 Cells , Humans , Immunoglobulin G/administration & dosage , Immunoglobulin G/immunology , Mice, Inbred C57BL , Mutation , Vero Cells , Viral Envelope Proteins/immunology , Viral Vaccines/administration & dosageABSTRACT
Repurposing FDA-approved drugs that treat respiratory infections caused by coronaviruses, such as SARS-CoV-2 and MERS-CoV, could quickly provide much needed antiviral therapies. In the current study, the potency and cellular toxicity of four fluoroquinolones (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) were assessed in Vero cells and A549 cells engineered to overexpress ACE2, the SARS-CoV-2 entry receptor. All four fluoroquinolones suppressed SARS-CoV-2 replication at high micromolar concentrations in both cell types, with enoxacin demonstrating the lowest effective concentration 50 value (EC50) of 126.4 µM in Vero cells. Enoxacin also suppressed the replication of MERS-CoV-2 in Vero cells at high micromolar concentrations. Cellular toxicity of levofloxacin was not found in either cell type. In Vero cells, minimal toxicity was observed following treatment with ≥37.5 µM enoxacin and 600 µM ciprofloxacin. Toxicity in both cell types was detected after moxifloxacin treatment of ≥300 µM. In summary, these results suggest that the ability of fluoroquinolones to suppress SARS-CoV-2 and MERS-CoV replication in cultured cells is limited.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Coronavirus Infections/drug therapy , Fluoroquinolones/pharmacology , Middle East Respiratory Syndrome Coronavirus/drug effects , SARS-CoV-2/drug effects , A549 Cells , Angiotensin-Converting Enzyme 2 , Animals , Cell Line , Chlorocebus aethiops , Ciprofloxacin/pharmacology , Enoxacin/pharmacology , Humans , Levofloxacin/pharmacology , Moxifloxacin/pharmacology , Vero CellsABSTRACT
Chikungunya virus (CHIKV) infections can cause severe and debilitating joint and muscular pain that can be long lasting. Current CHIKV vaccines under development rely on the generation of neutralizing antibodies for protection; however, the role of T cells in controlling CHIKV infection and disease is still unclear. Using an overlapping peptide library, we identified the CHIKV-specific T cell receptor epitopes recognized in C57BL/6 infected mice at 7 and 14 days post-infection. A fusion protein containing peptides 451, 416, a small region of nsP4, peptide 47, and an HA tag (CHKVf5) was expressed using adenovirus and cytomegalovirus-vectored vaccines. Mice vaccinated with CHKVf5 elicited robust T cell responses to higher levels than normally observed following CHIKV infection, but the vaccine vectors did not elicit neutralizing antibodies. CHKVf5-vaccinated mice had significantly reduced infectious viral load when challenged by intramuscular CHIKV injection. Depletion of both CD4+ and CD8+ T cells in vaccinated mice rendered them fully susceptible to intramuscular CHIKV challenge. Depletion of CD8+ T cells alone reduced vaccine efficacy, albeit to a lesser extent, but depletion of only CD4+ T cells did not reverse the protective phenotype. These data demonstrated a protective role for CD8+ T cells in CHIKV infection. However, CHKVf5-vaccinated mice that were challenged by footpad inoculation demonstrated equal viral loads and increased footpad swelling at 3 dpi, which we attributed to the presence of CD4 T cell receptor epitopes present in the vaccine. Indeed, vaccination of mice with vectors expressing only CHIKV-specific CD8+ T cell epitopes followed by CHIKV challenge in the footpad prevented footpad swelling and reduced proinflammatory cytokine and chemokines associated with disease, indicating that CHIKV-specific CD8+ T cells prevent CHIKV disease. These results also indicate that a T cell-biased prophylactic vaccination approach is effective against CHIKV challenge and reduces CHIKV-induced disease in mice.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chikungunya Fever/prevention & control , Chikungunya virus/immunology , Vaccination , Viral Vaccines/immunology , Animals , Chikungunya Fever/genetics , Chikungunya Fever/immunology , Chikungunya virus/genetics , Chlorocebus aethiops , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Vero Cells , Viral Vaccines/geneticsABSTRACT
Tripartite motif-containing protein 5α (TRIM5α) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5α is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we show that both human and rhesus macaque TRIM5α suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5α-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5α suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and proteasomal degradation. Importantly, TRIM5α contributes to the antiviral function of IFN-I against sensitive flaviviruses in human cells. Thus, TRIM5α possesses remarkable plasticity in the recognition of diverse virus families, with the potential to influence human susceptibility to emerging flaviviruses of global concern.
Subject(s)
Flavivirus Infections/metabolism , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex/metabolism , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Viral Proteins/metabolism , Virus Replication , Animals , Antiviral Restriction Factors , Cats , Chlorocebus aethiops , Dendritic Cells/metabolism , Dendritic Cells/virology , Flavivirus/pathogenicity , Flavivirus/physiology , Flavivirus Infections/virology , HEK293 Cells , Humans , Protein Binding , Proteolysis , Substrate Specificity , Ubiquitination , Vero CellsABSTRACT
Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10-8-10-6 M) or polyethylene glycol (PEG, molecular weight â¼8,000 Da, 10-7-10-4 M) increase the half-life of a green fluorescent protein expressing adenovirus from â¼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.